JP4086392B2 - Detection method of indicator substance - Google Patents

Description

[0001]
[Technical field to which the invention belongs]
The present invention relates to an indicator substance detection method for detecting an indicator substance in a collected specimen.
[0002]
[Prior art]
Artificial dialysis is widely used for patients whose renal function is reduced or lost. In this artificial dialysis, the blood purification action originally performed by the kidney is performed instead of the kidney.
[0003]
As artificial dialysis, peritoneal dialysis is known in addition to hemodialysis using a blood extracorporeal circuit including a dialyzer.
[0004]
In peritoneal dialysis, excess water and waste in the living body are removed by storing peritoneal dialysis fluid having high osmotic pressure in the abdominal cavity surrounded by the peritoneum. That is, in peritoneal dialysis, excess water in the living body moves from peritoneal capillaries to peritoneal dialysate in the peritoneal cavity due to the osmotic pressure difference generated between peritoneal dialysate and body fluid stored in the peritoneal cavity. Excess water and waste in the body are removed.
[0005]
By the way, diseases such as peritonitis, peritoneal sclerosis, peritoneal fibrosis, sclerosing peritonitis, sclerosing capsular peritonitis (SEP) may be seen in patients undergoing peritoneal dialysis.
[0006]
For example, when peritoneal sclerosis, peritoneal fibrosis, sclerosing peritonitis, SEP, etc. develop, peritoneal thickening may occur, resulting in a decrease in water removal ability. In such a case, the dialysis effect may be reduced.
[0007]
When peritoneal sclerosis, peritoneal fibrosis, sclerosing peritonitis, SEP, etc. develop, it is preferable to make an accurate diagnosis as early as possible before the disease progresses for patient safety.
[0008]
For example, when SEP develops, it is clinically known to exhibit intestinal obstruction symptoms such as loss of appetite, nausea, nausea, vomiting, underweight nutrition, abdominal pain, diarrhea, constipation, and bowel peristalsis. . In addition, as a result of autopsy, it is known that the small intestine adheres and becomes a lump and is wrapped with a thick peritoneum rich in collagenous fibers.
[0009]
At present, SEP is diagnosed by palpation of the abdomen (tactile palpation) in addition to findings of clinical symptoms.
[0010]
However, in such a method, an objective standard for making an appropriate diagnosis has not been established. For this reason, in order to make an appropriate diagnosis by such a method, a lot of experience is required, and it is difficult for a person other than an experienced doctor to make an appropriate diagnosis. Also, the accuracy of diagnosis is low.
[0011]
In some cases, diagnosis by image analysis is also performed, but it is uneconomical in terms of cost and time, and does not leave the scope of auxiliary diagnosis.
[0012]
Furthermore, even in SEPs in which the above clinical symptoms are confirmed, even if the medical condition progresses, typical symptoms may not occur.
[0013]
On the other hand, peritoneal dialysis, unlike hemodialysis, can be performed at home or at the workplace and requires less frequent visits, contributing to the rehabilitation of patients. In addition, peritoneal dialysis has the advantage that it has less influence on the circulatory system and internal body environment than hemodialysis.
[0014]
Therefore, if peritoneal sclerosis, peritoneal fibrosis, sclerosing peritonitis, SEP, and peritonitis, peritoneal thickening, etc. can be diagnosed early and reliably, peritoneal dialysis can be performed more safely, and patients with reduced or lost renal function Can provide immense benefits.
[0015]
[Problems to be solved by the invention]
An object of the present invention is to provide an index that enables easy and accurate diagnosis of peritoneal function, and further diagnosis of peritonitis, for example, peritoneal sclerosis, peritoneal fibrosis, sclerosing peritonitis, and SEP. It is to provide a method for detecting a substance.
[0016]
[Means for Solving the Problems]
  Such purposes are as follows (1) to (13This is achieved by the present invention.
[0017]
  (1)Specimens that are collected peritoneal dialysis dialysate drainageFrom insideFirst and secondAn indicator substance detection method for detecting an indicator substance,
  SaidFirstThe indicator substance is the organism from which the specimen is collectedA matrix metalloproteinase 9 that is detected or confirmed to increase in concentration at least at the onset of peritonitis,
The second indicator substance is a matrix metalloproteinase 2 that is detected or confirmed to increase in concentration at least at the onset of peritoneal thickening in the organism from which the specimen is collected,
Detection means capable of separately detecting the first indicator substance and the second indicator substance is used.A method for detecting an indicator substance characterized by the above.
[0019]
  (2)First and secondThe indicator substance has a correlation with the D / D0 glucose value of the peritoneum of the organism from which the specimen is collected.(1)A method for detecting an indicator substance described in 1.
[0020]
  (3) If the D / D0 glucose value decreases,First and secondThe concentration of the indicator substance has a tendency to increase (2) The method for detecting an indicator substance according to (1).
[0021]
  (4)First and secondThe indicator substance can be detected from a specimen having a volume of 1 mL or less (1) to (3) A method for detecting an indicator substance according to any one of the above.
[0031]
  (5The dialysate drainage liquid of the peritoneal dialysis is a sampling liquid of the drainage collected in the drainage container of the peritoneal dialysis.One of (1) to (4)A method for detecting an indicator substance described in 1.
[0032]
  (6) After adjusting the concentration of the collected sampleFirst and second(1) to (1) for detecting the indicator substance5) A method for detecting an indicator substance according to any one of the above.
[0033]
  (7) After adjusting the temperature of the collected sample,First and second(1) to (1) for detecting the indicator substance6) A method for detecting an indicator substance according to any one of the above.
[0034]
  (8) Adjust the temperature of the collected sample to room temperature or lower, and thenFirst and second(1) to (1) for detecting the indicator substance7) A method for detecting an indicator substance according to any one of the above.
[0035]
  (9)First and secondIn the detection of the indicator substance,First and second(1) to (1) for separating and visualizing the indicator substance8) A method for detecting an indicator substance according to any one of the above.
[0036]
  (10)First and secondThe detection of the indicator substance is performed by electrophoresis (1) to (9) A method for detecting an indicator substance according to any one of the above.
[0037]
  (11) The gel used for the electrophoresis isFirst and secondThe above having a substrate for visualizing the indicator substance (10) The method for detecting an indicator substance according to (1).
[0038]
  (12The above substrate is gelatin (above)11) The method for detecting an indicator substance according to (1).
[0039]
  (13)First and secondThe indicator substance is detected by the zymography method (1) to (12) A method for detecting an indicator substance according to any one of the above.
[0040]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail based on preferred embodiments.
[0041]
The present invention is characterized in that a sample is collected and then a predetermined index substance is detected from the sample.
[0042]
The index substance in the present invention is a substance that is detected in connection with a decrease in peritoneal function of an organism from which a specimen is collected or whose increase in concentration is confirmed. That is, the index substance in the present invention is a substance detected from a specimen or a substance whose increase in concentration is confirmed in relation to a decrease in the function of the peritoneum of the organism from which the specimen is collected.
[0043]
The decrease in peritoneal function occurs due to various causes, for example, when peritonitis or peritoneal thickening occurs.
[0044]
Here, peritonitis refers to inflammation of the peritoneum, and peritonitis used in the present specification is synonymous with peritonitis that is widely used.
[0045]
Further, as used herein, peritoneal thickening is a general term for peritoneal diseases such as peritoneal sclerosis, peritoneal fibrosis, sclerosing capsular peritonitis, sclerosing peritonitis, and at the onset of such peritoneal thickening, for example, A phenomenon in which the thickness of the peritoneum increases is observed.
[0046]
The present inventor conducted research and research on specimens collected from organisms with reduced peritoneal function, and found that when peritoneal functions declined, particularly when peritonitis or peritoneal thickening developed, Discovered that there are substances that can be detected automatically and substances that increase in concentration in the specimen.
[0047]
Therefore, the function of the peritoneum can be examined by detecting and identifying such a substance, that is, the index substance from the specimen, and further, objective judgment materials for diagnosing peritonitis and peritoneal thickening are obtained. be able to.
[0048]
The present inventor conducted various investigations and researches on specimens collected from organisms with reduced peritoneal function (particularly organisms that developed peritonitis or peritoneal thickening), and found various indicator substances.
[0049]
An example of such an indicator substance is protein. Examples of such proteins include enzymes such as proteinases, nucleases, ligases, isomerases, kinases, phosphatases, transcription factors (eg, repressors, activators, promoters, etc.), cell growth factors, cytokines and their receptors, immunity Globulin (antibody), extracellular matrix (such as fibronectin) and the like. Among these, from the viewpoint of ease of detection and sensitivity, enzymes, and in particular, various proteinases such as matrix proteinases, metalloproteinases, serine proteinases (plasmin, trypsin, t-PA, u-PA, kallikrein, etc.) Cysteine proteinase (such as cathepsin B and H), aspartic proteinase (such as calapsin-D) and the like are preferable.
[0050]
Examples of such an indicator substance include various genes such as a gene encoding a protein as described above (for example, mRNA, hnRNA, DNA, etc.) and a gene containing an operator region or a promoter region of such a gene.
[0051]
Examples of such indicator substances include various effectors such as an effector that regulates the activity of a protein (particularly an enzyme) as described above, and an inhibitor (inhibitor substance) that inhibits the activity of a protein (particularly an enzyme) as described above. Examples thereof include a fragment of a substrate substance cleaved and decomposed by a ligand and the proteinase.
[0052]
Examples of such indicator substances include plasminogen activator inhibitor (PAI), tissue inhibitor of MMPs (TIMP), and the like.
[0053]
The indicator substance is preferably a substance involved in the destruction or reconstruction of the extracellular matrix of the peritoneal tissue of the organism from which the specimen is collected.
[0054]
For example, when peritoneal function declines due to the development of peritonitis or thickening of the peritoneum, especially during the onset of peritoneal thickening, the destruction of the extracellular matrix and the formation of a new extracellular matrix are performed in the peritoneal tissue. It is thought that construction will take place.
[0055]
For this reason, if the substance involved in the destruction or reconstruction of the extracellular matrix of the peritoneal tissue of the organism from which the specimen is collected is used as an indicator substance, the peritoneal function can be examined, and further, the diagnosis of peritonitis and peritoneal thickening It can be performed. Moreover, by using such a substance as an indicator substance, it is possible to examine a more detailed cause of the decrease in peritoneal function, for example, which of peritonitis and peritoneal thickening has occurred.
[0056]
Furthermore, it is preferable that the indicator substance has a correlation with the D / D0 glucose value of the peritoneum of the organism from which the specimen is collected, and particularly is one that is detected prior to the decrease in the D / D0 glucose value. Here, the D / D0 glucose value means that a predetermined time has elapsed since a glucose-containing peritoneal dialysis solution having a predetermined glucose concentration D0 (for example, 2.5 W / V%) was injected into the abdominal cavity of an organism. This is the value obtained by dividing D by D0, where D is the glucose concentration of the peritoneal dialysate later (for example, after 90 minutes).
[0057]
When such a substance is used as an indicator substance, the peritoneal function of the organism from which the specimen is collected can be more easily examined. Further, if there is an abnormality such as a disease in the peritoneal tissue, the symptom is determined. It will be a judgment material when doing. In addition, when such a substance is used as an indicator substance, it becomes possible to accurately diagnose peritonitis and peritoneal thickening.
[0058]
Among them, it is preferable that the indicator substance has a tendency that the concentration of the indicator substance increases when the D / D0 glucose value decreases. When such a substance is used as an indicator substance, the peritoneal function can be performed more accurately, and further, diagnosis of diseases such as peritonitis and peritoneal thickening can be performed more accurately.
[0059]
Taking these viewpoints into consideration, matrix proteinase is particularly preferable as the indicator substance among the above-described substances. Matrix proteinases are associated with a decrease in peritoneal function, particularly at the onset of peritonitis and peritoneal thickening, and are particularly easy to detect. In addition, this substance is thought to be involved in the destruction of the extracellular matrix of peritoneal tissue. Furthermore, if the D / D0 glucose level decreases, an increase in the concentration is confirmed in the specimen prior to the decrease. It's easy to do. Therefore, by using matrix proteinase as an indicator substance, it is possible to more easily test for peritoneal function, and further diagnose peritonitis and peritoneal thickening.
[0060]
Examples of matrix proteinases include matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 7 (MMP7), matrix metalloproteinase 8 (MMP8), matrix metalloproteinase Matrix metalloproteinases such as 9 (MMP9).
[0061]
In addition, it is preferable that the indicator substance includes a plurality of types of indicator substances corresponding to the origin of the decrease in peritoneal function (a plurality of indicator substances corresponding to diseases of the peritoneum of the organism from which the specimen is collected). That is, the indicator substance preferably contains a plurality of kinds of indicator substances that tend to be detected or confirmed to increase in concentration when a specific disease develops in the peritoneum. This facilitates determination of the type of disease that has occurred in the peritoneum.
[0062]
In particular, the indicator substance includes an indicator substance that is detected or confirmed to increase in concentration at least at the onset of peritonitis, and an indicator substance that is detected or confirmed to increase in concentration at least at the onset of peritoneal thickening. It is preferable that When the index substance contains such a substance, it is possible to distinguish whether peritonitis or peritoneal thickening has occurred in the organism from which the specimen has been collected by simply detecting the index substance. In addition, when the indicator substance contains such a substance, it becomes possible to make a more accurate and detailed diagnosis of peritonitis and peritoneal thickening.
[0063]
Examples of an indicator substance that is detected or confirmed to increase in concentration at least at the onset of peritonitis include, for example, a matrix metalloproteinase 9, a matrix metalloproteinase of about 50 kDa, a matrix metalloproteinase 2 dimer, and the like. Can be mentioned. The “about 50 kDa matrix metalloproteinase” means, for example, a proteinase detected as a band in the vicinity of 50 kDa when an indicator substance is detected by a zymography method (especially gelatin zymography method) described later. In addition, “what is considered to be a dimer of matrix metalloproteinase 2” means that, for example, when an indicator substance is detected by a zymography method (especially gelatin zymography method) described later, the matrix metalloproteinase 2 is almost the same. It refers to a proteinase detected as a band at a position showing twice the molecular weight (see FIG. 3).
[0064]
As an indicator substance that is detected at least at the onset of peritoneal thickening or whose increase in concentration is confirmed, for example, matrix metalloproteinase 2, proteinase having a slightly higher molecular weight than matrix metalloproteinase 2, and matrix metalloproteinase 9 are larger. Examples include proteinases having a molecular weight. The “proteinase having a molecular weight slightly larger than that of the matrix metalloproteinase 2” is a little higher than that of the matrix metalloproteinase 2 when, for example, an indicator substance is detected by a zymography method (especially gelatin zymography method) described later. A proteinase detected as a band at a position showing a high molecular weight is referred to, and “a proteinase having a molecular weight larger than that of matrix metalloproteinase 9” is indicated by, for example, a zymography method (particularly a gelatin zymography method) described later. When a substance is detected, the proteinase is detected as a band at a position showing a molecular weight higher than that of the matrix metalloproteinase 9 (see FIG. 7).
[0065]
The indicator substance is preferably detectable from a sample having a volume of 1 mL or less, more preferably detectable from a sample having a volume of 0.1 mL or less, and detectable from a sample having a volume of 0.01 mL or less. More preferably. When such a substance is used as an indicator substance, the indicator substance can be detected with a small amount of specimen. Thereby, when carrying out the present invention, the amount of specimen to be collected can be reduced.
[0066]
The specimen used in the present invention is not particularly limited, and examples thereof include body fluid such as blood and lymph, excrement such as urine, and dialysate drainage such as peritoneal dialysis and hemodialysis. Among them, the specimen used in the present invention is preferably dialysate drainage, particularly peritoneal dialysis dialysate drainage. The inventor has found that the indicator substance is particularly easily detected from such a specimen. Therefore, when such a substance is used as a specimen, it becomes easy to detect the indicator substance.
[0067]
As the dialysis fluid drainage for peritoneal dialysis, a sample obtained by sampling a part of the drainage collected in the drainage container (drainage bag) for peritoneal dialysis is preferably used. The drainage container is separated from the catheter connected to the abdomen of the patient undergoing peritoneal dialysis and shielded by closing the flow path, so that when the sample is collected, bacteria or the like enter the patient's body. This can more reliably prevent infectious diseases from being induced in such patients.
[0068]
Any known method may be used to collect the sample. However, when a part of the drainage collected in the drainage container for peritoneal dialysis is sampled, for example, it is provided in the drainage container. Examples include a method of collecting from the sampling tube, a method of sealing and cutting a part of the tube communicating with the drainage container, and collecting the drainage remaining in the tube.
[0069]
Before performing the operation of detecting the index substance, it is preferable to adjust the sample amount in order to accurately detect the index substance. In particular, the adjustment of the sample amount is preferably performed using an instrument (for example, a pipetman, a syringe, etc.) that can accurately measure the volume of the sample. As a result, the indicator substance can be detected more accurately, and a test for peritoneal function, and further a diagnosis such as peritonitis and peritoneal thickening can be performed more accurately.
[0070]
When detecting the indicator substance from the collected specimen, the indicator substance is separated from the specimen and visualization thereof, that is, when detecting the indicator substance, the operation of separating the indicator substance, and the indicator substance It is preferable to perform an operation for visualizing. Thereby, even when many impurities exist in the specimen, the indicator substance can be easily detected and the indicator substance can be detected more accurately. Therefore, by separating the indicator substance and visualizing the indicator substance, it becomes possible to more accurately perform a test of peritoneal function and further a diagnosis of peritonitis and peritoneal thickening.
[0071]
Examples of the detection method (separation method) of the indicator substance include, for example, an electrophoresis method, an affinity chromatography, an ion exchange chromatography, a gel filtration chromatography, a chromatography method using reverse phase chromatography, and an indicator substance. Examples thereof include a precipitation method, a mass spectrum measurement method, and various immunological techniques such as an ELISA method.
[0072]
Among these, in particular, when the indicator substance is a protein or a gene, when the indicator substance is separated using electrophoresis, the indicator substance can be easily and accurately separated with a small amount of sample.
[0073]
The gel used for electrophoresis preferably contains polyacrylamide when the indicator substance is a protein. When the gel used for electrophoresis contains polyacrylamide, the resolution of the indicator substance is improved.
[0074]
Further, the gel used for electrophoresis preferably contains a surfactant such as SDS when the indicator substance is a protein. Thereby, the indicator substance can be detected more accurately.
[0075]
As a method for visualizing the indicator substance, for example, a method for staining the indicator substance, a method for checking the fluorescence by labeling the indicator substance with a fluorescent substance, and a labeling substance using a chromogenic substrate (alkaline phosphatase, HPR, etc.). Confirmation method, method of confirming the presence of radiation by labeling the indicator substance with a radioactive substance, method of measuring the absorption of visible light, ultraviolet rays, infrared rays, etc. of the indicator substance, method of measuring the activity of the indicator substance, indicator substance And a method for measuring the mass spectrum. Furthermore, examples of the method for visualizing the indicator substance include a method for visualizing the indicator substance by adsorbing, binding, or reacting the substance that can be visualized by the above-described method to the indicator substance.
[0076]
Furthermore, when the indicator substance is detected by a method in which only the substance group including the indicator substance is visualized, the indicator substance can be suitably detected. In particular, when the indicator substance is detected by electrophoresis, the gel used for electrophoresis promotes visualization of the indicator substance, particularly the indicator substance, and visualizes substances other than the indicator substance. It preferably contains a substrate that makes it difficult to occur. This facilitates the confirmation of the indicator substance when the indicator substance is visualized after electrophoresis. For this reason, it becomes possible to more accurately diagnose peritoneal function tests and further peritonitis and peritoneal thickening.
[0077]
For example, when the indicator substance is a proteinase such as a matrix proteinase, detection can be suitably performed by separating and visualizing the indicator substance by a zymography method using electrophoresis. This is because when the proteinase is detected by the zymography method, impurities other than the proteinase are difficult to detect, so that the indicator substance can be detected more accurately.
[0078]
The gel used for the zymography method preferably contains polyacrylamide for the same reason as described above. Further, the gel used in the zymography method preferably contains a surfactant such as SDS for the same reason as described above.
[0079]
In addition, the gel used in the zymography method preferably contains a substrate for visualizing an indicator substance such as gelatin, casein, elastin, fibrin, etc. in order to more suitably detect proteinase.
[0080]
In the case where the indicator substance is proteinase and the detection is performed by electrophoresis or zymography, and the gel used in such a method contains a substrate for visualizing the indicator substance The concentration of the substrate is not particularly limited, but is preferably about 0.01 to 1 W / V%, more preferably about 0.05 to 0.5 W / V%. When the concentration of the substrate is in this range, the indicator substance can be detected more suitably.
[0081]
In addition, when the indicator substance is matrix proteinase and the detection is performed by electrophoresis or zymography, and the gel used in such a method contains polyacrylamide, the content of polyacrylamide Is preferably about 2 to 30 W / V%, and more preferably about 5 to 15 W / V%. When the content of polyacrylamide is within this range, matrix proteinase can be detected more accurately.
[0082]
Further, when the indicator substance is a matrix proteinase and the detection is performed by electrophoresis or zymography, and the gel used in such a method contains a surfactant, such a surfactant is used. The content of is preferably about 0.0001 to 15 W / V%, more preferably about 0.01 to 5 W / V%. When the content of the surfactant is within this range, the matrix proteinase can be detected more accurately.
[0083]
When the indicator substance is DNA, the indicator substance may be detected after the DNA is amplified. Examples of the method for amplifying DNA include a PCR method and a method for cloning DNA.
[0084]
In addition, when the indicator substance is RNA such as mRNA or hnRNA, the cDNA of RNA may be synthesized using reverse enzyme, and such cDNA may be detected.
[0085]
When detecting the indicator substance, the indicator substance may be detected after adjusting the concentration of the collected specimen. As a result, the specimen can be adjusted to conditions suitable for detection of the indicator substance, and the indicator substance can be detected more accurately.
[0086]
Examples of the method for adjusting the concentration of the sample include a method of diluting the sample and a method of concentrating the sample.
[0087]
When diluting the specimen, it is preferable to adjust the liquid used for dilution to a pH suitable for detection of the specimen. Thereby, the specimen can be detected more accurately.
[0088]
Such pH is not particularly limited, but is preferably about 3 to 11 and more preferably about 5 to 9 when the indicator substance is proteinase.
[0089]
In addition, when the indicator substance is a protein and the indicator substance is detected by electrophoresis or zymography, and the sample is diluted before such detection, the liquid used for dilution is It preferably contains a surfactant such as SDS. Thereby, the indicator substance can be detected more accurately.
[0090]
In such a case, the surfactant content is preferably about 0.0001 to 20 W / V%, more preferably about 0.01 to 10 W / V%. When the content of the surfactant is within this range, the indicator substance can be detected more accurately.
[0091]
When detecting the indicator substance, the indicator substance may be detected after adjusting the temperature of the collected specimen to a predetermined temperature. Although there are various advantages, it can be adjusted to conditions suitable for the detection of the indicator substance, and the advantage that the indicator substance can be detected more accurately is great.
[0092]
For example, the index substance can be detected more accurately by detecting the index substance after adjusting the temperature of the collected specimen to room temperature or lower (for example, 0 to 25 ° C.).
[0093]
【Example】
“%” Indicating the concentration in the following examples means W / V% unless otherwise specified.
[0094]
1. Correlation of indicator substances with peritonitis and peritoneal function
[1.1] Onset of peritonitis
[0095]
The experimental animal developed peritonitis.
First, SPF / VAF rats (Crj: CD (SD) IGS, 6 weeks old, n = 4, male) were prepared as experimental animals.
[0096]
Next, after anesthetizing the rats with ether anesthesia, a silicone catheter was placed so that the peritoneal dialysate could be poured and drained into the abdomen.
[0097]
Next, peritoneal dialysis was performed. At this time, rats were injected with 50 mL / kg of peritoneal dialysis solution per rat body weight. The peritoneal dialysis solution was exchanged once a day via an indwelling silicon catheter. For peritoneal dialysate, Peritrick 135 (manufactured by Terumo) was used. During this experiment, rats were supplied with a sufficient amount of food and water.
[0098]
As a result of continuous peritoneal dialysis, all rats developed peritonitis due to tunnel infection at the catheter site from the 10th day after the start of peritoneal dialysis. The onset of peritonitis was judged by a marked increase in granulocytes in the dialysis drainage. Pseudomonas maltophilia was identified as a result of a bacterial test in the dialysis effluent to elucidate the cause of peritonitis. It is considered that this bacterium is likely to cause peritonitis.
[0099]
From the third day after the onset of peritonitis, the antibiotic permeant dialysis solution was mixed with bixillin (trade name: manufactured by Meiji Seika Co., Ltd.) and peritoneal dialysis was continued.
[0100]
Furthermore, on the 20th day after the onset of peritonitis, the rat peritoneum was sampled. The collected peritoneum was fixed with 10% formalin neutral buffer, then embedded in paraffin, and pathological analysis was performed. As a result, no peritoneal thickening was observed even 20 days after the onset of peritonitis, and only mild peritoneal mesothelial cell proliferation and extremely mild peritoneal mesothelial cell cubing were observed. FIG. 1 shows a paraffin section pathological image of the peritoneum at this time.
[0101]
[1.2] Peritoneal function evaluation test
In order to analyze the peritoneal function at the onset of peritonitis, in the experiment of [1.1] above, the peritoneal function of the rat was examined on the first day of the start of peritoneal dialysis and on the fifth day after the onset of peritonitis.
[0102]
Peritoneal function was evaluated by glucose permeability in the peritoneum, that is, D / D0 glucose value. The test for calculating the D / D0 glucose value was performed as follows.
[0103]
First, a peritoneal dialysis solution containing 2.5% glucose at 50 mL / kg body weight of rat (Terumo Corporation: “Peritric P250”) was injected into the abdominal cavity of the rat, and then after 0 minutes, 30 minutes, After 120 minutes and after 240 minutes, 0.5 mL of peritoneal dialysis solution injected into the abdominal cavity was collected.
[0104]
Next, the glucose concentration (D) of each collected sample was determined, and this was divided by the glucose concentration (D0) in the peritoneal dialysis solution at the time of intraperitoneal injection, thereby calculating the D / D0 glucose value. The glucose concentration was measured by the mulotase enzyme method.
The result is shown in FIG.
[0105]
As shown in FIG. 2, on the fifth day after the onset of peritonitis, glucose permeability of the rat peritoneum was remarkably enhanced. Thereby, it was confirmed that the peritoneal function fell on the 5th day after the onset of peritonitis.
[0106]
[1.3] Detection of indicator substance from dialysate drainage
The indicator substance was detected using the dialysate drainage obtained in the above experiment [1.1] as a specimen.
[0107]
In order to detect the indicator substance, the gelatin zymography method (electricity) was applied to the dialysate drainage fluid on the first day onset of the peritonitis obtained in the experiment [1.1] and on the first, third, and fifth days after the onset. Electrophoresis). As a comparative example, gelatin zymography was also performed on the dialysate drainage before the onset of peritonitis obtained in the experiment of [1.1] above. The method will be described in detail below.
[0108]
First, a sample sampled from the dialysate drainage (collected in a drainage container) obtained in the experiment of [1.1] above was used as a normal temperature (20 ° C.) electrophoresis buffer (0.09 M Tris-HCl ( (pH 6.8) / 3% SDS / 10% glycerol / bromophenol blue). As a result, the temperature of the specimen was adjusted to room temperature. For the comparative example, specimens were similarly prepared, and the following experiment was performed in the same manner as in this example.
[0109]
Next, 10 μL of the dialysate effluent diluted with this electrophoresis buffer was collected with a Pipetman, and this was electrophoresed on a polyacrylamide gel (14% gelatin / 0.1% SDS / 10% polyacrylamide). It was. The electrophoresis buffer (running buffer) was prepared with a composition of 0.025 M Tris-HCl / 0.19 M glycine / 0.001% SDS.
Thereby, the indicator substance was separated.
[0110]
After completion of electrophoresis, the electrophoresed gel was immersed in 50 mM Tris-HCl (pH 7.5) /0.1 M sodium chloride / 2.5% Triton X-100 solution (linaturation buffer). Shake for 5 hours.
[0111]
Next, this gel was washed three times with distilled water, subsequently immersed in 50 mM Tris-HCl (pH 7.5) / 10 mM calcium chloride solution (reaction buffer), and reacted at 37 ° C. for 18 hours.
[0112]
After completion of the reaction, the gel was washed three times with distilled water and then immersed in a staining solution (1.25% Coomassie Brilliant Blue R-250 / 45% ethanol / 10% acetic acid aqueous solution) for 20 minutes for staining.
[0113]
After staining, the gel is washed with a first washing solution (25% ethanol / 8% acetic acid aqueous solution), then the gel is washed with a second washing solution (5% ethanol / 7.5% acetic acid aqueous solution), and finally distilled water. The gel was washed with.
[0114]
Thereby, the indicator substance was visualized in the gel (zymography).
The results obtained by the zymography method are shown in FIG.
[0115]
As a result of observing the obtained zymography, as shown in FIG. 3, a marked increase in the concentration of matrix metalloproteinase 9 (MMP9) was observed at the onset of peritonitis. Furthermore, a matrix metalloproteinase of about 50 kDa, which was not confirmed at all before the onset of peritonitis, was also observed on the day of the onset of peritonitis in all individuals. In addition, proteinase having a higher molecular weight than MMP9 was also observed. This substance can also be an indicator substance.
[0116]
On the other hand, even when peritonitis developed, the concentration of matrix metalloproteinase 2 (MMP2) hardly changed.
[0117]
2. Correlation of indicator substances with peritoneal thickening and peritoneal function
[2.1] Development of peritoneal thickening
[0118]
[2.1.1] Method of developing peritoneal thickening
Prior to explaining the next experiment described in [2.1.2], a method of developing peritoneal thickening (a method of developing peritoneal thickening) will be described first.
[0119]
This method of developing peritoneal thickening is to cause peritoneal thickening by administering an inducer that induces thickening in the abdominal cavity of an experimental animal. As a result, peritoneal thickening can be artificially developed in experimental animals.
[0120]
Examples of the inducer include minerals such as talc (silicate mineral), sugar oxide derivatives such as chlorhexidine gluconate, and the like.
[0121]
From the viewpoint of generating only peritoneal thickening with high probability, the dosage of the inducer is preferably about 0.001 to 10 g / kg in the case of minerals, more preferably about 0.01 to 1 g / kg per body weight of the experimental animal. Preferably, in the case of a sugar oxide derivative, about 0.0001 to 1 g / kg is preferable, and about 0.001 to 0.1 g / kg is more preferable.
[0122]
The inducer may be administered directly into the peritoneal cavity or may be added to the peritoneal dialysis solution and administered while performing peritoneal dialysis.
[0123]
[2.1.2] Development of peritoneal thickening
Experimental animals developed peritoneal thickening.
[0124]
First, SPF / VAF rats (Crj: CD (SD) IGS, 8 weeks old, n = 4, male) were prepared as experimental animals.
[0125]
The rats were divided into a plurality of groups, specifically, a 7% GSP administration group, an infection group, a chlorhexidine gluconate administration group, a talc administration group, and a control group.
And the following operation was performed with respect to each divided group.
[0126]
In the 7% GSP administration group, 15 mL of peritoneal dialysis solution containing 7% glucose (the electrolyte composition was the same as “Peritrick” manufactured by Terumo Corporation) was administered intraperitoneally.
[0127]
In the infectious disease group, Pseudomonas maltophilia was cultured overnight in LB medium (1% bactotryptone / 5% yeast extract / 1% sodium chloride / pH 7.2), and the collected cells were collected in 15 mL of physiological saline. A suspension (1/100 dilution) in water (manufactured by Terumo) was administered intraperitoneally to the rat.
[0128]
In the chlorhexidine gluconate administration group, 3 mL of 0.1% chlorhexidine gluconate / 15% ethanol aqueous solution was administered intraperitoneally to rats.
[0129]
In the talc administration group, 1 g of talc was suspended in 15 mL of physiological saline (manufactured by Terumo), and then this was administered intraperitoneally to rats.
[0130]
These administration groups, except the talc administration group, were administered continuously for 3 weeks on a schedule of 5 days continuous administration / 2 days rest. The talc administration group was performed 3 cycles with a cycle of resting for 6 days after administration. In addition, administration of the above substance into the abdominal cavity of rats was performed by injection using a syringe. During this experiment, rats were supplied with a sufficient amount of food and water.
A non-administration group was provided as a control group.
[0131]
And in each group, the wall side peritoneum was extract | collected on the 22nd day after the experiment start, and peritoneal pathological analysis was performed. That is, the collected peritoneum was fixed with a formalin buffer solution in the same manner as described above, then embedded in paraffin, and the tissue image of the peritoneum was analyzed.
[0132]
Analyzed wall side peritoneal pathological slice images are shown in FIGS. 4 and 5, the arrow indicates the thickness of the peritoneum. 4 is a 7% GSP administration group, FIG. 4 b is a chlorhexidine gluconate administration group, FIG. 5 c is a talc administration group, and FIG. 5 d is a wall side peritoneal pathological section of an infection group. Each image is shown.
[0133]
As a result of this analysis, as shown in FIGS. 4 and 5, significant peritoneal thickening rich in fibrous matrix was confirmed only in the chlorhexidine gluconate administration group and the talc administration group. At the same time, infiltration such as macrophages was observed.
[0134]
In particular, peritoneal mesothelial cells were detached from the wall side peritoneum of rats in the chlorhexidine gluconate administration group, causing peritoneal sclerosis. Upon further examination, in this rat, the intestinal tract and the liver were wrapped in a thickened peritoneum, and encapsulated peritoneal sclerosis occurred.
[0135]
On the other hand, no peritoneal thickening was observed in the 7% GSP administration group, the infection group and the control group.
[0136]
[2.2] Peritoneal function evaluation test
In order to analyze the peritoneal function at the onset of peritoneal thickening, the peritoneal function of the rat was examined on the 22nd day from the start of the experiment in the experiment of [2.1.2] above.
[0137]
Peritoneal function was evaluated by glucose permeability in the peritoneum, that is, D / D0 glucose value. The test for calculating the D / D0 glucose value was performed as follows.
[0138]
17 mL of 2.5% glucose-containing peritoneal dialysis solution (manufactured by Terumo: “Peritric P250”) was intraperitoneally administered in the same manner as described above, and then injected into the peritoneal cavity 90 minutes later. 0.5 mL was collected.
[0139]
The glucose concentration and D / D0 glucose value were calculated in the same manner as in [1.2] above.
The result is shown in FIG.
[0140]
As shown in FIG. 6, in the 7% GSP administration group, the peritoneal function was slightly reduced as compared to the control group, and in the infection group, the peritoneal function was further reduced.
[0141]
Furthermore, in the chlorhexidine gluconate administration group and the talc administration group in which peritoneal thickening was observed, peritoneal function was significantly reduced as compared to the 7% GSP administration group, the infection group, and the control group.
[0142]
[2.3] Detection of indicator substance from drainage
Using the drainage of the peritoneal dialysis fluid of each rat obtained from the abdominal cavity in the experiment of [2.2] above as a specimen, the indicator substance was detected.
[0143]
In order to detect the indicator substance, a gelatin zymography method (electrophoresis method) was applied to a sample (peritoneal dialysate) collected from the abdominal cavity of each rat on the 22nd day of the experiment in the experiment [2.2]. ) The method will be described in detail below.
[0144]
First, the sample obtained in the experiment of [2.2] was diluted 200 times with the same electrophoresis buffer as in [1.3]. Thereby, the temperature of the specimen was adjusted to room temperature.
[0145]
Next, 10 μL of a sample diluted with this electrophoresis buffer was collected with Pipetman, and this was electrophoresed on a polyacrylamide gel in the same manner as in [1.3] above.
Thereby, the indicator substance was separated.
[0146]
Next, the indicator substance was visualized in the same manner as in [1.3].
The results obtained by the zymography method are shown in FIG.
[0147]
As a result of observing the obtained zymography, as shown in FIG. 7, in the chlorhexidine gluconate administration group and the talc administration group, the matrix metalloproteinase 2 significantly increased compared to the 7% GSP administration group, the infection group and the control group. (MMP2) increased. That is, a significant increase in MMP2 was confirmed in the group that caused peritoneal thickening.
[0148]
In addition, specific matrix proteinases other than MMP2 were observed in the group showing peritoneal thickening, but matrix metalloproteinase 9 (MMP9) was hardly confirmed.
[0149]
3. Summary
From the above results, it was confirmed that an increase in matrix metalloproteinase 9 was observed when peritonitis developed and peritoneal function decreased.
[0150]
Moreover, when peritoneal thickening developed and peritoneal function fell, it was confirmed that the increase in matrix metalloproteinase 2 is seen.
[0151]
Furthermore, it was confirmed that the matrix proteinase tends to increase when the D / D0 glucose value of the organism from which the specimen was collected decreases.
[0152]
【The invention's effect】
As described above, according to the present invention, it is possible to easily test the peritoneal function of the organism from which the specimen is collected.
[0153]
In addition, according to the present invention, it is only necessary to detect the indicator substance, so that the inspection can be easily performed and the accuracy thereof is high.
[0154]
Furthermore, according to the present invention, it is possible to obtain an objective judgment material when diagnosing a disease occurring in the peritoneum such as peritonitis and peritoneal thickening.
[0155]
In particular, when the indicator substance contains a plurality of indicator substances corresponding to the disease state of the peritoneum of the organism from which the specimen is collected, it becomes easy to specify the type of disease that has developed in the peritoneum.
[0156]
For example, by using matrix metalloproteinase 2 and matrix metalloproteinase 9 as indicator substances, it becomes possible to distinguish between peritonitis and peritoneal thickening for diagnosis.
In addition, early diagnosis of these diseases becomes possible.
[0157]
Further, according to the present invention, the indicator substance can be detected even when the amount of the specimen is small.
[0158]
In addition, if the dialysate drainage is used as a sample, it is not necessary to perform a special operation for collecting the indicator substance, and it is not necessary to perform any special treatment on the organism from which the sample is collected. There is little load on the organisms to be collected. In particular, if the dialysis fluid drainage of peritoneal dialysis is used as a sample, it becomes possible for a patient or the like to collect the sample at home or the like, which makes it very easy to collect the sample.
[0159]
Furthermore, if the drainage collected in the drainage container for peritoneal dialysis is sampled as a specimen, there is no risk of causing infections to the patient.
[0160]
Further, by detecting the indicator substance by electrophoresis or zymography, the indicator substance can be detected more easily and accurately.
[0161]
Since the present invention has the advantages as described above, by using the present invention, when performing peritoneal dialysis, the safety thereof can be improved, and for patients whose renal function is reduced or lost. It can bring immense benefits.
[Brief description of the drawings]
FIG. 1 is a photograph showing the form of a living organism.
FIG. 2 is a graph showing the change with time of the D / D0 glucose value in the example of the present invention.
FIG. 3 is a photograph of zymography.
FIG. 4 is a photograph showing the form of a living thing.
FIG. 5 is a photograph showing the form of an organism.
FIG. 6 is a graph showing D / D0 glucose values in an example of the present invention.
FIG. 7 is a photograph of zymography.

Claims (13)

A method for detecting an indicator substance for detecting first and second indicator substances from a sample which is a collected peritoneal dialysis dialysate ,
The first indicator substance is a matrix metalloproteinase 9 that is detected or confirmed to increase in concentration at least at the onset of peritonitis in the organism from which the specimen is collected ,
The second indicator substance is a matrix metalloproteinase 2 that is detected or confirmed to increase in concentration at least at the onset of peritoneal thickening in the organism from which the specimen is collected,
A detection method for an indicator substance, characterized by using a detection means capable of separately detecting the first indicator substance and the second indicator substance . The method for detecting an indicator substance according to claim 1 , wherein the first and second indicator substances have a correlation with the D / D0 glucose value of the peritoneum of the organism from which the specimen is collected. The method for detecting an indicator substance according to claim 2 , wherein when the D / D0 glucose value decreases, the concentrations of the first and second indicator substances tend to increase. The method for detecting an indicator substance according to any one of claims 1 to 3 , wherein the first and second indicator substances can be detected from a specimen having a volume of 1 mL or less. 5. The method for detecting an indicator substance according to claim 1, wherein the peritoneal dialysis dialysate drainage is a drainage sampling liquid collected in a peritoneal dialysis drainage container. Detection method of indicator material according to any one of claims 1 to 5 concentrations of the collected the specimen after adjusting the detection of said first and second indicator material. The method for detecting an indicator substance according to any one of claims 1 to 6 , wherein the first and second indicator substances are detected after adjusting the temperature of the collected specimen. The method for detecting an indicator substance according to any one of claims 1 to 7 , wherein the first and second indicator substances are detected after adjusting the temperature of the collected specimen to a room temperature or a temperature lower than that. The method for detecting an indicator substance according to any one of claims 1 to 8 , wherein in the detection of the first and second indicator substances, the first and second indicator substances are separated and visualized. Wherein the detection of the first and second indicator substance, the detection method of indicator material according to any one of claims 1 to 9 carried out by electrophoresis. The method for detecting an indicator substance according to claim 10 , wherein the gel used for electrophoresis has a substrate for visualizing the first and second indicator substances. The method for detecting an indicator substance according to claim 11 , wherein the substrate is gelatin. The method for detecting an indicator substance according to any one of claims 1 to 12 , wherein the first and second indicator substances are detected by a zymography method.

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