JPH0577028B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method and reagent for measuring the ratio of DNA to protein in cells, and more specifically, during mass screening for lung cancer, uterine cancer, etc.
The present invention provides a reagent consisting of a cell staining solution used for cell diagnosis performed using a cell quantification device or abnormal cell discovery (cytoscreening), and a method for measuring the ratio of DNA to protein in cells using the reagent. be. [Prior technology] The most important characteristics of cancer cells seen under a microscope are that, compared to normal cells, the nucleus is swollen to various degrees and stains deeply with hematoxylin staining, and there is less cytoplasm compared to the size of the nucleus. That is, it has a large nucleus/cytoplasm ratio, and this morphological feature is used for cytopathological diagnosis of cancer. In addition, generally, the more malignant a cancer cell is, the more poorly developed its cytoplasm is, so the nucleus/cytoplasm ratio is large, and this is sometimes used to estimate the malignancy of cancer. Furthermore, since the amount of protein in cancer cells varies somewhat depending on the type of cancer, this may be used to estimate the type of cancer. Looking at the characteristics of cancer cells from a cytochemical perspective, the swelling and intense staining of the nucleus is an increase in the amount of deoxyribonucleic acid (DNA) in the nucleus, and the relative decrease in the cytoplasm is a relative decrease in proteins, which are the main components of the cytoplasm. Furthermore, an increase in the nucleus/cytoplasm ratio can be understood as an increase in the DNA/protein ratio, so if these cytochemical characteristics are quantified using special equipment, they can be used to discover and diagnose cancer cells. Alternatively, it can be applied to estimate the malignancy and type of cancer. Around 1975, automatic cell analysis and separation equipment (hereinafter referred to as flow cytometry), which analyzes cells by flowing them through a thin stream of water at high speed and applying a laser beam to them, was developed in the United States and West Germany. The DNA of each cell was fluorescently stained with propidium iodide (hereinafter abbreviated as PI), and proteins in the cytoplasm were fluorescently stained with fluorescein isothiocyanate (hereinafter abbreviated as FITC), and each cell was analyzed by flow cytometry. Attempts have been made to diagnose cancer cells and estimate their type by simultaneously measuring DNA and protein. Around 1980, attempts to use this technology for cell diagnosis of uterine cancer and lung cancer began in the United States, and in Japan as well, in recent years,
Studies on uterine cancer are progressing, and studies have also begun on its application to mass cancer screening. The above cells were stained with PI and FITC (hereinafter,
The method of measuring by flow cytometry (abbreviated as PI/FITC staining) is based on the property that PI selectively stains cellular DNA and RNA, and FITC selectively stains proteins in the cytoplasm, and flow cytometry. Under the irradiation of the laser beam in the meter,
PI stained with DNA emits red fluorescence with a maximum wavelength of 620 nm, and FITC stained with protein emits red fluorescence with a maximum wavelength of 530 nm.
By utilizing the property of emitting yellow-green fluorescent light of m, each cell flowing at high speed inside the device is illuminated with a laser beam, and the amounts of red and yellow-green fluorescent light emitted from the cells are simultaneously detected, respectively. Processed with a computer, the amount of DNA and protein in the cell,
Alternatively, the DNA/protein ratio is determined. [Problems to be solved by the present invention] However, the conventional method of staining cells with PI/FITC and quantifying DNA and protein in the cells by flow cytometry and determining the DNA/protein ratio does not depend on the light source. Not only does it have the disadvantage of increasing diagnostic costs because it requires an expensive laser beam generator, but it also has red fluorescence with a maximum wavelength of 620 nm emitted from PI that dyes DNA, and red fluorescence with a maximum wavelength of 620 nm that is emitted from FITC that dyes protein. The wavelength difference between 530nm yellow-green fluorescence is relatively small at 90nm, and the accuracy of separating the two is somewhat low.More importantly, PI has the property of staining both DNA and RNA. Therefore, in order to stain only DNA, it is necessary to pre-digest the RNA using an RNA-digesting enzyme prior to the cell staining process, and this digestion process involves heating the cells together with the enzyme at 37°C. The entire staining process takes up to 2 hours, as it involves repeated washing and centrifugation multiple times, and it also causes cell damage and loss.Also, the titer of commercially available RNA-digesting enzymes is not constant. It has the disadvantage that accurate measurement results (diagnosis results) are difficult to obtain due to the fact that the effect of RNA removal is not constant. [Means for solving the problem] Therefore, the present inventors discovered that hematoporphyrin has the property of selectively staining proteins in cells and emitting red fluorescence with a maximum wavelength of 670 nm when irradiated with ultraviolet rays. and 2-[2-(4-hydroxyphenyl-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)-benzimidazole trihydrochloride or 4',6-diamidino2-
2 Phenylindole hydrochloride selectively stains DNA in cells and irradiates it with ultraviolet rays to detect the maximum wavelength.
Focusing on the property of emitting blue fluorescence at 470 nm, we discovered a method that utilizes these properties to detect the ratio of protein to DNA in cells easily, inexpensively, and in a short period of time with excellent accuracy. , we have completed the present invention. The present invention provides cells with hematoporphyrin and 2-
[2-(4-hydroxyphenyl-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)
-Benzimidazole trihydrochloride (so-called Hoechst reagent, hereinafter abbreviated as Hoechst reagent) or 4',6-diamidino 2-2 phenylindole hydrochloride (hereinafter abbreviated as DAPI)
A method for measuring the ratio of protein to DNA in cells by staining them with a cell fluorescent staining solution containing stained cells and measuring the blue and red fluorescence emitted by the stained cells, and the hematopoietic fluid used in this measuring method. This reagent consists of a cell fluorescent staining solution containing porphyrin and Hoechst-based reagents or DAPI. The reagents of the present invention include hematoporphyrin and Hoechst-based reagents or
In addition to those containing DAPI, staining solutions containing hematoporphyrin and Hoechst-based reagents or
It also includes those prepared separately and in combination with a staining solution containing DAPI. The cell fluorescent staining solution of the present invention requires an ultraviolet light source as a condition for use, and conventional measuring instruments include a fluorescent microscope equipped with an ultraviolet light source and a microspectroscopic quantitative device, or a simple type that operates only with an ultraviolet lamp. Flow cytometry (or flow cell analyzer) can be used, but even in the case of full-fledged flow cytometry using the laser beam mentioned above, the laser beam generating lamp can also be adjusted to emit ultraviolet rays, so it can also be used. . The Hoechst-based reagent in the cell fluorescent staining solution in the present invention is a benzimidazole-based compound, and its chemical structural formula is:

[R represents CH ₃ −N=, C ₂ H ₅ −N=, H ₂ C=, etc., and R′ represents −OH, −OCH ₃ , −OC ₂ H ₅ , −N(CH ₃ ) ₂ , − Cl, −NH ₂ , etc.]

It is indicated by. In addition, in the experimental examples and examples related to the present invention, Hoechst 33342 (in the chemical structural formula, R is CH ₃ -
A compound in which N and R' are -OC ₂ H ₅ was used. Also
DAPI is a phenyl indole compound and its chemical structure is:

Both are known substances that have the property of selectively binding to DNA in cells and emitting blue fluorescence with a maximum wavelength of 470 nm. Therefore, in the present invention, the Hoechst-based reagent or DAPI contained in the cell staining solution is used in exactly the same concept as the conventional DNA staining solution using each of these compounds, and the solvent and content are The same amount of staining solution as conventional staining solution can be used as is. On the other hand, the hematoporphyrin used in the cell fluorescent staining solution of the present invention has the chemical structural formula

〔Effect of the invention〕

The cell fluorescence staining solution of the present invention as a reagent for measuring the ratio of DNA to protein in cells is
Hoechst-based reagent or DAPI selectively stains DNA and emits blue fluorescence with a maximum wavelength of 470 nm under UV irradiation, and DAPI also selectively stains cytoplasmic proteins and emits blue fluorescence with a maximum wavelength of 670 nm under UV irradiation.
This method utilizes hematoporphyrin, which emits red fluorescence, and the cells are measured under ultraviolet irradiation after staining, so conventional PI/FITC staining after RNA digestion is used. Compared to measurements made under laser beam irradiation, the equipment used is simple and inexpensive, consumes less power, and does not require cooling, making it possible to measure the DNA/protein ratio in cells at a lower cost. In addition, the cell fluorescence staining solution of the present invention can be used to detect
In measuring the DNA/protein ratio, the wavelength difference between the fluorescence from DNA and protein under ultraviolet irradiation is
Since the DNA/protein ratio is measured using 200nm, PI/FITC is used after conventional RNA digestion.
Compared to the method of staining and measuring using the wavelength difference of 90 nm between DNA and protein under irradiation with a laser beam, the measurement accuracy is higher and highly accurate results can be obtained with simple operations. Furthermore, the cell fluorescence staining solution of the present invention can be used to
In measuring the DNA/protein ratio, the cell nucleus
Because it uses a substance that selectively stains DNA and a substance that selectively stains proteins in the cytoplasm, it is easier to measure the DNA/protein ratio prior to staining than with conventional PI/FITC staining. in advance
The step of digesting RNA with RNA-digesting enzymes is unnecessary, and therefore the sample does not need to be centrifuged multiple times37.
It eliminates heating to ℃, reduces the expense required for digestion, eliminates cell loss and damage caused by RNA digestion, and eliminates the hassle of RNA digestion, which is inherently uncertain in its effectiveness. Therefore, not only can more accurate measurement results (diagnosis results) be obtained, but the time required for the entire process of cell staining, which took approximately 2 hours with conventional methods, has been shortened to approximately 20 minutes. This is an extremely excellent method for mass medical examinations that involve processing a large number of samples.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the protein distribution map of lymphocytes as seen by hematoporphyrin single staining, and Figure 2 is a diagram showing the protein content distribution of lymphocytes and granular leukocytes as seen by hematoporphyrin Hoechst staining. , Figure 3 is
Figure 4 shows the protein content distribution of lymphocytes as seen by FICT single staining. Figure 4 shows the relationship between the concentration of hematoporphyrin staining solution and the red fluorescence intensity of lymphocytes. Figure 5
The figure shows the relationship between the concentration of Hoechst-based reagent staining solution and the blue fluorescence intensity of lymphocytes, Figure 6 shows the relationship between the concentration of DAPI staining solution and the blue fluorescence intensity of lymphocytes, and Figure 7 shows the relationship between the concentration of DAPI staining solution and the blue fluorescence intensity of lymphocytes. Figure 8 shows the effect of addition of mercaptoethylamine on the fading rate of red fluorescence caused by hematoporphyrin, Figure 8 shows the effect of addition of mercaptoethylamine on the fading rate of blue fluorescence caused by Hoechst reagents, and Figure 9 The figure shows the fluorescence wavelength characteristic curve of hematoporphyrin bound to cells when irradiated with ultraviolet rays, Figure 10 shows the fluorescence wavelength characteristic curve of Hoechst-based reagent bound to cells when irradiated with ultraviolet rays, and Figure 11 bound to the cell
Figure 12 shows the fluorescence wavelength characteristic curve of DAPI when irradiated with ultraviolet rays. Figure 12 shows the fluorescence wavelength characteristic curve of Hoechst-based reagent and hematoporphyrin bound to cells when irradiated with ultraviolet rays. Figure 13 shows the fluorescence wavelength characteristic curve bound to cells. FIG. 14 is a diagram showing the fluorescence wavelength characteristic curve of DAPI and hematoporphyrin that have been irradiated with ultraviolet rays, and FIG. 14 is a diagram showing the fluorescence wavelength characteristic curve of PI bound to cells and FITC that are irradiated with ultraviolet rays. In addition, Fig. 15 shows cancer cells treated with hematoporphyrin single staining, Fig. 16 shows a control group of cancer cells protein digestion test,
Figure 17 is a protein digestion test group of cancer cells, Figure 18 is hematoporphyrin single staining treatment of leukocytes, Figure 19 is
The figure shows FITC staining treatment of leukocytes, Figure 20 shows hematoporphyrin Hoechst staining treatment of cancer cells, and Figure 2
Figure 1 shows microscopic photographs of cancer cells treated with hematoporphyrin/DAPI staining.

Claims (1)

[Scope of Claims] 1 Cells are treated with hematoporphyrin and 2-[2-(4
-hydroxyphenyl-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)-benzimidazole trihydrochloride or 4',
A ratio of protein to DNA in a cell characterized by staining with a cell fluorescent staining solution containing 6-diamidino 2-2 phenylindole hydrochloride and measuring the blue fluorescence and red fluorescence emitted by the stained cell. measurement method. 2 Hematoporphyrin and 2-[2-(4-hydroxyphenyl-6-benzimidazolyl]-6
Cell fluorescence containing -(1-methyl-4-piperazyl)-benzimidazole trihydrochloride or 4',6-diamidino 2-2 phenylindole hydrochloride together with adjuvants selected from salt, mercaptoethylamine, and surfactants. The measuring method according to claim 1, characterized in that a staining solution is used. 3. The measuring method according to claim 1, wherein the concentration of hematoporphyrin is 0.01 to 0.03% by weight. 4 2-[2-(4-hydroxyphenyl)-6-
The concentration of benzimidazolyl]-6-(1-methyl-4-piperazyl)-benzimidazole trihydrochloride or 4',6-diamidino2-2phenylindole hydrochloride is 0.0005 to 0.001% by weight
The measuring method according to claim 1, characterized in that: 5. The measurement method according to claim 1, characterized in that the DNA/protein ratio of stained cells is measured using a fluorescence microscope, a flow cytometer, or a flow cytometer equipped with an ultraviolet light source and a microspectroscopic quantitative device. . 6 Hematoporphyrin and 2-[2-(4-hydroxyphenyl-6-benzimidazolyl]-6
-(1-methyl-4-piperazyl)-benzimidazole trihydrochloride or 4',6-diamidino-2-2 phenylindole hydrochloride. Reagent for measuring the ratio of protein to DNA. 7 The cell fluorescent staining solution contains hematoporphyrin and 2
-[2-(4-Hydroxyphenyl-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)-benzimidazole trihydrochloride or with 4',6-diamidino-2-2phenylindole hydrochloride The measuring reagent according to claim 6, characterized in that it contains an auxiliary agent selected from common salt, mercaptoethylamine, and a surfactant.8. 2-(4-hydroxyphenyl-6-
benzimidazolyl]-6-(1-methyl-4-piperazyl)-benzimidazole trihydrochloride or a staining solution containing 4',6-diamidino2-2phenylindole hydrochloride. A measuring reagent according to claim 6. 9. The reagent according to any one of claims 6 to 8, characterized in that the concentration of hematoporphyllin is 0.01 to 0.03% by weight. 10 2-[2-(4-hydroxyphenyl)-6
-benzimidazolyl]-6-(1-methyl-4-
(piperazyl)-benzimidazole trihydrochloride or 4',6-diamidino2-2phenylindole hydrochloride is from 0.0005 to 0.001% by weight. Any of the measurement reagents described above.