CN114323878A - Formalin fixation method for liver micronucleus test - Google Patents
Formalin fixation method for liver micronucleus test Download PDFInfo
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Abstract
The invention discloses a formalin fixation method for a liver micronucleus test. The method comprises the steps of grinding tissue blocks after alkali treatment to obtain dispersed cells, filtering by using a cell filter screen with the aperture of 30-50 mu m, carrying out centrifugation, then re-suspending the cells by using a neutral formalin solution to obtain a cell suspension, and dyeing the cell suspension by using SYBR GOLD dye solution diluted by 4000-6000 times by using deionized water. The experimental result of the formalin fixation method provided by the invention has high correlation with the traditional collagenase digestion method, the sensitivity for detecting liver carcinogens is equivalent to or higher than that of the collagenase digestion method, retrospective micronucleus analysis can be carried out on formalin-fixed liver tissues in general toxicological research, and the method has the advantages of simple experimental method, more harvested hepatocytes, low experimental cost and the like.
Description
Technical Field
The invention belongs to the field of hepatic micronucleus tests, and particularly relates to a method for preparing a hepatic cell suspension for the hepatic micronucleus test, namely a formalin fixation method.
Background
The Liver Micronucleus (LMN) assay is an effective method for detecting genotoxic compounds in vivo, particularly those that require metabolic activation. The method is suitable for detecting the precancerogen or the metabolite with the fragmentation effect, which is extremely unstable and can not reach the classical target cell of genetic research.
Although the liver is not the primary target tissue in conventional micronucleus assays, the liver is an important tissue in general toxicology studies and carcinogenic assays because the test compounds are metabolized and sometimes activated in the liver and thus have toxicological significance. Genotoxic rodent liver carcinogens that require metabolic activation or are not detectable in routine red blood cell micronucleus assays in rodents are reported to be detectable by the liver micronucleus assay.
In the prior art, a collagenase digestion method is generally used for preparing a Hepatocyte suspension, for example, the literature "rat in vivo multi-endpoint experiment evaluates the genotoxicity research of N-nitrosodiethylamine, the preparation method of the Hepatocyte suspension disclosed in Tonghen" belongs to the collagenase digestion method, DAPI-AO mixed staining solution is used for staining, then the number of Hepatocyte Micronucleus (MN) is observed by microscopic examination and the Hepatocyte micronucleus (MN-HEP%) is calculated. The collagenase digestion method was extensively validated by the Japanese Society of Environmental mutation research (CSGMT)/JEMS/MMS.
In the traditional collagenase digestion method, the preparation of hepatocyte suspension must be carried out in time after the liver is obtained; collagen contained in fresh liver tissue has great influence on cell suspension preparation, so that the obtained liver cells are less in quantity; the prepared cell suspension also contains blood cells to interfere the reading process.
Disclosure of Invention
The process for preparing the hepatocyte suspension in the prior art is complex, the prepared hepatocyte state is not good enough, microscopic examination is not facilitated, and the TE buffer solution is used for dyeing HEP specimens after being used for diluting SYBR GOLD staining solution, but the preparation process of the TE buffer is complicated. In view of the above disadvantages, the present invention provides a formalin fixation method which has a sensitivity comparable to or higher than that of collagenase digestion for detecting liver carcinogens. The formalin fixation method of the present invention also enables retrospective evaluation in formalin-fixed liver tissues in general toxicity studies.
In order to solve the technical problems, the invention provides a formalin fixation method which comprises the steps of grinding tissue blocks after alkali treatment to obtain dispersed cells, filtering the cells by using a cell filter screen with the aperture of 30-50 mu m, re-suspending the cells by using a neutral formalin solution after centrifugation to obtain a cell suspension, and dyeing the cell suspension by using SYBR GOLD staining solution diluted by 4000-6000 times by using deionized water.
The dilution factor of the SYBR GOLD dye solution is preferably 5000 times.
The methods of the invention can be used in conventional laboratory animals, such as rats and mice, as is conventional in the art.
Generally, the tissue obtained is cut into tissue pieces and washed prior to the alkaline treatment step, as is conventional in the art. After the alkaline treatment, the tissue mass after the alkaline treatment is ground before being filtered through a cell strainer, for example, it may be ground using a syringe plug.
The time and the rotating speed of the centrifugation in the invention can be conventional in the field, and preferably: the centrifugation time is 1-5min, and the rotation speed is 45-66 g.
In a preferred embodiment of the invention, the time of centrifugation is 2 min.
In a preferred embodiment of the invention, the rotation speed of the centrifuge is 50 g.
In a preferred embodiment of the present invention, the pore size of the cell strainer is 40 μm.
The dyeing is to mix diluted SYBR GOLD dye liquor and the cell suspension according to the volume ratio of 0.5-1.5: 1.
In a preferred embodiment of the invention, the volume ratio of diluted SYBR GOLD stain to the cell suspension is 1: 1.
The tissue of the present invention may be preserved in a conventional manner, and preferably, the tissue is preserved in a neutral formalin solution.
The volume percentage of the neutral formalin solution used in the present invention can be conventional in the art, and is preferably 4-10% by volume of formalin buffer.
In one embodiment of the present invention, the formalin buffer is used in a volume percentage of 10%.
The neutral formalin solution used in the present invention may be of any kind that is conventional in the art, and preferably a neutral formalin phosphate buffer.
The type of tissue described in the present invention may be conventional in the art, preferably liver tissue, more preferably left outer lobe tissue of the liver.
The alkaline treatment in the present invention may be conventional in the art and preferably comprises placing the tissue mass in a 10-13M KOH solution, treating at room temperature for 15-20h, and rinsing off residual KOH with water.
In one embodiment of the invention, the tissue is treated in 12M KOH solution at room temperature for about 16 hours.
To evaluate the effectiveness of the formalin fixation method of the present invention, micronucleus test was performed using formalin-fixed liver tissue and compared with the test results of the collagenase digestion method. The test was set up for a negative control group (deionized water and corn oil), a N-Nitrosopyrrolidine (NPYR) low, medium and high dose group (25/50/100mg kg. multidot.kg)-1·day-1) Quinoline (Quinoline) low-medium dose group (30/60/120 mg. kg)-1·day-1) And N-Nitrosodiethylamine (DEN) positive control group (12.5mg/kg), each group comprises 5-6 SPF (specific pathogen free) SD rats, the test substance is orally administered for 14 days, the animals are euthanized 24h after the last administration for liver sampling, 1g of fresh liver is taken for each samplePreparing a hepatocyte suspension by a collagenase digestion method; fixing the rest liver tissues in a neutral formalin solution, subsequently preparing a liver cell suspension by using a formalin fixing method, and then obtaining liver micronucleus indexes such as micronucleus rate (MN%), mitotic index (MI%), mitotic index and the like by using a microscope after SYBR Gold staining; the difference between the two methods is analyzed by a statistical method, and the correlation of the results of the formalin fixation method and the fresh tissue collagenase digestion method is compared.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and raw materials used in the invention are as follows:
DENN-nitrosopyrrolidine, all available from Chiloeia (Shanghai) chemical industry development, Inc.; quinoline and type VI collagenase were purchased from sigma; SYBR Gold dye liquors were purchased from Invitrogen; formaldehyde solution (37.0-40% containing blocking agent) was purchased from GENERAL-REGENT; PBS powder was purchased from Boshide biologies and dissolved in 2000mL deionized water per bag; the PBS solution and formaldehyde are prepared into 10 percent neutral formalin solution according to the volume ratio of 1: 9. The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
(1) the method has the advantages of simple process for preparing the hepatocyte suspension, convenient operation and hepatocyte yield far higher than that of the collagenase digestion method.
(2) The liver cells prepared by the method are in a perfect state, the liver cells can be observed clearly after flaking and dyeing, the quality of the slices is excellent, and the speed of microscope examination and slice reading is accelerated.
(3) The experimental result of the formalin fixation method provided by the invention has high relativity with the traditional collagenase digestion method, and the sensitivity for detecting liver carcinogens is equivalent to or higher than that of the collagenase digestion method.
(4) The SYBR Gold (SYGO) staining solution disclosed by the invention can be used after being diluted 5000 by using the purchased staining solution with deionized water, and can be used for reading after being mixed with a cell suspension 1:1 in a clean glass slide, so that a very clear staining effect can be obtained, and the method is more economical and convenient.
The method for evaluating the micronucleus in the formalin-fixed liver (namely the formalin-fixed method) is reproduced and optimized on the basis of the existing literature, retrospective micronucleus analysis can be carried out on formalin-fixed liver tissues in general toxicological research, and the method has the advantages of simple experimental method, high hepatocyte harvesting rate, low experimental cost and the like.
Drawings
FIG. 1 shows the comparison of the quality of the liver micronucleus images of the cell suspension observed under a fluorescence microscope. A liver micronucleus image (A picture) observed under a fluorescence microscope at 200 times by using a collagenase digestion method; an image of liver micronucleus observed under a fluorescence microscope at 200-fold magnification by formalin fixation (panel B); an image of liver micronucleus observed under a fluorescence microscope at 400-fold magnification using formalin fixation (panel C).
FIG. 2 shows the results of comparing the sizes of liver tissues used in the collagenase digestion method (Panel A) and the formalin fixation method (Panel B).
FIG. 3 is a diagram showing cell suspensions obtained by treating the livers of NPYR group rats by collagenase digestion (Panel A) and formalin fixation (Panel B).
FIG. 4 is a diagram showing cell suspensions obtained by treating the livers of rats in the quinoline group by collagenase digestion (Panel A) and formalin fixation (Panel B).
FIG. 5 shows MN-HEP% results of NPYR (Panel A) and quinoline (Panel B) collagenase digestion methods.
FIG. 6 shows the results of NPYR (panel A) and quinoline (panel B) formalin fixation with MN-HEP%.
FIG. 7 shows the HEP-MN% linear regression results of NPYR (Panel A) and quinoline (Panel B) formalin fixation and collagenase digestion.
FIG. 8 shows the results of collagenase digestion NPYR (panel A) and quinoline (panel B) MN-HEP%.
FIG. 9 shows the results of the formalin fixation method NPYR (Panel A) and quinoline (Panel B) MN-HEP%.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 drawing of liver from rat
The experiment adopts 2 test objects (NPYR and quinoline) and 1 positive control (DEN), and the experimental result of the test objects can be used as a verification result.
SPF grade male sd (sprague dawley) rats were randomly divided into 5 groups of 5 rats. NPYR (0,25,50,100 mg. kg) was administered by oral gavage for 14 consecutive days-1·day-1) The solvent is deionized water; quinoline (30/60/120mg kg)-1·day-1) The solvent is corn oil; DEN (12.5mg kg)-1·day-1) Used as a positive control group of liver micronucleus. After about 24 hours of the last administration of the test substance, the animals are treated with a mixed preparation of ketamine and xylazine (40-80 mg/kg)-1) Anesthesia, abdominal aorta exsanguination euthanasia and liver sampling. About 1g of liver left lobe was used for preparing hepatocyte suspension by collagenase digestion method, and the remaining liver tissue was preserved in neutral formalin.
Liver tissue preserved in neutral formalin for 1 week to 5 years or more may be used to prepare hepatocyte suspensions by formalin fixation.
EXAMPLE 2 preparation of hepatocyte suspensions by formalin fixation
Cutting the fixed liver tissue into 2-3 mm3Size, washing with water. About 10 pieces of tissue were placed in 15mL of 12M KOH solution, treated at room temperature for about 16h, and the residual KOH was rinsed with water. The liver pieces were crushed with a syringe plug, filtered through a 40 μm cell strainer, and suspended in water to disperse the hepatocytes. Centrifuge at 50g for 2min and resuspend with neutral formalin solution. The centrifugation and resuspension steps were repeated 3 more times. Finally, resuspending the hepatocytes by using a neutral formalin solution, and storing the obtained hepatocyte suspension at 2-8 ℃ for subsequent reading.
EXAMPLE 3 collagenase digestion method for preparation of hepatocyte suspension
The cut left lobe of liver of about 1g was rapidly transferred to pre-cooled HBSS for storage, then placed in preheated HBSS at 37 deg.C, shaken in a shaker at 37 deg.C and 130 revolutions for 10min, then HBSS was discarded, and liver tissue was washed with HBSS for 1 time. The liver tissue was washed 2 times with 10mL of red-yellow dye at 37 ℃ and 130 rpm, and the solution was discarded. Adding 10mL of 0.05% collagenase solution, digesting at 37 deg.C for 30min in a 130-rotary shaker, discarding the collagenase solution, transferring the liver pieces to a 50mL centrifuge tube, adding 10% FBS HBSS, cutting the tissue into small pieces of about 0.5mm with surgical scissors, and vortexing for 3min to separate the hepatocytes. And (3) precipitating larger liver blocks (about 10 to 20 seconds), quickly pouring the cell suspension (avoiding any large liver blocks) into a new 15ml centrifuge tube, centrifuging for 2min at 50g, repeating for 3 times until the supernatant is clear and transparent, finally resuspending the liver cells by using neutral formalin, and storing the obtained liver cell suspension at 2-8 ℃ for subsequent slide reading.
Example 4 microscopic slide analysis
SYBR Gold (SYGO) stain at 5000-fold dilution was dropped onto glass slides along with an equal amount of hepatocyte suspension and sectioned for microscopic slide analysis. 2000 normal hepatocytes were counted during reading. The SYGO stained slide specimen was observed under a fluorescence microscope using a B-excitation filter (wavelength 420-490nm) and the results are shown in panel B of FIG. 1 (200-fold mirror) and panel C of FIG. 1 (400-fold mirror); FIG. 1A is a diagram of a cell suspension obtained by collagenase digestion. Because the formalin fixation method uses fixed liver tissues, the shape of the liver cells is fixed by formalin solution in a better state during dissection, and the final liver cell suspension can be obtained after alkali treatment and grinding; the collagenase digestion method uses fresh liver tissues, and the fresh liver tissues are firstly stored in an HBSS buffer solution, then are fixed after being digested by collagenase, centrifuged and washed by formalin solution, so that the state of the obtained liver cells is not as good as that of the liver cells obtained by the formalin fixation method. The hepatocytes with good state prepared by the formalin fixation method can be counted more intuitively; the hepatocyte prepared by the collagenase digestion method has a poor state, and many interfering cells are generated during reading, so that the reading speed is influenced.
FIG. 2 is a comparison of the sizes of liver tissues used in the collagenase digestion method and the formalin fixation method, wherein FIG. 2A is a diagram of the left lobe of the liver of a normal rat, about 3g, and 1g of cell suspension is taken during the experiment; FIG. 2B is a view of fixed liver tissue, 10 small blocks of 3mm are taken3Fixed liver tissue of size was used to prepare cell suspensions. Negative control, NPYR and quinoline low and medium dosage groups, and 1 positive control group are set in the experiment, 8 groups are provided in total, and 6 animals in each group are subjected to the experiment.All samples from each dose group were separately prepared for hepatocyte suspension by collagenase digestion and formalin fixation. The amount of the cell suspension obtained is shown in FIGS. 3 and 4.
In conjunction with fig. 2 to 4, it can be seen visually that more hepatocytes can be harvested using less liver tissue by the formalin fixation method. Meanwhile, in the process of preparing cell suspension by a formalin fixation method, the hepatic cells are more easily dispersed by alkali treatment, and after the blood cells are fixed, all the hepatic cells can be used during grinding, so that the hepatic cells are hardly lost, and the yield of the hepatic cells is high; in the process of preparing the hepatic cells by the collagenase digestion method, more hepatic cells are lost in the steps of the collagenase digestion process, the liver tissue shearing, the centrifugation and the like, so the yield is low.
2000 parenchymal Hepatocytes (HEPs) were analyzed, and the number of micronucleus-containing hepatocytes (MN-HEPs) was recorded. The results of the collagenase digestion method by MN-HEP% and the results of the formalin fixation method by MN-HEP% are shown in FIGS. 5 and 6, and FIGS. 8 and 9, respectively.
Example 5 statistical analysis
The MN-HEP% of each dose group of the test sample is compared with the MN-HEP% of the vehicle control group by statistical analysis, and the statistical analysis is carried out by adopting the following method:
firstly, carrying out normal distribution analysis (Shapiro-Wilk test, P is more than 0.05); if P is less than or equal to 0.05 in Shapiro-Wilk test, data conversion (Log (10), square root of arcsine, rank conversion, etc.) is carried out to make the data normally distributed (P >0.05 in Shapiro-Wilk test); the ANOVA test was then performed on the transformed data.
② if the ANOVA test result is significant (P is less than or equal to 0.05), further using Dunnett' st test (0.05 and 0.01 level); if the ANOVA test result has no significant difference (P >0.05), the statistics are over. The positive control group is compared with the vehicle control group by adopting independent sample t test (the difference of significance P is less than or equal to 0.05).
And analyzing the correlation between the results of the collagenase digestion method and the formalin fixation method by adopting a regression analysis method.
Results of the experiment
As shown in fig. 5 and 6 and fig. 8 and 9, the MN-HEP% was significantly increased in each of NPYR and quinoline dose groups compared to the vehicle control group. The MN-HEP% result of the NPYR formalin fixation method is slightly higher than that of the collagenase digestion method, and the MN-HEP% result of the quinoline formalin fixation method is equivalent to that of the collagenase digestion method.
As shown in FIG. 7, the NPYR and quinoline dose groups obtained by formalin fixation and collagenase digestion showed better MN-HEP% correlation (correlation coefficients 0.8614 and 0.9279, respectively).
Conclusion of the experiment
Similar results are obtained by a quinoline and NPYR dosage group formalin fixation method and a collagenase digestion method, and the sensitivity of the formalin fixation method for detecting liver carcinogens is equivalent to or higher than that of the collagenase digestion method; and the hepatocytes with good state prepared by the formalin fixation method can be counted more intuitively, and more hepatocytes can be harvested by using less hepatic tissues. In the process of preparing cell suspension by the formalin fixation method, the hepatocyte is more easily dispersed by alkali treatment, and after the blood cell is fixed, all the hepatocyte can be used during grinding, so that the hepatocyte is hardly lost, and the yield of the hepatocyte is high.
A formalin-fixed rat liver micronucleus test method is initially established and recommended, and can be used for retrospective micronucleus analysis of formalin-fixed liver tissues in general toxicology studies.
Claims (10)
1. A formalin fixation method is characterized by comprising the steps of grinding tissue blocks after alkali treatment to obtain dispersed cells, filtering the cells by using a cell filter screen with the aperture of 30-50 mu m, carrying out centrifugation, then carrying out heavy suspension on the cells by using a neutral formalin solution to obtain a cell suspension, and dyeing the cell suspension by using SYBR GOLD staining solution diluted by 4000-6000 times by using deionized water.
2. Formalin fixation according to claim 1, characterised in that the speed of the centrifugation is 45-66g, preferably 50 g; the centrifugation time is 1-5min, preferably 2 min.
3. The formalin fixation method according to claim 1, wherein the cell strainer has a pore size of 40 μm.
4. The formalin fixation method according to claim 1, wherein the staining is mixing a diluted SYBR GOLD staining solution with the cell suspension in a volume ratio of 0.5 to 1.5: 1;
the dilution multiple of the SYBR GOLD dye solution is preferably 5000 times;
the volume ratio is preferably 1: 1.
5. The formalin fixation method according to claim 1, wherein the neutral formalin solution is formalin buffer solution with a volume percentage of 4% to 10%;
preferably, the formalin buffer is 10% by volume.
6. The formalin fixation method according to claim 1, wherein the neutral formalin solution is a neutral formalin phosphate buffer.
7. The formalin fixation method according to claim 1, wherein the tissue is preserved in a neutral formalin phosphate buffer solution at a volume percentage of 4% to 10%; preferably, the tissue is preserved in a 10% by volume solution of neutral formalin in phosphate buffer.
8. The formalin fixation method according to claim 1, wherein the tissue is liver tissue, preferably left outer lobe tissue of the liver.
9. The formalin fixation method according to claim 1, wherein the alkali treatment comprises placing the tissue block in 10-13M KOH solution, treating at room temperature for 15-20h, and washing away the residual KOH with water.
10. The formalin fixation method according to claim 9, wherein the KOH solution has a concentration of 12M; and/or the room temperature treatment time is 16 h.
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