CN112616775A - Method for establishing hepatic fibrosis of healthy SD male rat by adopting compound factor method - Google Patents
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Abstract
The invention relates to the technical field of medical animal breeding, and discloses a method for establishing hepatic fibrosis of a healthy SD male rat by adopting a compound factor method, which comprises the following steps: s1: culturing the blood stem cells; s2: preparing stem cell active matters; according to the following steps: 1: 1, mixing A, B with blood cells of schistosome to obtain active substance of blood stem cells; s3: feeding and culturing adult SD male rats; s4: feeding for 1 week, measuring body weight of the rat, feeding high-fat CDAA feed, and injecting blood stem cell active matter; the compound factor is formed by mixing the blood stem cells, the stem cell active matter and the schistosome and is injected into a healthy SD male rat, the animal model prepared by the rat conforms to the pathological process of conversion from NASH to HCC, the operation is easy, the time consumption is short, the stability and the effectiveness are realized, the modeling and the technical support are provided for developing and developing a new medicine for treating the non-alcoholic steatohepatitis, and the compound factor has great economic and social benefits.
Description
Technical Field
The invention relates to the technical field of medical animal cultivation, in particular to a method for establishing hepatic fibrosis of healthy SD male rats by adopting a compound factor method.
Background
Hepatic fibrosis is a pathophysiological process, which refers to abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver injury has liver fibrosis in the process of liver repair and healing, and if the injury factor cannot be removed for a long time, the fibrosis process can be continuously developed into liver cirrhosis for a long time. It is therefore not an independent disease; a composite factor is a collective term for a plurality of factors related to functions or sources.
The model animals currently used for researching NASH, hepatic fibrosis, liver cirrhosis and HCC mainly have chemical poison induction, physical injury, pathological diet, gene knockout and the like. These techniques all suffer from the following problems:
(1) the induction of chemical poison is to inject DEN or carbon tetrachloride (CCl4) into rats or mice through digestive tract and abdominal cavity to induce liver cells to be acutely or chronically damaged, so that liver fibrosis, liver cirrhosis and liver cancer are formed, although liver cancer can be induced, the natural evolution process of human from simple fatty liver, NASH to HCC cannot be completely simulated, and the basic model parameters required by NASH-HCC research and new drug development are not provided.
(2) Physical injury modeling is to ligate the hepatic bile duct of rat or mouse to cause mechanical intrahepatic cholestasis, which leads to progressive liver injury, hepatic fibrosis, cirrhosis and liver cancer formation. However, the modeling is usually used for basic research and drug development of hepatic fibrosis and liver cirrhosis, and is still not suitable for basic model parameters required by NASH-HCC research and new drug development.
(3) Pathologic diet induction modeling is the induction of fatty liver, NASH and HCC formation by feeding rats or mice on a high-fat, choline-deficient, high-fructose diet. Although the model is close to the pathological process of human NASH-HCC, the period is long, HCC does not appear in all animals finally, the observation time is about more than 24 months, and the model is not beneficial to experimental research.
(4) The gene knockout induction of liver cancer modeling is realized by knocking out the combination of cancer suppressor genes. The modeling can form fatty liver, hepatic fibrosis, NASH and HCC, but the gene knockout method is expensive in cost, difficult to operate and not beneficial to smooth experiment.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for establishing hepatic fibrosis of a healthy SD male rat by adopting a compound factor method, which has the advantages of easy operation and short time consumption and solves the problem of difficult operation.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: a method for establishing hepatic fibrosis of healthy SD male rats by adopting a compound factor method comprises the following steps:
s1: culture of blood Stem cells
Adding PBS into intestinal blood for dilution, adding 10ml of lymphocyte separation liquid into a 50ml centrifuge tube, slowly adding the diluted intestinal blood, slowly sucking out the white cloudy lymphocyte layer in the middle of layering, adding PBS, centrifuging for 5 minutes, removing supernatant after centrifuging, adding fetal calf serum again, mixing uniformly, and repeatedly centrifuging once; discarding the supernatant, adding culture medium, mixing, and standing at 100cm2Culturing in the culture bottle;
s2: preparation of Stem cell active
Taking cultured stem cells for digestion and counting, washing fetal calf serum for 3 times, dissolving in 30ml of physiological saline, and ultrasonically breaking the cells; dissolving out intracellular protein and nutrients, centrifuging for 10 min, collecting supernatant, removing cell debris, concentrating with ultrafiltration membrane, removing immunoglobulin, collecting filtrate, filtering with filter membrane to obtain sterile solution with concentration of 200 μ g/ml, and storing at 4 deg.C to obtain solution A;
measuring the pH value of the upper layer culture solution of 30ml when the pH value of the culture medium for culturing the cells is 5.5, filtering by using a filter membrane to concentrate the volume of the upper layer culture solution to about 5ml, taking out the upper layer culture solution, adding 5ml of normal saline, and fixing the volume to 10 ml; filtering with 0.22 μm filter membrane to make it sterile, measuring protein concentration, determining concentration to be 200 μ g/ml, and storing at 4 deg.C to obtain solution B;
according to the following steps: 1: 1, mixing A, B with blood cells of schistosome to obtain active substance of blood stem cells;
s3: feeding and culturing adult SD male rats;
s4: feeding the rats after the weight measurement for 1 week, feeding high-fat CDAA feed, injecting blood stem cell active substances, continuously culturing for 8 weeks, wherein the injection of the blood stem cell active substances is carried out for 2 days in one week, and the common drinking water is carried out for the other 5 days;
s5: observing the mental state and hair condition of the rat every day during feeding, measuring the food intake and water intake, measuring the body weight of the rat every week, starting from the first feeding of high-fat CDAA diet and injection of blood stem cell active substances, continuously feeding for 8 weeks, and obtaining the rat with hepatic fibrosis.
Preferably, S3 further comprises subjecting SD male rats to 25-30 deg.C room temperature and 55% -65% humidity cage, adaptively feeding for 1 week in 12 hr light/dark cycle, and measuring body weight of the rats.
Preferably, the high fat CDAA feed in S4, wherein, by weight: fat 32%, carbohydrate 55%, protein 13%, choline 0; the carbohydrate is starch, dextrin, sugar, and protein as amino acid premix, the fat is oleum Maydis and hydrogenated vegetable oil, and the calorie is 4.3 kcal/g.
Preferably, the step S1 further includes:
s11: when the cells grow to 80-90% of the bottom of the culture bottle, sucking out the culture medium, adding PBS for washing, sucking out PBS, adding pancreatin, adding alpha-MEM culture medium containing 20% fetal calf serum, neutralizing the pancreatin, sucking out the neutralization solution, putting into a centrifuge tube, adding PBS, centrifuging for 5 minutes, removing the supernatant after centrifugation, adding PBS, mixing uniformly, re-centrifuging once, counting the cells, adding 2X 105 cells into each bottle, and placing into a culture bottle of 100cm2 for culture.
Preferably, the method further comprises the step of S5:
the liver puncture tissue specimen of the SD male rat is directly provided with collagen specificity excitation spectrum to directly excite isolated liver tissue without any chemical special staining, and the inherent fluorescence characteristics of the liver tissue are observed by using a special fluorescence excitation block system of a fluorescence microscope to diagnose the liver fibrosis degree.
Preferably, it is characterized in that: the excitation light wavelength of the special fluorescence excitation block in S5 is distributed in the lower range of 300nm-460nm, and the liver tissue section for liver fibrosis diagnosis is a liver tissue section without any staining.
Preferably, it is characterized in that: the liver fibrosis diagnosis system aims at liver tissue sections including frozen sections and paraffin-embedded sections, and the inherent fluorescence of the liver tissue is generated after an optical filter in a lower wavelength range of 300nm-460nm or the excitation light of other corresponding light sources is excited without any chemical staining or fluorescence labeling.
Preferably, the liver fibrosis diagnosis system of S5 comprises any means for determining the degree of liver fibrosis based on the inherent fluorescence characteristics of the liver tissue specimen and the wavelength range of 300nm-460 nm.
(III) advantageous effects
Compared with the prior art, the invention provides a method for establishing hepatic fibrosis of healthy SD male rats by adopting a compound factor method, which has the following beneficial effects:
the method for establishing the hepatic fibrosis of the healthy SD male rat by adopting the compound factor method comprises the steps of mixing the blood stem cells, the stem cell active matter and the schistosome to form the compound factor, injecting the compound factor into the healthy SD male rat, wherein an animal model prepared by the rat accords with the pathological process of conversion from NASH to HCC, is easy to operate, short in time, stable and effective, provides modeling and technical support for developing and developing a new medicine for treating nonalcoholic steatohepatitis, and has huge economic and social benefits.
Drawings
FIG. 1 is a schematic view of the structure of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, a method for establishing hepatic fibrosis in healthy SD male rats by using a complex factor method comprises the following steps:
s1: culture of blood Stem cells
Diluting intestinal blood with PBS, adding 10ml lymphocyte separation solution into 50ml centrifuge tube, slowly adding diluted intestinal blood, slowly sucking out white nebulous lymphocyte layer in middle of layer, addingCentrifuging the PBS for 5 minutes, removing the supernatant after centrifugation, adding fetal bovine serum again, mixing uniformly, and repeatedly centrifuging once; discarding the supernatant, adding culture medium, mixing, and standing at 100cm2Culturing in the culture bottle;
s2: preparation of Stem cell active
Taking cultured stem cells for digestion and counting, washing fetal calf serum for 3 times, dissolving in 30ml of physiological saline, and ultrasonically breaking the cells; dissolving out intracellular protein and nutrients, centrifuging for 10 min, collecting supernatant, removing cell debris, concentrating with ultrafiltration membrane, removing immunoglobulin, collecting filtrate, filtering with filter membrane to obtain sterile solution with concentration of 200 μ g/ml, and storing at 4 deg.C to obtain solution A;
measuring the pH value of the upper layer culture solution of 30ml when the pH value of the culture medium for culturing the cells is 5.5, filtering by using a filter membrane to concentrate the volume of the upper layer culture solution to about 5ml, taking out the upper layer culture solution, adding 5ml of normal saline, and fixing the volume to 10 ml; filtering with 0.22 μm filter membrane to make it sterile, measuring protein concentration, determining concentration to be 200 μ g/ml, and storing at 4 deg.C to obtain solution B;
according to the following steps: 1: 1, mixing A, B with blood cells of schistosome to obtain active substance of blood stem cells;
s3: feeding and culturing adult SD male rats;
s4: feeding the rats after the weight measurement for 1 week, feeding high-fat CDAA feed, injecting blood stem cell active substances, continuously culturing for 8 weeks, wherein the injection of the blood stem cell active substances is carried out for 2 days in one week, and the common drinking water is carried out for the other 5 days;
s5: observing the mental state and hair condition of the rat every day during feeding, measuring the food intake and water intake, measuring the body weight of the rat every week, starting from the first feeding of high-fat CDAA diet and injection of blood stem cell active substances, continuously feeding for 8 weeks, and obtaining the rat with hepatic fibrosis.
In this example, S3 further comprises measuring the body weight of SD male rats by placing them in cages at room temperature of 25-30 deg.C and humidity of 55% -65%, and adaptively feeding them for 1 week in 12-hour light/dark cycle.
In this embodiment, specifically, the high-fat CDAA feed in S4, wherein by weight: fat 32%, carbohydrate 55%, protein 13%, choline 0; the carbohydrate is starch, dextrin, sugar, and protein as amino acid premix, the fat is oleum Maydis and hydrogenated vegetable oil, and the calorie is 4.3 kcal/g.
In this embodiment, specifically, the step S1 further includes the following steps:
s11: when the cells grow to 80-90% of the bottom of the culture bottle, sucking out the culture medium, adding PBS for washing, sucking out PBS, adding pancreatin, adding alpha-MEM culture medium containing 20% fetal calf serum, neutralizing the pancreatin, sucking out the neutralization solution, putting into a centrifuge tube, adding PBS, centrifuging for 5 minutes, removing the supernatant after centrifugation, adding PBS, mixing uniformly, re-centrifuging once, counting the cells, adding 2X 105 cells into each bottle, and placing into a culture bottle of 100cm2 for culture.
In this embodiment, specifically, the method further includes S5:
the liver puncture tissue specimen of the SD male rat is directly provided with collagen specificity excitation spectrum to directly excite isolated liver tissue without any chemical special staining, and the inherent fluorescence characteristics of the liver tissue are observed by using a special fluorescence excitation block system of a fluorescence microscope to diagnose the liver fibrosis degree.
In this embodiment, specifically, the following features are provided: the excitation light wavelength of the special fluorescence excitation block in S5 is distributed in the lower range of 300nm-460nm, and the liver tissue section for liver fibrosis diagnosis is a liver tissue section without any staining.
In this embodiment, specifically, the following features are provided: the liver fibrosis diagnosis system aims at liver tissue sections including frozen sections and paraffin-embedded sections, and the inherent fluorescence of the liver tissue is generated after an optical filter in a lower wavelength range of 300nm-460nm or the excitation light of other corresponding light sources is excited without any chemical staining or fluorescence labeling.
In this embodiment, the liver fibrosis diagnosis system in S5 includes any means for determining the degree of liver fibrosis based on the inherent fluorescence characteristics of the liver tissue specimen and the wavelength range between 300nm and 460 nm.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A method for establishing hepatic fibrosis of healthy SD male rats by adopting a compound factor method is characterized by comprising the following steps: the method comprises the following steps:
s1: culture of blood Stem cells
Adding PBS into intestinal blood for dilution, adding 10ml of lymphocyte separation liquid into a 50ml centrifuge tube, slowly adding the diluted intestinal blood, slowly sucking out the white cloudy lymphocyte layer in the middle of layering, adding PBS, centrifuging for 5 minutes, removing supernatant after centrifuging, adding fetal calf serum again, mixing uniformly, and repeatedly centrifuging once; discarding the supernatant, adding culture medium, mixing, and standing at 100cm2Culturing in the culture bottle;
s2: preparation of Stem cell active
Taking cultured stem cells for digestion and counting, washing fetal calf serum for 3 times, dissolving in 30ml of physiological saline, and ultrasonically breaking the cells; dissolving out intracellular protein and nutrients, centrifuging for 10 min, collecting supernatant, removing cell debris, concentrating with ultrafiltration membrane, removing immunoglobulin, collecting filtrate, filtering with filter membrane to obtain sterile solution with concentration of 200 μ g/ml, and storing at 4 deg.C to obtain solution A;
measuring the pH value of the upper layer culture solution of 30ml when the pH value of the culture medium for culturing the cells is 5.5, filtering by using a filter membrane to concentrate the volume of the upper layer culture solution to about 5ml, taking out the upper layer culture solution, adding 5ml of normal saline, and fixing the volume to 10 ml; filtering with 0.22 μm filter membrane to make it sterile, measuring protein concentration, determining concentration to be 200 μ g/ml, and storing at 4 deg.C to obtain solution B;
according to the following steps: 1: 1, mixing A, B with blood cells of schistosome to obtain active substance of blood stem cells;
s3: feeding and culturing adult SD male rats;
s4: feeding the rats after the weight measurement for 1 week, feeding high-fat CDAA feed, injecting blood stem cell active substances, continuously culturing for 8 weeks, wherein the injection of the blood stem cell active substances is carried out for 2 days in one week, and the common drinking water is carried out for the other 5 days;
s5: observing the mental state and hair condition of the rat every day during feeding, measuring the food intake and water intake, measuring the body weight of the rat every week, starting from the first feeding of high-fat CDAA diet and injection of blood stem cell active substances, continuously feeding for 8 weeks, and obtaining the rat with hepatic fibrosis.
2. The method for establishing hepatic fibrosis of healthy SD male rats by using the compound factor method according to claim 1, wherein the method comprises the following steps: s3 also comprises placing SD male rat in cage with room temperature of 25-30 deg.C and humidity of 55% -65%, adaptively feeding for 1 week in 12 hr light/dark cycle, and measuring weight of rat.
3. The method for establishing hepatic fibrosis of healthy SD male rats by using the compound factor method according to claim 2, wherein the method comprises the following steps: a high fat CDAA feed in S4, wherein, by weight: fat 32%, carbohydrate 55%, protein 13%, choline 0; the carbohydrate is starch, dextrin, sugar, and protein as amino acid premix, the fat is oleum Maydis and hydrogenated vegetable oil, and the calorie is 4.3 kcal/g.
4. The method for establishing hepatic fibrosis of healthy SD male rats by using the compound factor method according to claim 1, wherein the method comprises the following steps: the step of S1 further includes:
s11: when the cells grow to 80-90% of the bottom of the culture bottle, sucking out the culture medium, adding PBS for washing, sucking out PBS, adding pancreatin, adding alpha-MEM culture medium containing 20% fetal calf serum, neutralizing the pancreatin, sucking out the neutralization solution, putting into a centrifuge tube, adding PBS, centrifuging for 5 minutes, removing the supernatant after centrifugation, adding PBS, mixing uniformly, re-centrifuging once, counting the cells, adding 2X 105 cells into each bottle, and placing into a culture bottle of 100cm2 for culture.
5. The method for establishing hepatic fibrosis of healthy SD male rats by using the compound factor method according to claim 1, wherein the method comprises the following steps: further comprising S5:
the liver puncture tissue specimen of the SD male rat is directly provided with collagen specificity excitation spectrum to directly excite isolated liver tissue without any chemical special staining, and the inherent fluorescence characteristics of the liver tissue are observed by using a special fluorescence excitation block system of a fluorescence microscope to diagnose the liver fibrosis degree.
6. The method for establishing hepatic fibrosis of healthy SD male rats by using the compound factor method according to claim 5, wherein the method comprises the following steps: the excitation light wavelength of the special fluorescence excitation block in S5 is distributed in the lower range of 300nm-460nm, and the liver tissue section for liver fibrosis diagnosis is a liver tissue section without any staining.
7. The method for establishing hepatic fibrosis of healthy SD male rats by using the compound factor method according to claim 6, wherein the method comprises the following steps: the liver fibrosis diagnosis system aims at liver tissue sections including frozen sections and paraffin-embedded sections, and the inherent fluorescence of the liver tissue is generated after an optical filter in a lower wavelength range of 300nm-460nm or the excitation light of other corresponding light sources is excited without any chemical staining or fluorescence labeling.
8. The method for establishing hepatic fibrosis of healthy SD male rats by using the compound factor method according to claim 1, wherein the method comprises the following steps: the liver fibrosis diagnosis system of S5 comprises any means for determining the degree of liver fibrosis by means of the inherent fluorescence characteristics of the liver tissue specimen and the wavelength range of 300nm-460 nm.
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