Method for constructing animal model of hepatic cell fibrosis induced by chronic cadmium exposure

Abstract

The invention provides a construction method of an animal model of chronic low-dose cadmium exposure induced hepatic fibrosis, and provides a research basis for the reason of chronic cadmium exposure induced hepatic fibrosis and the drug efficacy analysis of the chronic cadmium exposure induced hepatic fibrosis. The method simulates a chronic low-dose cadmium exposure condition, is convenient to operate and stable, can help scientific researchers to visually observe the occurrence and development process of chronic liver injury and hepatic fibrosis induced by heavy metal exposure, and can provide a powerful basis for mechanism research of heavy metal poisoning. According to experimental results, in the body shape, living state, liver appearance and liver tissue pathological change process of a mouse, the mouse shows the mouse liver chronic injury and fibrosis change process under the chronic low-dose cadmium exposure environment, the direct relation between the cadmium exposure time and the liver fibrosis progress can be directly obtained, and the obtained animal model is stable. Researchers can create a disease process similar to human diseases through construction of the animal model in the future, the disease process can be quickly simulated, mass copying can be performed in a short time, the research period can be shortened, the animal model can be used for large-scale experimental research, and powerful support is provided for drug research and development and treatment strategies.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for constructing an animal model of chronic cadmium exposure-induced hepatic fibrosis. Background Art

[0002] Hepatic fibrosis is an important pathophysiological process in the development of liver diseases, referring to the abnormal proliferation of intrahepatic connective tissue caused by various pathogenic factors. Almost any liver injury has a process of hepatic fibrosis during the liver repair and healing process. The hepatic fibrosis caused by short-term injury can be reversed with the self-repair of the liver. If the injury factors cannot be removed for a long time and the fibrosis process persists for a long time, it will be irreversible and develop into liver cirrhosis and even liver cancer.

[0003] Cadmium is a heavy metal that can enter the human body through various routes, such as inhalation and ingestion. Long-term exposure to cadmium may cause various health problems, including damage to the kidneys, skeletal system, and liver. Regarding the relationship between cadmium and hepatic fibrosis, studies have shown that cadmium can cause liver injury and may thus promote the occurrence and development of hepatic fibrosis. Therefore, research on the causes of cadmium-induced hepatic fibrosis and its animal models will contribute to the treatment of liver diseases caused by various reasons. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing an animal model of chronic cadmium exposure-induced hepatic fibrosis, which simulates a chronic cadmium poisoning environment to induce hepatic fibrosis in mice and is used for analyzing the causes of cadmium-induced hepatic fibrosis and evaluating the efficacy of liver cancer drugs.

[0005] To solve the above technical problem, the present invention provides a method for constructing an animal model of chronic cadmium exposure-induced hepatic fibrosis, comprising the following steps:

[0006] (1) Selection of animal samples: Select 6-week-old male C57BL / 6J mice with a body weight of 20-25 g;

[0007] (2) Adaptive feeding: All mice are raised in an individually ventilated cage (IVC) system that meets the cleaning agent standard for 7 days. The room temperature is controlled at 23±2°C, the humidity is controlled at 60±5%, the light illumination is 12h:12h light-dark alternation, and the diet is free access to autoclaved water and sterilized feed;

[0008] (3) Drinking water intoxication: Twenty male mice are grouped and numbered for individual feeding. The feed and drinking water are provided in a free-feeding manner. The experiment is divided into 4 groups (n = 5 in each group), namely different feeding time periods of 30d, 60d, and 150d. The mice drink an aqueous cadmium chloride solution containing 0.01% (0.1 mg / ml) of cadmium every day, and the control group is given deionized water;

[0009] (4) Appearance behavior record: Observe and record the changes in the hair, body weight, weekly water intake, and daily behavior activities of the mice every day after exposure to the poison.

[0010] (5) Tissue and organ separation: After the experimental group of mice were exposed to cadmium chloride for 30d / 60d / 150d, they were sacrificed by cervical dislocation. The control group of mice were sacrificed by cervical dislocation after 150d. The livers of the mice were taken respectively to measure their organ coefficients, and part of them was fixed in neutral buffered formalin for standby.

[0011] (6) Histopathological observation: The liver tissues of the mice were fixed, paraffin-embedded, sectioned, routinely stained with HE, Masson, and immunohistochemistry (SMA). The pathological changes were observed and photographed under a microscope.

[0012] (7) Statistical processing: The original data were calculated for the mean and standard deviation, and the results were expressed in the form of X±S. Tests were performed using spss27.0. The body weight, organ coefficient, and water intake were analyzed. P<0.05 was considered to be significantly different, and the animal model of chronic cadmium exposure-induced hepatocyte fibrosis was obtained.

[0013] Furthermore, the application of the method for constructing an animal model of chronic cadmium exposure-induced hepatocyte fibrosis in the evaluation of the efficacy of drugs for liver cancer cells.

[0014] The advantages and beneficial effects of the present invention are as follows:

[0015] The present invention provides a method for constructing an animal model of chronic cadmium exposure-induced hepatocyte fibrosis, which provides a research basis for the causes of chronic cadmium exposure-induced hepatocyte fibrosis and the analysis of the efficacy of drugs. The present invention simulates the condition of chronic low-dose cadmium exposure, is convenient to operate, and the method is stable. It can help scientific research workers intuitively observe the occurrence and development process of chronic liver injury and hepatocyte fibrosis diseases induced by heavy metal exposure, and can provide a strong basis for the mechanism research of heavy metal poisoning. From the experimental results of the present invention, in the process of the body shape, living state, liver shape, and liver tissue pathological changes of the mice, the process of chronic liver injury and fibrosis changes of the mice under the condition of chronic low-dose cadmium exposure is reflected. The direct relationship between cadmium exposure time and the progression of hepatocyte fibrosis can be directly obtained, and the obtained animal model is stable. Future researchers can construct this animal model to cause a pathological process similar to human diseases, can quickly simulate the disease process, and can be replicated in large numbers in a short time, shortening the research cycle. It can be used for large-scale experimental research and provides strong support for drug R & D and treatment strategies. Description of the Drawings

[0016] Figure 1 It is a diagram of the morphological changes of the liver organs in the embodiment;

[0017] Figure 2 It is a HE staining diagram of the liver tissue in the embodiment;

[0018] Figure 3 Masson staining diagram of liver tissue for the example;

[0019] Figure 4 SMA expression diagram for the example. Detailed implementation manners

[0020] The present invention will be further described in detail below through examples. These examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0021] A method for constructing an animal model of chronic cadmium exposure-induced hepatocyte fibrosis, comprising the following steps:

[0022] (1) Selection of animal samples: Select 6-week-old male C57BL / 6J mice with a body weight of 20-25 g;

[0023] (2) Adaptive feeding: All mice are raised in an individually ventilated cage (IVC) system meeting the cleaner standard for 7 days. The room temperature is controlled at 23±2°C, the humidity is controlled at 60±5%, the light illumination is 12h:12h light-dark alternation, and the diet is free access to autoclaved water and sterilized feed;

[0024] (3) Drinking water intoxication: Twenty male mice are grouped and numbered for individual feeding. The feed and drinking water are provided in a free-feeding manner. The experiment is divided into 4 groups (n = 5 in each group), namely different feeding time periods of 30d, 60d, and 150d. The mice drink an aqueous cadmium chloride solution containing 0.01% cadmium (0.1 mg / ml) every day, and the control group is given deionized water;

[0025] (4) Recording of appearance and behavior: After intoxication, observe and record the changes in the hair, body weight, weekly water intake, and daily behavior activities of the mice every day;

[0026] (5) Isolation of tissues and organs: After the experimental group mice are exposed to cadmium chloride for 30d / 60d / 150d, they are decapitated and sacrificed. The control group mice are decapitated and sacrificed after 150d. The livers of the mice are taken to measure their organ coefficients, and part of them is fixed in neutral buffered formalin for standby;

[0027] (6) Histopathological observation: The liver tissues of the mice are fixed, paraffin-embedded, sectioned, routinely stained with HE, Masson stained, and immunohistochemically stained (SMA). The pathological changes are observed and photographed under a microscope;

[0028] (7) Statistical processing: The original data is calculated for the mean and standard deviation, and the results are expressed in the form of X±S. The spss27.0 is used for testing. The body weight, organ coefficient, and water intake are analyzed. P<0.05 indicates significant difference, and the animal model of chronic cadmium exposure-induced hepatocyte fibrosis is obtained.

[0029] Preferably, the method for constructing an animal model of chronic cadmium exposure-induced hepatocyte fibrosis is applied to the evaluation of the efficacy of drugs on liver cancer cells.

[0030] The specific operation of the histopathological observation in step 6 is as follows:

[0031] 1. H&E staining:

[0032] ⑴ The specimens fixed with paraformaldehyde are dehydrated successively through four gradients of 75% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol.

[0033] ⑵ Clear in xylene for 1 min.

[0034] ⑶ Immerse in soft wax, 20 min for the first time and 40 min for the second time.

[0035] ⑷ Embed in paraffin, 20 min for the first time and 20 min for the second time.

[0036] ⑸ Cut sections (section thickness is about 4 - 6 μm), and place them in an oven at 60 °C for 2 - 3 h.

[0037] ⑹ Remove paraffin in xylene, 10 min each time, repeat 2 times.

[0038] ⑺ Elute successively through four gradients of 100% alcohol, 95% alcohol, 80% alcohol, and 75% alcohol, 5 min each time.

[0039] ⑻ Stain with hematoxylin for 5 min, then wash with water, and differentiate with hydrochloric acid alcohol for 5 - 10 s.

[0040] ⑼ Wash with running water to blue back for 10 min.

[0041] ⑽ Stain with eosin solution for 5 - 30 s.

[0042] ⑾ Dehydrate successively through four gradients of 75% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol, 5 min each time.

[0043] ⑿ Clear in xylene 3 times, 5 min each time.

[0044] ⒀ After air-drying, seal the sections with neutral gum.

[0045] 2. Masson staining

[0046] ⑴ Dewax the paraffin sections to water.

[0047] ⑵ Stain the nuclei with Weigert iron hematoxylin staining solution for 5 - 10 min (when in use, take equal amounts of solution A1 and A2 and mix well to form Weigert iron hematoxylin staining solution, and do not prepare it in advance and store).

[0048] ⑶ Differentiate with acidic ethanol differentiation solution for 5 - 15 s, and wash thoroughly with water;

[0049] ⑷ Blue with Masson bluing solution for 3 - 5 min, and wash thoroughly with water;

[0050] ⑸ Wash with distilled water for 1 min;

[0051] ⑹ Stain with ponceau fuchsin staining solution for 5 - 10 min;

[0052] ⑺ During the above operation process, prepare a weak acid working solution according to the ratio of distilled water: weak acid solution = 2:1, and wash with the weak acid working solution for 1 min; ⑻ Differentiate with 1% phosphomolybdic acid solution for 1 - 2 min; wash with the prepared weak acid working solution for 1 min;

[0053] ⑼ Without washing with water, directly stain with aniline blue staining solution for 1 - 2 min;

[0054] ⑽ Wash with the prepared weak acid working solution for 1 min;

[0055] ⑾ Dehydrate quickly with 95% ethanol;

[0056] ⑿ Dehydrate with absolute ethanol 3 times, 5 - 10 s each time;

[0057] ⒀ Clear with xylene 3 times, 1 - 2 min each time;

[0058] ⒁ Mount with neutral balsam;

[0059] ⒂ Staining result: The cell nucleus, collagen fiber or protein are blue; the cytoplasm, muscle, and red blood cells are red.

[0060] 3. Histochemical staining of liver tissue (SP three-step method)

[0061] ⑴ Paraffin sections, dewax to water routinely.

[0062] ⑵ Incubate with 0.3% or 3% H2O2 deionized water (colorless liquid) for 10 - 30 minutes to inactivate endogenous peroxidase activity. Rinse with distilled water and soak in PBS for 5 minutes

[0063] ⑶ Candidate steps: Antigen retrieval: microwave (it is recommended to use medium heat 4 times within 30 minutes), high pressure, enzyme retrieval method. Cool naturally, and then use for 3 minutes × 3 times.

[0064] ⑷ Serum blocking: at room temperature for 15 - 30 minutes, preferably consistent with the source of the secondary antibody. Pour off, do not wash.

[0065] ⑸ Dropwise add the primary antibody diluted in an appropriate ratio, incubate at 37°C for 2 - 3 hours or overnight at 4°C (it is best to rewarm). Rinse with PBS, 3 minutes × 5 times.

[0066] ⑹ Dropwise add the biotin-labeled secondary antibody, incubate at room temperature or 37°C for 30 minutes - 1 h.

[0067] ⑺ BS rinse, 3 minutes × 5 times.

[0068] ⑻ Add SP (streptavidin-peroxidase), incubate at room temperature or 37 °C for 30 minutes - 1 h.

[0069] ⑼ PBS rinse, 3 minutes × 5 times.

[0070] ⑽ Color development with color developer (such as DAB).

[0071] ⑾ Rinse thoroughly with tap water.

[0072] ⑿ Counterstaining, dehydration, and clearing can be carried out.

[0073] ⒀ Select an appropriate mounting medium for mounting the slides.

[0074] The specific operation of step 7 statistical analysis is as follows:

[0075] Use the SPSS 22.0 software package to perform statistical analysis on the data. The data is expressed as mean ± standard deviation For comparison of means among multiple groups, one-way ANOVA is used. For correlation analysis, Spearman correlation coefficient test is used. p < 0.05 indicates statistically significant differences.

[0076] Main reagents used in the present invention

[0077] Immunohistochemistry (SP) Kit Beijing Zhongshan Jinqiao Co., Ltd. Hematoxylin Fuzhou Maixin Biotechnology Co., Ltd. Eosin Fuzhou Maixin Biotechnology Co., Ltd. 10% Neutral Gum Fuzhou Maixin Biological Development Co., Ltd. Masson Staining Kit Beijing Solarbio Science & Technology Co., Ltd. Phosphate Buffered Saline (PBS) Beijing Solarbio Science & Technology Co., Ltd. 4% Paraformaldehyde Beijing Solarbio Science & Technology Co., Ltd.

[0078] Main instruments used in the present invention

[0079] Refrigerators at 4℃ and -20℃ Haier Company Pathological Microtome German SLEE CUT4062 Type Inverted Fluorescence Microscope Japanese Olympus IX71 DRM+Q550 Type Pathological Image Analyzer German LEI Company Optical Microscope Japanese Olympus BX53 Micropipette German Eppendorf Company

[0080] The experimental results of the examples are as follows:

[0081] 1. Observation of physiological state

[0082] The mice in the experimental group were in good spirits, with normal hair color. The water intake of each cage of mice was 70 - 80 mL per week, and the body weight increased slowly (Table 1). Under the condition of drinking water containing cadmium chloride at a concentration of 1 g / L, the changes in the growth state (body weight, hair) of the mice in the cadmium poisoning group were not obvious; however, the liver weight showed a slow increasing change, which was in line with chronic liver damage and hepatic fibrosis changes.

[0083] 2. Organ morphology

[0084] Compared with the control group, in the groups with different exposure times, the weight of the liver showed an increasing trend with the modeling time, and the liver weight / body weight ratio in the exposure groups was higher than that in the control group. At all different time points, it indicated that liver damage occurred in mice in a cadmium environment, and the degree of damage was positively correlated with the intervention time (Table 1). By observing the livers of mice, it was found that the liver color of the control group was bright red, the liver surface was smooth and shiny, and it felt elastic when touched. Dense liver tissue could be seen through the outer membrane. In the mice in the exposure group, as the exposure time prolonged, the liver color gradually faded from bright red, irregular deformation occurred on the liver surface, the liver surface became rough and lacked luster, the liver elasticity decreased, and the gap between liver tissues could be observed to become larger through the capsule, and irregular depressions appeared at the edge. Specifically, as shown in Figure 1 the changes indicated that the exposure time was positively correlated with the degree of liver damage (P<0.05).

[0085] Table 1 Differences in organ coefficients and physiological indexes between different exposure times and the control group

[0086]

[0087] *, P<0.05

[0088] 3. Histomorphological observation (HE),

[0089] For liver tissue samples with different exposure times, through HE staining, it was found that when the exposure time was short, it could first trigger an inflammatory response in the liver. A large number of inflammatory cells could be observed scattered in the area of liver blood vessel distribution, indicating that in the microenvironment of cadmium overload, the liver inflammatory response was obvious and could lead to hepatocyte damage. As the exposure time increased, the inflammatory stimulation intensified. In the tissue exposed for 2 months, a large number of hepatocytes showed edema, and a large number of hepatocytes necrosed, and a post-injury hyperplastic structure was formed. In the liver tissue exposed for 5 months, significant remodeling of hepatocytes and portal structures could be observed. The hepatocytes were swollen, the cell gaps were widened, the portal structures proliferated, fibrous tissue proliferated, and the liver lobe structure showed mild changes ( Figure 2 ).

[0090] 4. Masson staining

[0091] Masson staining method: (1) Routinely dewax paraffin sections to water; (2) Stain with iron hematoxylin for 7 minutes; (3) Wash with distilled water; (4) Differentiate with 1% hydrochloric acid alcohol for 30 seconds; (5) Rinse with running water for 5 minutes and then wash with distilled water; (6) Immerse in Ponceau acid fuchsin staining solution for 5 minutes; (7) Wash with distilled water; (8) Differentiate with phosphomolybdic acid aqueous solution for 5 minutes; (9) Stain with aniline blue for 5 minutes; (10) Differentiate with phosphomolybdic acid aqueous solution for 1 minute; (11) Treat with 1% glacial acetic acid for 1 minute; (12) Dehydrate with 95% alcohol and absolute alcohol, clear with xylene, and mount with resin ( Figure 3);The results of Masson staining showed that collagen fibers were blue, cytoplasm, muscle fibers and red blood cells were red, and cell nuclei were blue-brown. The comparison of the fibrotic areas between the control group and the poisoned group suggested a significant difference in the fibrotic area after statistical analysis (P<0.05, Figure 4 );In addition, the comparison of the fibrotic areas of each group at different poisoning time periods showed a significant difference in the fibrotic area after statistical analysis (P<0.05).

[0092] 5. Expression of SMA in liver tissue by immunohistochemistry

[0093] The results of SMA staining were the same as those of Masson, suggesting that with the prolongation of cadmium exposure time, the expression of SMA in liver tissue showed an increasing trend, and the fibrous tissue also increased significantly, but it was still in the early stage of fibrosis and did not progress to the stage of liver cirrhosis.

[0094] The above examples were practiced at Guangxi Medical University. This technology originated from the Guangxi Science and Technology Plan "Multi-center Study on Carcinogenic Effects and Spatial Prediction Models of Heavy Metal Exposure in High-incidence Areas of Primary Liver Cancer in Guangxi" (Contract No.: Guangxi Science and Technology AB23026041). Based on the above research content, the project team found that under the condition of chronic low-dose cadmium exposure, the changes in the body shape, living state, liver appearance and liver tissue pathological state of mice were all consistent with the process of chronic liver injury and fibrosis.

Claims

1. A method for constructing an animal model of liver fibrosis induced by chronic cadmium exposure, characterized in that: The following steps are involved: (1) Animal sample selection: 6-week-old C57BL / 6J male mice weighing 20–25 g were selected; (2) Adaptive feeding: All mice were housed in an independent ventilation cage (IVC) system that met detergent standards for 7 days. The room temperature was controlled at 23±2°C, the humidity was controlled at 60±5%, the lighting was alternating between 12h and 12h light and dark, and the diet was free access to autoclaved sterilized water and edible sterilized feed. (3) Drinking water poisoning: 20 male mice were grouped and raised individually, with free access to feed and drinking water. The experiment was divided into 4 groups (n = 5 in each group), i.e., 30 days, 60 days, and 150 days of different feeding periods. They drank 0.01% (0.1 mg ml-1) cadmium chloride aqueous solution every day. The control group was given deionized water; (4) Appearance and behavior records: After exposure, the mice’s hair, body weight, weekly water intake, and daily behavior were observed and recorded every day; (5) Tissue and organ separation: The mice in the experimental group were killed by cervical dislocation 30 days, 60 days, and 150 days after exposure to cadmium chloride, and the mice in the control group were killed by cervical dislocation 150 days after exposure. The livers of the mice were taken to determine their organ coefficients and then fixed in neutral buffered formalin for later use; (6) Histopathological observation: The mouse liver tissue was fixed, embedded in paraffin, sectioned, and stained with conventional HE, Masson staining, and immunohistochemistry (SMA). The pathological changes were observed and recorded under a microscope and photographed; (7) Statistical processing: The mean value and standard deviation of the original data were calculated, and the results were expressed in the form of X±S. SPSS 27.0 was used for analysis of body weight, organ coefficient and water intake. P<0.05 was considered significant. The animal model of chronic cadmium exposure-induced liver fibrosis was obtained.

2. Application of the method for constructing an animal model of chronic cadmium exposure-induced liver fibrosis according to claim 1 in the evaluation of drug efficacy of liver cancer cells.

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