动物造模,药效评价,肝癌转基因 z-bio 2024-08-09 608

　　The experimental animals used to establish liver cancer transgenic animal models are often CD1, C57BL/6 × DBA, C57BL/6 × SJL and other strains of mice. The exogenous genes transferred into animal bodies can be single genes such as HBx, SV40, C-myc, or dual genes such as c-myc/TGF - α, C-myc/TGF - β 1, HBx/myc. The liver cancer animal model established by transferring a single gene has simpler operation steps and higher modeling success rate than the liver cancer animal model established by transferring a dual gene. However, the experimental period of the animal model established by transferring a dual gene is shorter than that of the animal model established by transferring a single gene.

　　The most common transgenic animal model of hepatitis B virus (I-IBV) in primary liver cancer is hepatocellular carcinoma, and 90% of hepatocellular carcinoma is related to viral hepatitis. HBV generally does not infect cultured cells or commonly used experimental animals, which to some extent limits the study of its mechanism of liver cancer. The genetically modified liver cancer animal model provides a new approach and method for studying hepatitis virus associated liver cancer. Currently, foreign scholars have conducted extensive research and reporting in this area.

　　Method of replication: The 1.15kb HBV subtype ADR DNA fragment was microinjected into CD1 mouse single-cell fertilized eggs and integrated into the host genome. The expression levels of HBx transgenic RNA in different tissues of the mice were analyzed. Alternatively, a 0.87kb HBV subtype ADR DNA fragment (located at nucleotide 988-1860 in the genome) can be introduced into the pUC118 vector, and the DNA fragment can be microinjected into the nucleus of the fertilized egg of female (C57BL/6 × DBA) F1 mice, and then transferred into pseudopregnant female mice to analyze the expression levels of HBx transgenic RNA in different tissues of the mice. Kim et al. established a HBx transgenic mouse liver cancer model on CD1 mice and found that HBx transgenic RNA was expressed at relatively high levels in liver, kidney, and testicular tissues. The selective expression of this HBV DNA sequence in liver and kidney tissues was similar to that in humans infected with HBV. Liver histological examination revealed multiple lesions with abnormal changes in liver cells at 4 months of age, with no significant increase in gallbladder cell proliferation. At 8-10 months of age, tumor nodules appear in the liver with characteristics of hepatocellular carcinoma. HBx protein in liver cells is highly expressed in the nodules, and alpha fetoprotein (AFP) is detected as positive. The survival period of this transgenic mouse varies by gender, with most male transgenic mice dying between 11-15 months of age, while female mice only die between 17-21 months of age. Anatomical analysis revealed that all animals had tumors in their livers, and the microscopic histopathological diagnosis was hepatocellular carcinoma. Cancer tissue still has high expression of HBx protein and AFP, and some animals have liver cancer metastasis. The incidence of liver tumors in this model has obvious gender differences. The incidence of liver cancer in male mice is 90%, and that in female mice is 60%. Yu et al. established a HBx transgenic mouse liver cancer model using C57BL/6 × DBA mice. The study found that atypical hyperplasia occurred in the liver at 6 months of age and developed into hepatocellular carcinoma at 11-18 months of age. The tumor incidence rate of this model was 86%. Detection of HBx expression revealed that HBx protein is not expressed in non transgenic mouse liver cells, but is highly expressed in underdeveloped and atypical proliferating liver cells of transgenic mice; No proliferating cell nuclear antigen (PCNA) positive cells were detected in the liver tissue of non transgenic mice, while the number of PCNA positive cells in atypical proliferative liver nodules and hepatocellular carcinoma tissues of transgenic mice significantly increased, indicating that HBx antigen, as a viral transactivation factor encoded by HBV, initiates the formation of liver cancer and is significantly correlated with the occurrence of liver cancer.

　　2. Monkey virus 40 (SV40) transgenic animal model The monkey virus 40 (SV40) transgenic animals with different promoters can have cell abnormalities in different parts, such as the SV40 gene regulated by the insulin gene promoter can cause pancreatic cancer, and the SV40T gene regulated by the antithrombin III (AT III) gene enhancer can cause liver cancer.

　　Method of replication: Using human AT Ⅲ gene and SV40T antigen (SV40 Tags) sequence to construct exogenous DNA, a SV40 transgenic mouse liver cancer model was established. Gillet et et al. found that the liver exhibits mitotic abnormalities before 2 months of age, many well-defined tumor nodules appear at 4 months of age, and hepatocellular adenoma develops at 6 months of age, with underdeveloped liver cells around the nodules. Unlike hepatocellular adenomas in the human body, this model did not exhibit any signs of cirrhosis. Seven months of age is the late stage of liver cancer development, and its liver weight can be 15 times that of a normal liver. The liver structure is completely destroyed, accompanied by many necrotic areas. The survival period of these genetically modified mice rarely exceeds 10 months, and most of them die at 8 months of age. Their tumor incidence rate is 100%. Unlike other genetically modified liver cancer animal models, some animals in this model showed lung metastasis, indicating that the tumor malignancy induced by AT-SV40T transgenic is relatively high. Analyzing the expression levels of genetically modified organisms, it was found that the expression of Tag mRNA was very low at birth and sharply increased after one week, and was only limited to the liver. The mRNA and protein expression levels of genetically modified organisms in proliferative nodules were higher than those in non proliferative areas. In the late stage of liver cancer, the expression level in the liver lesion area is significantly reduced, but the expression level in lung metastatic tumors is significantly increased. Ornithine transaminase (OTC), as a sensitive marker of liver differentiation, can be detected in tumor nodules and surrounding cells before 7 months of age for its mRNA expression. Subsequently, its expression level decreases, but it still shows significant expression in lung metastatic tumors. Dupuy et al. established an SV40 transgenic mouse liver cancer model to study tumor angiogenesis and tissue factor expression. Research has found that there is significant abnormal angiogenesis in liver cancer, and during the adenoma stage, there is proliferation, remodeling, and arterialization of the hepatic sinusoids; In some sinusoidal spaces, liver cells with endothelial cell phenotype can be found implanted into endothelial cells, indicating a pattern of local angiogenesis. Through this transgenic model, it was found that these abnormal angiogenesis are mainly related to platelet activation, and it was found that this platelet activation is activated by tissue factor (TF) produced by endothelial cells and invasive macrophages. HIF-1a/VEGF and TF play key roles in liver cancer angiogenesis. This model can be used as a screening model for drugs such as anti-tumor angiogenesis or anti-tumor thrombosis.

　　3. The transgenic mouse liver cancer model with dual transfer of transforming growth factor alpha (TGF-a) and c-myc often co expresses oncogenes TGF-a and C-myc in human primary liver cancer tissues, indicating that their interaction may be an important process in cellular malignancy. Compared with transgenic animals expressing Alb-c-myc or MT-TGF-a genes separately, the simultaneous expression of these two genes in mouse liver significantly accelerates liver tumor formation.

　　Method of replication: Firstly, the constructed 4.5kb Alb/c-myc gene was transferred into (C57BL/6j × CBA/j) F1 fertilized eggs to cultivate Alb/c-myc transgenic mice; Transfer the constructed 2.3kb EcoRI MT TGF-a gene into CD1 fertilized eggs and cultivate MT/TGF-a transgenic mice; Mating two types of transgenic mice to obtain TGF-a/c-myc transgenic mice. Research has shown that c-myc transgenic mice with Alb enhancer/promoter integrated separately exhibit varying degrees of liver cell dysplasia before 2 months of age, no liver hyperplasia nodules were found at 12 months of age, and some animals developed liver cell adenomas at 15-18 months. MT/TGF-a transgenic mice can only induce well differentiated multifocal hepatocellular carcinoma at 10-15 months of age. At 6 weeks of age, TGF-a/c-myc transgenic mice showed significant developmental abnormalities in liver cells, manifested as dense nucleoli, condensed chromatin, and other signs of cell apoptosis. By 9 weeks of age, approximately 80% of genetically modified mice exhibit such damage and develop many underdeveloped cell foci. After 19 weeks of age, 70% of transgenic male mice showed single or multiple focal tumor nodules in the liver, which were pathologically analyzed as well differentiated hepatocellular carcinoma. At this stage, there was no corresponding pathological damage in the liver of transgenic mice with either c-myc or TGF-a. Throughout the entire experimental period, no tumor formation was observed in the liver of (C57BL/6j × CBA/j) × CD1 wild-type (WT) mice of the same age.