注射突变p53基因CRISPR/Cas9质粒和肝癌细胞建立肝癌小鼠模型  

Construction of a hepatocellular carcinoma mouse model by injection of a mutant p53 gene CRISPR/Cas9 plasmid and hepatoma cells

作　　者：王燕鸽 高子涵[1] 李宗霖 王康龙 白雪 李瑞芳[1] 有曼 王红伟[1] WANG Yange;GAO Zihan;LI Zonglin;WANG Kanglong;BAI Xue;LI Ruifang;YOU Man;WANG Hongwei(Department of Pharmacy,School of Basic Medicine,Henan University of Science and Technology,Henan Engineering Technology Center of Liver Disease Prevention and Control,Luoyang 471023,China)

机构地区：[1]河南科技大学基础医学院药学系,河南省肝病防治工程技术中心,河南洛阳471023

出　　处：《中国实验动物学报》2021年第2期190-196,共7页Acta Laboratorium Animalis Scientia Sinica

基　　金：河南省科技攻关项目(202102310486)。

摘　　要：目的采用尾静脉注射突变p53基因CRISPR/Cas9质粒和H22细胞建立肝癌模型。方法将健康雄性BALB/c小鼠随机空白组(生理盐水)、质粒组(质粒)、对照组(H22细胞+盐水)、实验组(H22细胞+质粒),每组25只。尾静脉注射建立肝癌模型。分别在注射后的2、3、4、5周中取材,眼眶采血检测小鼠血清转氨酶(ALT/AST)水平。观察肝、肺表面变化及结节数目,计算各组小鼠的成模率、死亡率以及脏器指数。HE染色观察小鼠肝的组织病理形态学改变。免疫组化、Western Blot等方法检测小鼠肝中p53及PCNA蛋白的表达情况。结果实验组和对照组均能成功构建转移性肝癌小鼠模型,其中实验组和对照组小鼠的成模率分别为60.87%和45.83%,死亡率为4.35%和29.17%。两组小鼠血清ALT、AST水平均有显著增加,肝表面按时间顺序也出现大小不等的囊肿和白色颗粒状结节。HE结果显示5周后实验组和对照组小鼠肝小叶结构均有不同程度的破坏,呈明显肿瘤病理学改变。质粒组与实验组小鼠肝中p53蛋白的表达量明显降低(P<0.05);实验组和对照组的肝组织中PCNA蛋白表达显著增加,且实验组表达量明显高于对照组(P<0.05)。结论通过尾静脉注射突变p53基因CRISPR/Cas9质粒和H22细胞的方法能够成功快速构建转移性肝癌小鼠模型。Objective To establish a mouse model of liver cancer by injecting mutant p53 gene CRISPR/Cas9 plasmid and H22 cells into the tail vein.Methods Healthy male BALB/c mice were randomly divided into blank group(normal saline),plasmid group(Plasmid),control group(H22 cells+saline)and experimental group(H22 cells+Plasmid),each with 25 mice.Tail vein injection was used to establish the liver cancer model.Samples were taken at 2,3,4 and 5 weeks after the injection,and blood was collected from the orbit to detect serum aminotransferase(ALT/AST).Liver and lung surface changes and the number of nodules were observed,and successful modeling rate,mortality and organ index in each group were assessed.HE staining was used to observe the pathological and morphological changes in the liver.Immunohistochemistry,western blot and other method were used to evaluate the expression of p53 and PCNA protein in the liver.Results Both the experimental group and the control group showed successful generation of a mouse model of metastatic liver cancer.The rates of successful modeling in the experimental and control groups were 60.87% and 45.83%,respectively,and the mortality rate was 4.35% and 29.17%,respectively.The serum ALT and AST levels in the two groups of mice increased significantly,and cysts and white granular nodules of varying sizes appeared on the liver surface over time.The HE result showed that the liver lobules in the experimental and control groups were damaged to varying degrees after 5 weeks,showing obvious tumor-related pathological changes.The expression of p53 protein in the liver of the plasmid and experimental groups was significantly reduced(P<0.05).The expression of PCNA protein in the liver tissue of the experimental and control groups was increased significantly,and expression was significantly higher in the experimental group compared with the control group(P<0.05).Conclusions The method of injecting mutant p53 gene CRISPR/Cas9 plasmid and H22 cells through the tail vein can successfully and rapidly construct a m

关 键 词：尾静脉注射 基因编辑 突变p53基因 H22细胞 转移性肝癌模型 

分 类 号：Q95-33[生物学—动物学]