摘要
背景:研究表明,2型糖尿病啮齿类动物模型及人体的丙酮酸脱氢酶复合体表达量降低,PDHA1基因缺陷也是导致丙酮酸脱氢酶复合体酶活性改变最常见的原因。目的:构建并鉴定胰岛β细胞特异性敲除PDHA1基因小鼠,为后续探究PDHA1基因在糖尿病发病机制方面作用提供模型基础。方法:实验方案经南方医科大学伦理委员会批准。利用Cre/lox P系统理论,将PDHA1^loxp/loxp小鼠与胰岛β细胞特异性表达Cre重组酶的小鼠进行数代杂交,3-4周龄小鼠通过PCR法鉴定筛选基因型,子2代中基因型为PDHA1^loxp/loxpCre^+/-小鼠即为实验所需要构建的模型小鼠。取4周龄PDHA1^flox/floxCre^+/-小鼠(βKO小鼠组)与Cre^+/-小鼠(对照组)各5只,给予相同的水和饲料,待8周龄时连续7d注射腹腔注射他莫昔芬50mg/kg诱导PDHA1基因敲除,观察3周后腹腔注射麻醉,取胰腺组织、脂肪组织、肝脏组织。采用q PCR法、Western Blot法、免疫组织化学法鉴定PDHA1基因敲除效果,观察小鼠表型。结果与结论:(1)PCR基因扩增筛选出基因型为PDHA1^loxp/loxpCre^+/-小鼠;(2)q PCR法检验小鼠胰腺、脂肪、肝脏组织,证实敲除效果具有组织特异性,胰岛中βKO组小鼠PDHA1 mRNA的相对表达较对照组明显下降;(3)Western Blot检测及免疫组织化学法观察显示,βKO组小鼠PDHA1蛋白表达较对照组小鼠明显降低,证实敲除PDHA1基因效果明显;(4)结果说明,利用Cre/loxP系统成功构建胰岛β细胞特异性敲除PDHA1基因的小鼠模型,为动物水平上探究糖尿病发病机制提供新的靶点。
BACKGROUND: In rodent models of type 2 diabetes mellitus and human, the expression level of pyruvate dehydrogenase complex is decreased, and PDHA1 deficiency is the most common cause of changed pyruvate dehydrogenase complex activity.OBJECTIVE: To construct and identify the inducible islet-specific PDHA1 knockout mice, so as to provide basis for the study on the role of PDHA1 in pathogenesis of diabetes mellitus.METHODS: The study was approved by the Ethics Committee of Southern Medical University. By using Cre-loxP recombination system,PDHA1^flox/floxCre^+/-mice were generated by crossing PDHA1^flox/flox mice with Cre^+/-mice. Genotypic identification was performed by PCR at the age of 3-4 weeks and the PDHA1^flox/floxCre^+/-mice were the required mouse model. PDHA1flox/floxCre+/-mice(βKO group, n=5) and Cre+/-mice(control group, n=5) at the age of 4 weeks were selected and received the same water and feed. Tamoxifen(50 mg/kg) was intraperitoneally injected at the age of 8 week to induce gene knockout. Three weeks later, pancreatic tissue, adipose tissue, and liver tissue were removed under anesthesia. qPCR, western blot and immunohistochemical staining were applied to identify the PDHA1 knockout effect and to observe the mouse phenotype.RESULTS AND CONCLUSION:(1) PCR analysis selected PDHA1^flox/floxCre^+/-mice.(2) qPCR method was used to test the pancreas, fat and liver tissues of mice, and it was confirmed that the knockout effect had tissue specificity. The expression level of PDHA1 mRNA in islet in theβKO group was significantly lower than that in the control group.(3) The results of western blot and immunohistochemical scanning showed a significant decrease in PDHA1 protein in the βKO group compared with the control group, suggesting the PDHA1 knockout effect was obvious.(4) In summary, the β cell-specific deletion of the PDHA1 gene is successfully constructed by Cre-loxP system, which provides a novel target for studying the pathogenesis of diabetes at animal level.
作者
王肖
赖舒畅
叶艳诗
许雪娟
白晓春
沈洁
Wang Xiao;Lai Shuchang;Ye Yanshi;Xu Xuejuan;Bai Xiaochun;Shen Jie(Department of Endocrine and Metabolism,the Third Affiliated Hospital of Southern Medical University,Guangzhou 510631,Guangdong Province,China;Central Laboratory,the Third Affiliated Hospital of Southern Medical University,Guangzhou 510631,Guangdong Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2019年第31期5023-5029,共7页
Chinese Journal of Tissue Engineering Research
基金
广州市科技计划项目(201604020007),项目负责人:沈洁~~
