STAT5b靶控报告基因检测IR激酶活性细胞模型的建立

发布时间：2018-04-17 04:30

本文选题：胰岛素受体激酶 + STAT5b　；参考：《西华大学》2006年硕士论文

【摘要】：目的根据胰岛素受体介导的信号转导通路,构建STAT5b应答元件靶控报告基因的细胞模型,通过检测报告基因表达水平间接监测胰岛素受体(IR)激酶活性,从而为高通量地筛选出以IR激酶为靶标的药物提供新的细胞模型。方法 (1)转染方案的选择:通过对pEGFP-3与脂质体不同的转染比例,转染HepG2细胞表达GFP效率比较,确定最佳的转染方案。(2)转染细胞模型的建立和验证:STAT5b靶控报告基因(联合或不联合IR基因及联合或不联合STAT5b基因)对HepG2细胞或CHO细胞的转染,检测胰岛素对转染细胞STAT5b靶控报告基因表达的影响,并比较分析联合或不联合IR基因及联合或不联合STAT5b基因对细胞模型灵敏性的影响,选择最佳的细胞转染模型。(3)考察胰岛素对细胞模型的量效和时效的关系。(4)通过IR激酶抑制剂考察细胞模型的特异性。(5)通过Z′因子考察细胞模型的稳定性。(6)利用MTT法考察胰岛素对细胞增殖功能的影响。结果 (1)用pEGFP-C3转染HepG2细胞来确定质粒DNA与lipofectamine~(TM)2000最佳转染方案为质粒DNA(μg):lipofectamine~(TM)2000(μ1)=1:2。(2)HepG2细胞株与质粒组合的考察,结果表明转染外源性的人胰岛素受体pRC/CMV·hIR质粒可以显著提高细胞模型的灵敏度。(3)CHO细胞株与质粒组合的考察,结果表明STAT5b蛋白在诱导Luc表达中发挥着重要作用的,本实验通过共转染pBS-SK-STAT5b质粒可以达到此目的。分析比较两种细胞株的实验结果,本实验选用pSTAT5b_3-TK-LUC、pSV-μ-Gal、DRC/CMV·hIR与pBS-SK-STAT5b四种质粒共转染CHO细胞建立细胞模型。在10~(-7)mol/L胰岛
[Abstract]:Aim to construct a cell model of target control reporter gene of STAT5b response element according to the signal transduction pathway mediated by insulin receptor, and to indirectly monitor the activity of insulin receptor (IRR) kinase by detecting the expression level of reporter gene.This provides a new cell model for high throughput screening of IR kinase targeted drugs.Methods 1) selection of transfection scheme: the efficiency of GFP expression in HepG2 cells was compared by different transfection ratio of pEGFP-3 and liposome.Establishment and verification of the cell model of transfection with the optimal transfection method. The transfection of HepG2 cells or CHO cells by the target control reporter gene (combined or not combined IR gene and combined or uncombined STAT5b gene) was verified.To detect the effect of insulin on the expression of target control reporter gene in STAT5b transfected cells, and to compare and analyze the effects of combined or uncombined IR gene and combined or uncombined STAT5b gene on the sensitivity of the cell model.Select the best cell transfection model. (3) investigate the relationship between the dose effect of insulin on the cell model and the effect of time. 4) examine the specificity of the cell model by IR kinase inhibitor. 5) study the stability of the cell model by Z' -factor.The effect of insulin on cell proliferation was investigated by MTT.Results 1) pEGFP-C3 transfection of HepG2 cells was used to determine that the optimal transfection scheme of plasmid DNA and lipofectamine~(TM)2000 was the combination of plasmid DNA (渭 g: lipofectamine2) TM2000 (渭 1)=1:2.(2)HepG2 cell line) and plasmid.The results showed that transfection of exogenous human insulin receptor pRC/CMV hIR plasmid could significantly improve the sensitivity of the cell model. The results showed that STAT5b protein played an important role in the induction of Luc expression.This experiment can achieve this goal by co-transfection of pBS-SK-STAT5b plasmid.The results of two cell lines were analyzed and compared. The cell model was established by co-transfection of pSTAT5bti3-TK-LUCnpSV- 渭 -Gal-DRC / CMV hIR and pBS-SK-STAT5b into CHO cells.In 10~(-7)mol/L islets
【学位授予单位】：西华大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R-331

【相似文献】