STAT5b靶控报告基因检测IR激酶活性细胞模型的建立
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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
OBJECTIVE This research was designed to develop a cell system to detect IR kinase, according to the signal transduction pathway on insulin receptor, based on activity of insulin receptor kinase assayed by STAT5b targeting reporter gene. Activity of insulin receptor kinase was examined indirectly by assaying the expression level of reporter gene. The cell model utilized insulin receptor kinase as target to obtain high throughput screening of the effective drugs. METHODS (1) The optimal transfection formula was determined by comparing the expression level of GFP in HepG2 cells transfected by the different ratio of pEGFP-C3 to liposome. (2) The optimal cell model of transfection formula was screened by comparing the inductive expression level of reporter gene when HepG2 cells or CHO cells co-transfected by STAT5b targeting reporter gene (combined with IR, STAT5b gene or not) was treated by insulin and analyzed the influence of the transfection formula on the sensitivity of cell model. (3) The effect of insulin on cell model in dose and time dependent manner was examined. (4) The specificity of cell model was tested by the Tyrphostin of insulin receptor kinase. (5) The stability of cell model was examined by Z' factor. (6) The effect of insulin on the proliferation function of cells was determined by MTT method. RESULTS (1) The optimal transfection formula was the ratio of one to two of plasmid DNA (μg) to lipofectamine~(TM) 2000 (μl) by transfecting HepG2 cells. (2) The result of examining combination of HepG2 cells with plasmids indicated that
the plasmid expressing functional human insulin receptor was indispensable. (3) The result of CHO cell model suggested that STAT5b protein was indispensable for cell model. The cell model in the experiment, cotransfected pSTAT5b3-TK-LUC, pSV-6-Gal, pRC/CMVhIR and pBS-SK-STAT5b into CHO cells, was established by comparing the result of the HepG2 cell model with the CHO cell model. The highest expression level of reporter gene in CHO cells, induced by insulin at 10'7mol/L for 12 hours, cotransfected the above-mentioned four plasmids was up to 6.10 fold. The Luc expression level inducted by insulin was dose and time dependent manner. The cell model possessing the wide range of treatment concentration and the superior sensitivity was well suited for the requirement of high throughput screening. (4) The expression of reporter gene induced by insulin was down to 84.53% after being treated with the inhibitive reagent of receptor tyrosine kinase phosphorylated, Tyrphostin AG 1024 at lOfimolfL. The induction ratio of reporter gene decreased from 6.10 fold to 1.24 fold after the cells cotransfected pMT2-wtlB expressing the PTP1B protein, preventing receptor tyrosine kinase being phoshporylated, was induced by insulin. (5) Z' factor responses the repetition and reliability of experimentation and was independent on the experimental objects. Z' factor is good range between 0.5 and 1. The Z1 factor of the cell model established in the experiment was 0.61, which indicated that the cell model was very stable. (6) The cells were treated by insulin at 1013~10"4mol/L for the different time, and then tested absorbance by MTT at 570nm. The results showed no effect of the proliferation of CHO cells. The accuracy and reliability of the cell model was fine when the cells were treated by insulin at 10'7mol/L for 12 hours.CONCLUTION After examining the multiple parameters of the cell model established, assayed by STAT5b targeting reporter gene assaying activity of IR kinase, the cell model has good stability, fine repetition, splendid specificity, superior sensitivity, short period, high automatization, high throughput, low cost and no radioactivity, and so on. Consequently, the ceil model is great valuable in
screening anti-diabetes drugs.
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
OBJECTIVE This research was designed to develop a cell system to detect IR kinase, according to the signal transduction pathway on insulin receptor, based on activity of insulin receptor kinase assayed by STAT5b targeting reporter gene. Activity of insulin receptor kinase was examined indirectly by assaying the expression level of reporter gene. The cell model utilized insulin receptor kinase as target to obtain high throughput screening of the effective drugs. METHODS (1) The optimal transfection formula was determined by comparing the expression level of GFP in HepG2 cells transfected by the different ratio of pEGFP-C3 to liposome. (2) The optimal cell model of transfection formula was screened by comparing the inductive expression level of reporter gene when HepG2 cells or CHO cells co-transfected by STAT5b targeting reporter gene (combined with IR, STAT5b gene or not) was treated by insulin and analyzed the influence of the transfection formula on the sensitivity of cell model. (3) The effect of insulin on cell model in dose and time dependent manner was examined. (4) The specificity of cell model was tested by the Tyrphostin of insulin receptor kinase. (5) The stability of cell model was examined by Z' factor. (6) The effect of insulin on the proliferation function of cells was determined by MTT method. RESULTS (1) The optimal transfection formula was the ratio of one to two of plasmid DNA (μg) to lipofectamine~(TM) 2000 (μl) by transfecting HepG2 cells. (2) The result of examining combination of HepG2 cells with plasmids indicated that
the plasmid expressing functional human insulin receptor was indispensable. (3) The result of CHO cell model suggested that STAT5b protein was indispensable for cell model. The cell model in the experiment, cotransfected pSTAT5b3-TK-LUC, pSV-6-Gal, pRC/CMVhIR and pBS-SK-STAT5b into CHO cells, was established by comparing the result of the HepG2 cell model with the CHO cell model. The highest expression level of reporter gene in CHO cells, induced by insulin at 10'7mol/L for 12 hours, cotransfected the above-mentioned four plasmids was up to 6.10 fold. The Luc expression level inducted by insulin was dose and time dependent manner. The cell model possessing the wide range of treatment concentration and the superior sensitivity was well suited for the requirement of high throughput screening. (4) The expression of reporter gene induced by insulin was down to 84.53% after being treated with the inhibitive reagent of receptor tyrosine kinase phosphorylated, Tyrphostin AG 1024 at lOfimolfL. The induction ratio of reporter gene decreased from 6.10 fold to 1.24 fold after the cells cotransfected pMT2-wtlB expressing the PTP1B protein, preventing receptor tyrosine kinase being phoshporylated, was induced by insulin. (5) Z' factor responses the repetition and reliability of experimentation and was independent on the experimental objects. Z' factor is good range between 0.5 and 1. The Z1 factor of the cell model established in the experiment was 0.61, which indicated that the cell model was very stable. (6) The cells were treated by insulin at 1013~10"4mol/L for the different time, and then tested absorbance by MTT at 570nm. The results showed no effect of the proliferation of CHO cells. The accuracy and reliability of the cell model was fine when the cells were treated by insulin at 10'7mol/L for 12 hours.CONCLUTION After examining the multiple parameters of the cell model established, assayed by STAT5b targeting reporter gene assaying activity of IR kinase, the cell model has good stability, fine repetition, splendid specificity, superior sensitivity, short period, high automatization, high throughput, low cost and no radioactivity, and so on. Consequently, the ceil model is great valuable in
screening anti-diabetes drugs.
引文
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