FrzA基因转导干预Wnt信号通路减轻TNF-α诱导心肌细胞炎症损伤的研究

发布时间：2018-07-07 21:22

本文选题：双链重组9型腺相关病毒 + Frz　；参考：《新疆医科大学》2017年硕士论文

【摘要】：目的:以双链重组AAV9(double strand AAV9,ds AAV9)型腺相关病毒为载体介导的Frz A基因转染心肌细胞炎症损伤模型,观察是否能抑制心肌细胞Wnt通路表达活性,从而减轻心肌细胞凋亡,促进细胞存活。方法:利用TNF-α刺激H9c2心肌细胞建立心肌细胞炎症损伤模型,模拟体内心肌缺血状态,MTT法检测细胞活性及Annexin V-FITC/PI双染法检测心肌细胞凋亡率,确定TNF-α最佳浓度。ds AAV9介导Frz A基因转染心肌细胞,Western blot检测各组细胞Wnt通路关键分子蛋白的表达,RT-PCR检测各组细胞Wnt通路关键分子m RNA的表达。Western blot检测各组细胞凋亡相关蛋白的表达,Annexin V-FITC/PI双染法检测各组心肌细胞凋亡率的变化。结果:(1)免疫荧光结果显示心肌细胞的纯度可达到98%以上。(2)MTT与Annexin V-FITC/P I双染法结果显示TNF-α浓度对心肌细胞活性的影响是以剂量依赖的方式,随着TNF-α的浓度增加,心肌细胞活性降低,细胞凋亡率增高,应选择浓度50μg/L的TNF-α来建立心肌细胞炎症损伤模型。(3)Western blot及RT-PCR结果均显示TNF-α可有效激活Wnt信号通路,且Frz A基因转染可抑制Wnt信号通路的活性。(4)Western blot及Annexin V-FITC/PI双染法结果显示,TNF-α激活Wnt信号通路后,细胞凋亡增多。而ds AAV9介导Frz A基因转导可有效减轻TNF-α诱导的心肌细胞凋亡,并且这种抗凋亡作用有可能正是通过抑制Wnt信号通路来实现的。结论:CMV启动子介导的ds AAV9载体可高效的转染心肌细胞,TNF-α刺激体外培养的心肌细胞可建立心肌细胞炎症损伤模型,模拟体内心力衰竭状态,激活Wnt信号通路,诱导心肌细胞凋亡。Ds AAV9介导Frz A基因转染可抑制心肌细胞Wnt信号通路的表达活性,可有效减轻心肌细胞的凋亡。
[Abstract]:Aim: to investigate whether the expression of Wnt pathway in cardiomyocytes can be inhibited and the apoptosis of cardiomyocytes can be alleviated by transfection of Frz A gene into cardiomyocytes with double-stranded recombinant AAV9 (double strand AAV9ds AAV9 adeno-associated virus as a vector. Promote cell survival. Methods: the myocardial inflammatory injury model was established by TNF- 伪 stimulation of H9c2 cardiomyocytes. The cell activity was detected by MTT assay and the apoptosis rate of cardiomyocytes was detected by Annexin V-FITC / Pi double staining. Determination of the optimal concentration of TNF- 伪 .DS AAV9 mediated Frz-A gene transfection into cardiomyocytes Western blot detection of Wnt pathway key molecule protein expression in each group; RT-PCR detection of Wnt pathway key molecule mRNA expression; Western blot detection of apoptosis in each group of cells The expression of related protein was detected by Annexin V-FITC / Pi double staining. Results: (1) Immunofluorescence showed that the purity of cardiomyocytes was over 98%. (2) the results of MTT and Annexin V-FITC / P I double staining showed that the effect of TNF- 伪 concentration on cardiomyocyte activity was dose-dependent. With the increase of TNF- 伪 concentration, cardiomyocyte activity decreased. TNF- 伪 concentration of 50 渭 g / L should be selected to establish myocardial inflammatory injury model. (3) Western blot and RT-PCR results showed that TNF- 伪 could effectively activate Wnt signaling pathway. FrzA gene transfection inhibited the activity of Wnt signaling pathway. (4) Western blot and Annexin V-FITC / Pi double staining showed that TNF- 伪 activated Wnt signaling pathway and apoptosis increased. DS AAV9 mediated Frz A gene transduction can effectively attenuate the apoptosis induced by TNF- 伪, and this anti-apoptosis effect may be achieved by inhibiting the Wnt signaling pathway. Conclusion DS AAV9 vector mediated by 1: CMV promoter can efficiently transfect TNF- 伪 into cultured cardiomyocytes to establish myocardial inflammatory injury model, simulate heart failure in vivo and activate Wnt signaling pathway. Inducing cardiomyocyte apoptosis. DS-AAV9 mediated FrzA gene transfection can inhibit the expression of Wnt signal pathway and attenuate the apoptosis of cardiomyocytes.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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