β3-AR对心肌细胞凋亡的影响及介导机制研究

发布时间：2018-01-02 01:18

本文关键词：β3-AR对心肌细胞凋亡的影响及介导机制研究　出处：《石河子大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:采用去甲肾上腺素(noradrenaline,NE)诱导心肌细胞凋亡,分别应用β3-AR激动剂及阻滞剂进行干预,观察β3-AR对心肌细胞凋亡的影响,探讨β3-AR与PI3K/Akt和p38MAPK信号通路对心肌细胞凋亡的作用,为β3-AR的信号通路研究提出新的理论。方法:使用优化的Ⅱ型胶原酶和差速贴壁法分离培养心肌细胞,分别用总蛋白法、超速离心法、试剂盒法提取心肌细胞β3-AR膜蛋白。BCA法进行蛋白定量,Western blot法检测蛋白样品中β3-AR提取蛋白的特异性。实验分组:正常对照组;NE诱导组:NE培养48 h;BRL组:β3-AR激动剂(BRL37344)干预+NE培养48 h;SR组:β3-AR阻滞剂(SR59230A)干预+NE培养48 h。TUNEL法及流式细胞仪双染法检测心肌细胞凋亡率;Western blot检测各组凋亡相关因子Bcl-2、Bax和caspases-3蛋白的表达;Western blot和q RT-PCR检测各组PI3K/Akt和p38MAPK信号通路蛋白和m RNA的表达。结果:1.优化心肌细胞的培养方法可获得产量大、浓度高的细胞,能满足后续实验;2.与其他组比较,试剂盒法提取蛋白的浓度最高,差异均有统计学意义(P0.05);Western blot检测结果示:与其他组比较,试剂盒提取法所得β3-AR膜蛋白含最高,差异均有统计学意义(P0.05);3.TUNEL法及流式细胞仪检测心肌细胞凋亡率:BRL组细胞凋亡率明显增高,与NE诱导组比较,差异有统计学意义(P0.05);4.Western blot检测结果示:BRL组caspases-3和Bax/Bcl-2比值增高,与NE诱导组比较,差异有统计学意义(P0.05);5.Western blot检测结果示:BRL组磷酸化的p38MAPK蛋白表达增高,与NE诱导组比较,差异有统计学意义(P0.05);SR组磷酸化的Akt蛋白表达增高,与NE诱导组比较,差异有统计学意义(P0.05)。结论:1.试剂盒提取法可有效提高β3-AR膜蛋白的浓度和特异性;2.β3-AR可促进体外培养NE诱导的心肌细胞凋亡;3.β3-AR过度激活可通过下调PI3K/Akt信号和上调p38MAPK信号通路促进心肌细胞凋亡。
[Abstract]:Objective: using norepinephrine (noradrenaline, NE) were used to induce apoptosis of myocardial cells, beta 3-AR agonists and antagonists intervention, to observe the effect of beta 3-AR on myocardial cell apoptosis, explore the beta 3-AR and PI3K/Akt and p38MAPK signal pathway on apoptosis of myocardial cells with new theory for the study of beta 3-AR signal pathway. Methods: using the optimized type II collagenase and differential adherent cultured myocardial cells, respectively, with the total protein, ultracentrifugation, protein quantitative extraction of myocardial cell beta 3-AR membrane protein.BCA kit method, specific protein beta 3-AR extraction protein samples Western blot method in the experimental groups. Normal control group; NE group: NE culture induced by 48 h; group BRL: 3-AR beta agonists (BRL37344) intervention +NE cultured for 48 h; group SR: 3-AR beta blockers (SR59230A) intervention +NE 48 h.TUNEL culture method and flow cytometry double staining The apoptosis rate of myocardial cells; apoptosis related factor blot detection of Western Bcl-2, the expression of Bax and caspases-3 protein expression of Western and Q; blot RT-PCR was used to detect PI3K/Akt and p38MAPK signal pathway of M protein and RNA. Results: 1. optimized culture of myocardial cells can be obtained from the large amount, high concentration of cells, can satisfy the follow-up experiment 2.; compared with the other groups, the kit method for extracting protein was the highest, the differences were statistically significant (P0.05); Western blot assay results showed that: compared with the other groups, extraction kit method the beta 3-AR membrane protein containing the highest, the differences were statistically significant (P0.05); 3.TUNEL method and flow detection of myocardial cell apoptosis type cell rate: the apoptosis rate in BRL group increased significantly, compared with the induction of NE, the difference was statistically significant (P0.05); 4.Western blot assay results showed: in group BRL, caspases-3 and Bax/Bcl-2 ratio increased, and NE induced group Comparison, the difference was statistically significant (P0.05); 5.Western blot assay results showed: in BRL group the expression of phosphorylated p38MAPK protein increased, compared with the induction of NE, the difference was statistically significant (P0.05); SR group the expression of phosphorylated Akt protein increased, compared with the induction of NE, the difference was statistically significant (P0.05) conclusion: 1.. Extraction Kit method can effectively improve the beta 3-AR membrane protein concentration and specificity; 2. beta 3-AR can promote cultured myocardial cell apoptosis induced by NE in vitro; 3. beta excessive activation of 3-AR can downregulate the PI3K/Akt signal and the up regulation of p38MAPK signaling pathway to promote myocardial cell apoptosis.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R54

【参考文献】