高血糖促心脏连接蛋白43磷酸化及其对连接通道功能的影响

发布时间：2018-11-21 10:07

【摘要】： 心脏间隙连接通道是心肌细胞之间的特殊连接通道,其功能是完成心肌细胞之间的电化学信息交换,其功能的异常与心律失常的发生有密切关系。心室间隙连接通道主要由连接蛋白43组成。间隙连接通道的功能主要受胞内pH值、胞内Ca2+浓度、连接蛋白磷酸化状态、跨通道电压的调控。新近的一些研究表明高血糖可以促间隙连接通道连接蛋白43磷酸化并使其功能发生改变。但高血糖促连接蛋白43磷酸化的具体位点及其机理尚不清楚。高血糖可以激活蛋白激酶C,而蛋白激酶C又可以使连接蛋白43羧基末端丝氨酸磷酸化,据此推测高血糖可能通过使连接蛋白43羧基末端丝氨酸磷酸化而改变其调控功能。由此,本课题组拟应用基因点突变、抗原再现技术来研究高血糖对间隙连接通道连接蛋白43的促磷酸化作用及其机制,如果能阐明高血糖致心脏间隙连接通道功能异常的机制,那将有助于阐明糖尿病患者心律失常的发生机理并为预防及治疗糖尿病并发心律失常开辟另一崭新的方向。本研究首先通过构建pcDNA3-野生型连接蛋白43质粒(Wt-Cx43-pcDNA3)、pcDNA3-突变型连接蛋白43质粒(S262F-Cx43-pcDNA3,S368A -Cx43-pcDNA3),分别转染HeLa细胞,经G418筛选后得到表达相应连接蛋白43的细胞株(Wt-Cx43-HeLa细胞,S262F-Cx43-HeLa细胞,S368A -Cx43-HeLa细胞)。应用上述细胞株分两部分进行细胞学实验。第一部分是在筛选后细胞株的培养基中加入不同浓度的葡萄糖(5mmol/L,25mmol/L),应用抗磷酸化Cx43特异性抗体或应用Cx43位点磷酸化抗体通过细胞免疫化学,细胞免疫荧光,及免疫印迹分析Cx43磷酸化情况。对比分析不同葡萄糖浓度对Cx43磷酸化的影响及磷酸化位点。第二部分通过应用Lucifer Yellow划痕试验检测高血糖间隙连接通道功能的改变。结果表明:1,高血糖能促使Cx43磷酸化;2,应用抗连接蛋白43磷酸化特异性位点的抗体(p-connexin43-Ser368和p-connexin43-Ser262)进行细胞免疫荧光,Western bloting,结果表明高血糖促使连接蛋白43的Ser368位点磷酸化,而对Ser262的无磷酸化作用;3,应用Lucifer Yellow划痕试验检测间隙连接通道功能的改变,结果表明高血糖能降低间隙连接通道传导。综上所述,本实验研究表明(1)高血糖可以促进Cx43的磷酸化,提高Cx43的磷酸化程度;(2)高血糖促使连接蛋白43的Ser368位点磷酸化;(3)高血糖能降低间隙连接通道传导,降低细胞GJ功能。
[Abstract]:The gap junction channel is a special junction channel between cardiomyocytes, whose function is to complete the electrochemical information exchange between cardiomyocytes. The abnormal function is closely related to the occurrence of arrhythmia. The ventricular gap junction channel is mainly composed of connexin 43. The function of gap junction channel is mainly regulated by intracellular pH, intracellular Ca2 concentration, phosphorylation of connexin, and transchannel voltage. Recent studies have shown that hyperglycemia stimulates gap junction channel junction protein 43 phosphorylation and changes its function. However, the specific site and mechanism of hyperglycemic connexin 43 phosphorylation are unclear. Hyperglycemia can activate protein kinase C, and protein kinase C can phosphorylate connectin 43 carboxyl terminal serine, which suggests that hyperglycemia may change its regulatory function by phosphorylation of connectin 43 carboxyl terminal serine. Therefore, our team intends to use gene point mutation and antigen reproduction technique to study the phosphorylation of gap junction protein 43 by hyperglycemia and its mechanism, if we can clarify the mechanism of hyperglycemia induced cardiac gap junction pathway dysfunction. It will help to clarify the mechanism of arrhythmia in patients with diabetes and open up a new direction for the prevention and treatment of arrhythmia. In this study, pcDNA3- wild-type connexin 43 plasmid (Wt-Cx43-pcDNA3) and pcDNA3- mutant connexin 43 plasmid (S262F-Cx43-pcDNA3S368A-Cx43-pcDNA3) were constructed and transfected into HeLa cells. After G418 screening, the cell lines expressing the corresponding connexin 43 (Wt-Cx43-HeLa cells, S262F-Cx43-HeLa cells, S368A-Cx43-HeLa cells) were obtained. The above cell lines were divided into two parts to carry out cytological experiments. The first part is to add different concentrations of glucose (5 mmol / L ~ 25 mmol 路L ~ (-1) to the culture medium of the screened cell line, and then apply anti-phosphorylated Cx43 specific antibody or Cx43 site phosphorylated antibody through cellular immunocytochemistry to make cell immunofluorescence. The phosphorylation of Cx43 was analyzed by Western blot. The effects of glucose concentration on Cx43 phosphorylation and phosphorylation sites were analyzed. In the second part, Lucifer Yellow scratch test was used to detect the change of gap junction channel function. The results showed that: 1) hyperglycemia could promote the phosphorylation of Cx43; 2. The results of cellular immunofluorescence, Western bloting, showed that hyperglycemia induced the phosphorylation of Ser368 site of connexin 43 by using antibodies (p-connexin43-Ser368 and p-connexin43-Ser262) to the phosphorylated site of connexin 43. No phosphorylation of Ser262 was observed. 3. Lucifer Yellow scratch test was used to detect the change of gap junction channel function. The results showed that hyperglycemia could reduce gap junction channel conduction. To sum up, the present study shows that (1) hyperglycemia can promote the phosphorylation of Cx43 and increase the phosphorylation of Cx43, (2) hyperglycemia can promote the phosphorylation of Ser368 site of connexin 43; (3) hyperglycemia can decrease gap junction channel conduction and GJ function.
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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