真核表达载体S368A-Cx43-pcDNA3的构建及其在HeLa细胞中的表达
发布时间：2019-05-09 00:47
【摘要】： 研究背景:连接蛋白43(connexin43,Cx43)是心脏表达最丰富的连接蛋白,主要分布在心室,其构成的缝隙连接(gapjunction,GJ)在心肌细胞中的电偶联及化学信息交流(缝隙连接通道介导的细胞间通讯)中起着非常重要的作用。GJ通道功能的正常是心脏正常功能发挥的重要保证。近年来的研究表明细胞间电偶联障碍是心律失常的另一个重要原因,其所起的致心律失常作用甚至比兴奋性异常及膜离子通道功能紊乱起了更重要的作用。己知Cx43构成的GJ通道的功能受到胞内pH值、胞内Ca2+浓度、ATP浓度、Cx的磷酸化状态、跨通道电压和一些神经体液因子等多因素的调节,且大多数的功能调节位点位于胞浆内羧基末端。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。因此,研究高血糖对连接蛋白43磷酸化程度的影响及其分子机制,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。 研究目的:构建携带S368A -Cx43-pcDNA3的真核表达载体,转染HeLa细胞,观察间隙连接蛋白Cx43在HeLa细胞中的表达.为进一步研究高血糖促连接蛋白43磷酸化的具体位点及对缝隙连接通道的功能调控作用打下基础。 研究方法:1.构建pcDNA3-突变型连接蛋白43质粒(S368A -Cx43-pcDNA3),分别转染HeLa细胞,用G418筛选得到高表达的细胞;(S368A -Cx43-HeLa细胞)。对照组为pcDNA3.0、pcDNA3. 0-Cx43直接转染HeLa细胞.2.应用抗连接蛋白43抗体进行细胞免疫荧光、免疫印迹,观察S368A-Cx43的表达;3.应用Lucifer Yellow划痕试验检测间隙连接通道功能的改变。 研究结果:1,正确构建了S368A-Cx43-pcDNA3真核表达载体。2. S368A-Cx43可以在HeLa细胞中表达.3. Ser368位点突变能改变间隙连接通道传导功能。
[Abstract]:Background: connexin 43 (connexin43,Cx43) is the most abundant connexin expressed in the heart. It is mainly distributed in the ventricle and forms a gap junction (gapjunction,). GJ plays an important role in electrical coupling and chemical information exchange (gap junction channel mediated intercellular communication) in cardiomyocytes. The normal function of GJ channel is an important guarantee of normal cardiac function. Recent studies have shown that cell-to-cell coupling disorder is another important cause of arrhythmias, and its arrhythmogenic effects are even more important than abnormal excitability and membrane ion channel dysfunction. It is known that the function of GJ channels composed of Cx43 is regulated by many factors, such as intracellular pH, intracellular Ca2 concentration, ATP concentration, phosphorylated state of Cx, cross-channel voltage and some neurohumoral factors, etc. Most of the functional regulatory sites are located at the end of the cytosolic carboxyl group. Recent studies have shown that hyperglycemia can promote the phosphorylation of gap junction channel junction protein 43 and change its function. However, the specific site and mechanism of hyperglycemic connexin 43 phosphorylation are still unclear. Therefore, the study of the effect of hyperglycemia on the phosphorylation of connexin 43 and its molecular mechanism will help to clarify the mechanism of arrhythmias in diabetic patients and open up another new direction for the prevention and treatment of diabetes mellitus complicated with arrhythmias. Objective: to construct eukaryotic expression vector carrying S368A-Cx43-pcDNA3 and transfect HeLa cells to observe the expression of gap junction protein Cx43 in HeLa cells. It lays a foundation for further study on the specific sites of phosphorylation of hyperglycemic junction protein 43 and the regulation of gap junction channel function. Methods of study: 1. PcDNA3- mutant connexin 43 plasmid (S368A-Cx43-pcDNA3) was constructed and transfected into HeLa cells respectively. The highly expressed cells were screened by G418. (S368A-Cx43-HeLa cells). The control group was pcDNA3.0,pcDNA3.. HeLa cells were directly transfected with 0-Cx43. 2. The expression of S368A-Cx43 was observed by immunofluorescence and immunoblotting with anti-junction protein 43 antibody. Lucifer Yellow scratch test was used to detect the change of gap junction channel function. The results were as follows: 1. The eukaryotic expression vector of S368A-Cx43-pcDNA3 was constructed correctly. 2. S368A-Cx43 can be expressed in HeLa cells. Mutation of Ser368 site can change gap junction channel conduction function.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346;R587.2
[Abstract]:Background: connexin 43 (connexin43,Cx43) is the most abundant connexin expressed in the heart. It is mainly distributed in the ventricle and forms a gap junction (gapjunction,). GJ plays an important role in electrical coupling and chemical information exchange (gap junction channel mediated intercellular communication) in cardiomyocytes. The normal function of GJ channel is an important guarantee of normal cardiac function. Recent studies have shown that cell-to-cell coupling disorder is another important cause of arrhythmias, and its arrhythmogenic effects are even more important than abnormal excitability and membrane ion channel dysfunction. It is known that the function of GJ channels composed of Cx43 is regulated by many factors, such as intracellular pH, intracellular Ca2 concentration, ATP concentration, phosphorylated state of Cx, cross-channel voltage and some neurohumoral factors, etc. Most of the functional regulatory sites are located at the end of the cytosolic carboxyl group. Recent studies have shown that hyperglycemia can promote the phosphorylation of gap junction channel junction protein 43 and change its function. However, the specific site and mechanism of hyperglycemic connexin 43 phosphorylation are still unclear. Therefore, the study of the effect of hyperglycemia on the phosphorylation of connexin 43 and its molecular mechanism will help to clarify the mechanism of arrhythmias in diabetic patients and open up another new direction for the prevention and treatment of diabetes mellitus complicated with arrhythmias. Objective: to construct eukaryotic expression vector carrying S368A-Cx43-pcDNA3 and transfect HeLa cells to observe the expression of gap junction protein Cx43 in HeLa cells. It lays a foundation for further study on the specific sites of phosphorylation of hyperglycemic junction protein 43 and the regulation of gap junction channel function. Methods of study: 1. PcDNA3- mutant connexin 43 plasmid (S368A-Cx43-pcDNA3) was constructed and transfected into HeLa cells respectively. The highly expressed cells were screened by G418. (S368A-Cx43-HeLa cells). The control group was pcDNA3.0,pcDNA3.. HeLa cells were directly transfected with 0-Cx43. 2. The expression of S368A-Cx43 was observed by immunofluorescence and immunoblotting with anti-junction protein 43 antibody. Lucifer Yellow scratch test was used to detect the change of gap junction channel function. The results were as follows: 1. The eukaryotic expression vector of S368A-Cx43-pcDNA3 was constructed correctly. 2. S368A-Cx43 can be expressed in HeLa cells. Mutation of Ser368 site can change gap junction channel conduction function.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346;R587.2
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