真核表達(dá)載體S368A-Cx43-pcDNA3的構(gòu)建及其在HeLa細(xì)胞中的表達(dá)
發(fā)布時(shí)間：2019-05-09 00:47
【摘要】： 研究背景:連接蛋白43(connexin43,Cx43)是心臟表達(dá)最豐富的連接蛋白,主要分布在心室,其構(gòu)成的縫隙連接(gapjunction,GJ)在心肌細(xì)胞中的電偶聯(lián)及化學(xué)信息交流(縫隙連接通道介導(dǎo)的細(xì)胞間通訊)中起著非常重要的作用。GJ通道功能的正常是心臟正常功能發(fā)揮的重要保證。近年來的研究表明細(xì)胞間電偶聯(lián)障礙是心律失常的另一個(gè)重要原因,其所起的致心律失常作用甚至比興奮性異常及膜離子通道功能紊亂起了更重要的作用。己知Cx43構(gòu)成的GJ通道的功能受到胞內(nèi)pH值、胞內(nèi)Ca2+濃度、ATP濃度、Cx的磷酸化狀態(tài)、跨通道電壓和一些神經(jīng)體液因子等多因素的調(diào)節(jié),且大多數(shù)的功能調(diào)節(jié)位點(diǎn)位于胞漿內(nèi)羧基末端。新近的一些研究表明高血糖可以促間隙連接通道連接蛋白43磷酸化并使其功能發(fā)生改變。但高血糖促連接蛋白43磷酸化的具體位點(diǎn)及其機(jī)理尚不清楚。因此,研究高血糖對連接蛋白43磷酸化程度的影響及其分子機(jī)制,將有助于闡明糖尿病患者心律失常的發(fā)生機(jī)理并為預(yù)防及治療糖尿病并發(fā)心律失常開辟另一嶄新的方向。 研究目的:構(gòu)建攜帶S368A -Cx43-pcDNA3的真核表達(dá)載體,轉(zhuǎn)染HeLa細(xì)胞,觀察間隙連接蛋白Cx43在HeLa細(xì)胞中的表達(dá).為進(jìn)一步研究高血糖促連接蛋白43磷酸化的具體位點(diǎn)及對縫隙連接通道的功能調(diào)控作用打下基礎(chǔ)。 研究方法:1.構(gòu)建pcDNA3-突變型連接蛋白43質(zhì)粒(S368A -Cx43-pcDNA3),分別轉(zhuǎn)染HeLa細(xì)胞,用G418篩選得到高表達(dá)的細(xì)胞;(S368A -Cx43-HeLa細(xì)胞)。對照組為pcDNA3.0、pcDNA3. 0-Cx43直接轉(zhuǎn)染HeLa細(xì)胞.2.應(yīng)用抗連接蛋白43抗體進(jìn)行細(xì)胞免疫熒光、免疫印跡,觀察S368A-Cx43的表達(dá);3.應(yīng)用Lucifer Yellow劃痕試驗(yàn)檢測間隙連接通道功能的改變。 研究結(jié)果:1,正確構(gòu)建了S368A-Cx43-pcDNA3真核表達(dá)載體。2. S368A-Cx43可以在HeLa細(xì)胞中表達(dá).3. Ser368位點(diǎn)突變能改變間隙連接通道傳導(dǎo)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.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：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.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R346;R587.2
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