skMLCK基因对成肌细胞C2C12肌纤维成分及信号通路的影响

发布时间：2018-04-24 02:39

本文选题：C2C12 + skMLCK　；参考：《锦州医科大学》2017年硕士论文

【摘要】：目的通过分别建立sk MLCK基因高、低表达的小鼠成肌细胞C2C12稳转细胞株,采用实时定量PCR及Western blot技术分析sk MLCK基因对小鼠成肌细胞C2C12肌纤维及其相关信号通路中关键成分的影响,初步探讨sk MLCK基因影响肌纤维的分子机制,为后续研究奠定基础。方法复苏本实验室前期获得的sk MLCK高表达稳转株进行扩大培养;将最佳干扰效率的sh RNA重组载体转染C2C12中,经嘌呤霉素筛选后获得低表达稳转细胞株。采用实时定量PCR及Western blot方法分别检测高表达和低表达细胞株中肌纤维组成成分(MLC、α-actin、Myhc slow、Myhc 2a、Myhc 2x和Myhc 2b)和信号通路关键因子(sk MLCK、CFL2、MEF2C、Ca M、p38、AMPKα2、Rho A)的m RNA及蛋白质的表达情况统计,并进行统计分析。结果1、实时定量PCR检测sk MLCK高表达细胞株m RNA表达的结果表明:与空白对照组相比,肌纤维组成成分中Myhc slow、Myhc 2a、MLC显著或极显著升高(p0.05或p0.01),α-actin、Myhc 2x、Myhc 2b极显著降低(p0.01);信号通路关键分子MEF2C、Ca M、p38、AMPKα2、Rho A显著或极显著降低(p0.05或p0.01);CFL2各组呈现显著或极显著降低(p0.05或p0.01)。Western blot检测sk MLCK高表达细胞株中蛋白质表达的表达情况结果表明:与空白对照组相比,肌纤维组成成分中Myhc 2a与MLC极显著升高(p0.01);α-actin、Myhc slow、Myhc 2x、Myhc 2b显著或极显著降低(p0.05或p0.01);信号通路关键分子中,MAPKp38、Rho A极显著升高(p0.01);MEF2C、Ca M、AMPKα2显著或极显著降低(p0.05或p0.01);CFL2各组呈现显著或极显著降低(p0.05或p0.01)。2、实时定量PCR检测sk MLCK低表达细胞株m RNA表达的结果表明:与空白对照组相比,肌纤维组成成分中α-actin、MLC、Myhc 2x、Myhc 2b显著或极显著升高(p0.05或p0.01);Myhc slow、Myhc 2a极显著降低(p0.05或p0.01);信号通路关键分子中,AMPKα2、Rho A极显著升高(p0.01).CFL2各组呈现显著或极显著升高(p0.05或p0.01)。Western blot检测sk MLCK低表达细胞株中蛋白质表达的表达情况结果表明:与空白对照组相比,肌纤维组成成分中,α-actin、MLC、Myhc 2x、Myhc 2b显著或极显著升高(p0.05或p0.01);信号通路的关键成分中,AMPKα2、Rho A极显著升高(p0.01);CFL2各组呈现显著或极显著升高(p0.05或p0.01)。3、将本实验中sk MLCK高、低表达稳转细胞株所有蛋白表达数据合并进行统计学相关分析发现,sk MLCK仅与信号通路Rho A显著(p0.05)高度相关,相关系数为0.818,呈曲线关系。结论1、C2C12中,sk MLCK基因通过改变Rho A信号通路的变化来影响肌纤维的组成。2、sk MLCK对肌纤维的生长发育具有抑制作用;3、sk MLCK抑制CFL2的表达,同时对其它信号通路(AMPK、Ca M和MAPK)也产生影响。
[Abstract]:Objective to establish mouse myoblast stable transformation cell lines with high and low expression of SK MLCK gene, and to analyze the effect of SK MLCK gene on C2C12 muscle fiber and key components of signal pathway in mouse myoblast by real-time quantitative PCR and Western blot. The molecular mechanism of SK MLCK gene affecting muscle fiber was preliminarily discussed, which laid a foundation for further study. Methods after resuscitation of SK MLCK high expression stable transgenic strain obtained in our laboratory, the sh RNA recombinant vector with the best interference efficiency was transfected into C2C12, and the low expression stable transfer cell line was obtained after screening by purine mycin. Real-time quantitative PCR and Western blot were used to detect the expression of m RNA and protein in high expression and low expression cell lines (MLC, 伪 -actinine Myhcslow-Myhc2aHc2x and Myhc 2b) and signal pathway key factor (MLCKL2MEF2CU Ca Mp38AMPK 伪 2Rho A), respectively. Results 1. The expression of m RNA in SK MLCK high expression cell line was detected by real time quantitative PCR. In muscle fiber components, Myhc slow-regulated Myhc2aAMLC significantly or extremely increased p0.05or p0.01a, 伪 -actinine Myhc2xMHC2b significantly decreased p0.01a; signal pathway key molecule MEF2CU Ca M38AMPK 伪 2Rho A significantly or very significantly decreased p0.05or p0.01CFL2 groups showed significant or extremely significant decrease in p0.05 or p0.01).Western blot detection. The results of protein expression in SK MLCK overexpression cell line showed that: compared with the blank control group, Myhc 2a and MLC in muscle fiber components increased significantly (p0.01a); 伪 -actinine Myhc slow (Myhc2x) Myhc2b significantly or extremely significantly decreased p0.05 or p0.01C; MAPKp38 Rho A in signal pathway key molecules increased significantly (p0.01MEF2CU Ca MAMPK 伪 2) or decreased significantly (p0.05 or p0.01) CFL2. The expression of m RNA in SK MLCK low expression cell line was detected by real time quantitative PCR. In the composition of myofiber, 伪 -actinin MLC (MLC) Myhc2x (Myhc2b) significantly or extremely significantly increased p0.05 or p0.01 Myhc2a significantly decreased p0.05 or p0.01a, and the key molecule of signal pathway, AMPK 伪 _ 2 Rho A, increased p0.01g 路CFL2 significantly or significantly increased p0.05 or p0.01).Western blot detection of SK MLCK low. The expression of protein in the expressed cell line showed that: compared with the blank control group, In the composition of myofiber, 伪 -actinine MLC (MLC) Myhc2x (Myhc2x) Myhc2b significantly or extremely increased p0.05 or p0.01a, and the key component of signal pathway, AMPK 伪 _ 2o _ (Rho A) significantly increased p0.01C ~ (2 +) CFL2. The results showed that the MLCK of these two groups was significantly higher than that of control group (p 0.05 or p 0.01 ~ (-1) 路3), and that of the key components of the signal pathway was significantly higher than that of the control group (P < 0.05 or P 0.01U 路3). All the protein expression data of low expression stable cell line were analyzed by statistical correlation analysis. It was found that MLCK was highly correlated with signal pathway Rho A (P 0.05), and the correlation coefficient was 0.818, showing a curve relationship. Conclusion (1) MLCK gene in C2C12 can inhibit the growth and development of muscle fiber by changing the signal pathway of Rho A, and inhibit the expression of CFL2 by MLCK. It also affects the expression of AMPK Ca M and MAPK in other signaling pathways.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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