PKC和Rab GTPase在钙信号调节骨骼肌细胞GLUT4胞内运输机制中的作用

发布时间：2018-05-21 17:25

  本文选题：骨骼肌 + 蛋白激酶C　； 参考：《天津医科大学》2015年硕士论文

【摘要】：目的:应用稳定过表达GLUT4myc的L6-GLUT4myc大鼠骨骼肌细胞探讨传统型PKC(conventional PKC,cPKC)、新颖型PKC(novel PKC,nPKC)以及Rab蛋白在钙信号调节骨骼肌细胞葡萄糖转运子4(glucose transporter 4,GLUT4)胞内运输机制中的作用。方法:第一部分,用1mM钙离子载体ionomycin孵育L6-GLUT4myc成肌细胞,Western blot检测ionomycin对PKC底物磷酸化的影响,并用1mM cPKC和nPKC抑制剂G?6983和1mM cPKC抑制剂G?6976预孵育L6-GLUT4myc成肌细胞,ELISA测定细胞表面GLUT4水平及GLUT4的内吞和外排,Western blot检测PKC底物的磷酸化,探讨cPKC和nPKC是否参与ionomycin促进骨骼肌细胞GLUT4转位的作用。第二部分,应用RNA干扰技术分别下调PKCα、PKCζ和PKCε的蛋白表达,随机分为对照组和ionomycin组,ELISA测定ionomycin作用下的GLUT4转位以及GLUT4的内吞和外排,进一步探讨何种PKC亚型参与ionomycin促进骨骼肌细胞GLUT4转位的作用。第三部分,应用RNA干扰技术分别下调Rab8a、Rab13和Rab14的蛋白表达,随机分为对照组和ionomycin组,ELISA测定细胞表面GLUT4水平,探讨哪个Rab参与ionomycin促进骨骼肌细胞GLUT4转位的作用。结果:我们前期的研究结果显示:ionomycin升高L6-GLUT4myc成肌细胞胞浆Ca2+浓度,显著增加细胞表面GLUT4水平。第一部分结果显示:ionomycin显著增加PKC底物的磷酸化水平。G?6983和G?6976均显著抑制ionomycin促进的GLUT4myc转位(p0.05,p0.001)和PKC底物的磷酸化。Ionomycin抑制GLUT4myc内吞,促进GLUT4myc外排,G?6983既抑制ionomycin抑制的GLUT4myc内吞(p0.01)也抑制ionomycin刺激的GLUT4myc外排(p0.001)。G?6976抑制ionomycin抑制的GLUT4myc的内吞(p0.05)但不影响ionomycin促进的GLUT4myc的外排。第二部分结果显示:siPKCα和siPKCζ均显著抑制ionomycin刺激的GLUT4myc转位(p0.001),而siPKCε不影响ionomycin促进的GLUT4myc转位。siPKCζ既抑制ionomycin抑制的GLUT4myc内吞(p0.001)也抑制ionomycin促进的GLUT4myc外排(p0.001)。siPKCα抑制ionomycin抑制的GLUT4myc的内吞(p0.001),但不影响ionomycin促进的GLUT4myc的外排。第三部分结果显示:siRab13显著抑制ionomycin促进的GLUT4myc转位(p0.05),而siRab8a和siRab14不影响ionomycin刺激的GLUT4myc转位。结论:1.Ionomycin激活骨骼肌细胞PKC。2.cPKC和nPKC参与ionomycin促进GLUT4转位的作用,PKCα和PKCζ是起作用的PKC亚型。3.cPKC介导ionomycin抑制GLUT4内吞的作用,PKCα是起作用的cPKC亚型。nPKC参与ionomycin促进GLUT4外排及抑制其内吞的作用,PKCζ是起作用的nPKC亚型。4.Rab13介导ionomycin促进GLUT4转位的作用。综上,ionomycin通过PKCα/PKCζ-Rab13信号转导通路促进骨骼肌细胞GLUT4转位。
[Abstract]:Aim: to investigate the role of traditional PKC(novel PKCnPKCand Rab protein in regulating intracellular transport of glucose transporter 4(glucose transporter 4 (GLUT4) in skeletal muscle cells of L6-GLUT4myc rats with stable overexpression of GLUT4myc. Methods: in the first part, 1mM calcium carrier ionomycin was used to incubate L6-GLUT4myc myoblasts with Western blot to detect the effect of ionomycin on the phosphorylation of PKC substrate. The level of GLUT4 on the cell surface and the phosphorylation of PKC substrate were detected by 1mM cPKC and nPKC inhibitor Gn6983 and 1mM cPKC inhibitor Gf6976 by Elisa, and the endocytosis and efflux of GLUT4 were detected by Western blot. To investigate whether cPKC and nPKC are involved in the role of ionomycin in promoting GLUT4 translocation of skeletal muscle cells. In the second part, RNA interference technique was used to down-regulate the protein expression of PKC 伪 -PKC 味 and PKC 蔚, respectively. The GLUT4 translocation induced by ionomycin and the endocytosis and efflux of GLUT4 were determined by Elisa in control group and ionomycin group. To further explore the subtype of PKC involved in the role of ionomycin in promoting GLUT4 transposition of skeletal muscle cells. In the third part, RNA interference technique was used to down-regulate the expression of Rab8aI Rab13 and Rab14, and the GLUT4 level on the cell surface was determined by Elisa in control group and ionomycin group. The aim of this study was to explore the role of ionomycin in promoting GLUT4 translocation of skeletal muscle cells. Results: the results of our previous study showed that the concentration of Ca2 in the cytoplasm of L6-GLUT4myc myoblasts was increased and the level of GLUT4 on the surface of the cells was significantly increased by 1% ionomycin. The results of the first part showed that: ionomycin significantly increased the phosphorylation level of PKC substrates. Gn6983 and Gn6976 significantly inhibited the GLUT4myc transposition promoted by ionomycin (p0.05, p0.001) and the phosphorylation of PKC substrates. Ionomycin inhibited GLUT4myc endocytosis. Promoting GLUT4myc efflux Gn6983 inhibited both ionomycin inhibited GLUT4myc endocytosis p0.01) and ionomycin stimulated GLUT4myc efflux p0.001N. GC6976 inhibited ionomycin inhibited GLUT4myc endocytosis p0.05) but did not affect ionomycin induced GLUT4myc efflux. The results of the second part showed that both ionomycin stimulated GLUT4myc transposition (p0.001) was significantly inhibited by siPKC 味 and siPKC 伪, while siPKC 蔚 had no effect on GLUT4myc transposition induced by ionomycin. SiPKC 味 inhibited ionomycin inhibited GLUT4myc endocytosis p0.001) and ionomycin induced GLUT4myc efflux p0.001n. SiPKC 伪 inhibited ionomycin inhibited GLUT4myc endocytosis. However, the efflux of GLUT4myc promoted by ionomycin was not affected. The results of the third part showed that: siRab13 significantly inhibited GLUT4myc transposition (p0.05) promoted by ionomycin, while siRab8a and siRab14 did not affect GLUT4myc translocation stimulated by ionomycin. Conclusion 1. Ionomycin activates PKC.2.cPKC and nPKC of skeletal muscle cells and participates in the role of ionomycin in promoting GLUT4 translocation. PKC 伪 and PKC 味 are the PKC subtypes. 3. 3. C PKC mediates ionomycin to inhibit GLUT4 endocytosis. PKC 伪 is an active cPKC subtype. N PKC participates in ionomycin to promote GLUT4 efflux and inhibit its internals. PKC 味 is a subtype of nPKC. 4. Rab13 mediates the role of ionomycin in promoting GLUT4 translocation. Ionomycin promotes GLUT4 translocation of skeletal muscle cells through PKC 伪 / PKC 味 -Rab13 signal transduction pathway.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

【相似文献】

相关期刊论文 前10条

1 胡俊;张琳琳;夏晶;陈新山;;固定不同时间对兔骨骼肌肌动蛋白免疫组化染色影响的研究[J];中国组织化学与细胞化学杂志;2007年06期

2 袁建琴;刘承宜;徐晓阳;;骨骼肌肌酸转运及其分子机制[J];中国运动医学杂志;2009年03期

3 井上勋;;骨骼肌兴奋——收缩耦联的进化[J];泸州医学院学报;1993年04期

4 张晓峰;石松;魏永旺;;骨骼肌的运动适应机制[J];中国组织工程研究与临床康复;2011年46期

5 郭宏波;转基因的骨骼肌细胞及其在I型糖尿病治疗中的应用[J];临床和实验医学杂志;2002年04期

6 都爱莲,任惠民,吕传真,卢家红,赵重波,朱雯华;25kD蛋白在人骨骼肌中的定位及在人其他组织中的表达[J];神经解剖学杂志;2004年02期

7 蒙碧辉,刘红,梁莹,黄松,冼苏;1型糖尿病树，

本文编号：1920099