本文選題：骨骼肌 + 蛋白激酶C　； 參考：《天津醫(yī)科大學》2015年碩士論文

【摘要】：目的:應用穩(wěn)定過表達GLUT4myc的L6-GLUT4myc大鼠骨骼肌細胞探討傳統(tǒng)型PKC(conventional PKC,cPKC)、新穎型PKC(novel PKC,nPKC)以及Rab蛋白在鈣信號調節(jié)骨骼肌細胞葡萄糖轉運子4(glucose transporter 4,GLUT4)胞內運輸機制中的作用。方法:第一部分,用1mM鈣離子載體ionomycin孵育L6-GLUT4myc成肌細胞,Western blot檢測ionomycin對PKC底物磷酸化的影響,并用1mM cPKC和nPKC抑制劑G?6983和1mM cPKC抑制劑G?6976預孵育L6-GLUT4myc成肌細胞,ELISA測定細胞表面GLUT4水平及GLUT4的內吞和外排,Western blot檢測PKC底物的磷酸化,探討cPKC和nPKC是否參與ionomycin促進骨骼肌細胞GLUT4轉位的作用。第二部分,應用RNA干擾技術分別下調PKCα、PKCζ和PKCε的蛋白表達,隨機分為對照組和ionomycin組,ELISA測定ionomycin作用下的GLUT4轉位以及GLUT4的內吞和外排,進一步探討何種PKC亞型參與ionomycin促進骨骼肌細胞GLUT4轉位的作用。第三部分,應用RNA干擾技術分別下調Rab8a、Rab13和Rab14的蛋白表達,隨機分為對照組和ionomycin組,ELISA測定細胞表面GLUT4水平,探討哪個Rab參與ionomycin促進骨骼肌細胞GLUT4轉位的作用。結果:我們前期的研究結果顯示:ionomycin升高L6-GLUT4myc成肌細胞胞漿Ca2+濃度,顯著增加細胞表面GLUT4水平。第一部分結果顯示:ionomycin顯著增加PKC底物的磷酸化水平。G?6983和G?6976均顯著抑制ionomycin促進的GLUT4myc轉位(p0.05,p0.001)和PKC底物的磷酸化。Ionomycin抑制GLUT4myc內吞,促進GLUT4myc外排,G?6983既抑制ionomycin抑制的GLUT4myc內吞(p0.01)也抑制ionomycin刺激的GLUT4myc外排(p0.001)。G?6976抑制ionomycin抑制的GLUT4myc的內吞(p0.05)但不影響ionomycin促進的GLUT4myc的外排。第二部分結果顯示:siPKCα和siPKCζ均顯著抑制ionomycin刺激的GLUT4myc轉位(p0.001),而siPKCε不影響ionomycin促進的GLUT4myc轉位。siPKCζ既抑制ionomycin抑制的GLUT4myc內吞(p0.001)也抑制ionomycin促進的GLUT4myc外排(p0.001)。siPKCα抑制ionomycin抑制的GLUT4myc的內吞(p0.001),但不影響ionomycin促進的GLUT4myc的外排。第三部分結果顯示:siRab13顯著抑制ionomycin促進的GLUT4myc轉位(p0.05),而siRab8a和siRab14不影響ionomycin刺激的GLUT4myc轉位。結論:1.Ionomycin激活骨骼肌細胞PKC。2.cPKC和nPKC參與ionomycin促進GLUT4轉位的作用,PKCα和PKCζ是起作用的PKC亞型。3.cPKC介導ionomycin抑制GLUT4內吞的作用,PKCα是起作用的cPKC亞型。nPKC參與ionomycin促進GLUT4外排及抑制其內吞的作用,PKCζ是起作用的nPKC亞型。4.Rab13介導ionomycin促進GLUT4轉位的作用。綜上,ionomycin通過PKCα/PKCζ-Rab13信號轉導通路促進骨骼肌細胞GLUT4轉位。
[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 偽 -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 偽, 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 偽 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 偽 and PKC 味 are the PKC subtypes. 3. 3. C PKC mediates ionomycin to inhibit GLUT4 endocytosis. PKC 偽 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 偽 / PKC 味 -Rab13 signal transduction pathway.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R587.1

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