SOCS干预对IUGR大鼠骨骼肌细胞代谢的影响及机制研究

发布时间：2018-02-28 23:15

本文关键词：宫内发育迟缓胰岛素受体底物-1 细胞信号转导抑制因子胰岛素抵抗细胞因子信号转导抑制因子短发夹RNA RNA干扰宫内发育迟缓细胞因子信号转导抑制因子短发夹RNA RNA干扰葡萄糖转运体4　出处：《华中科技大学》2011年博士论文　论文类型：学位论文

【摘要】：目的宫内发育迟缓大鼠骨骼肌细胞的原代培养及鉴定,研究其胰岛素信号通路中胰岛素受体底物-1(IRS-1),细胞信号转导抑制分子-1(SOCS-1)和-3(SOCS-3)的表达变化,初步探讨胰岛素抵抗发生发展的机制。方法采用母孕期饥饿法建立IUGR大鼠模型。以正常新生鼠作为对照,组织块翻转干涸培养法体外原代培养大鼠骨骼肌细胞,运用运用SABC法对骨骼肌细胞作肌动蛋白免疫组织化学染色进行鉴定。通过Real-time PCR和Western blot检测新生和12周龄IUGR大鼠骨骼肌细胞IRS-1、SOCS-1、SOCS-3mRNA和蛋白水平表达变化。结果IUGR大鼠出生时体质量、身长显著小于对照组(P均0.05)。组织块培养法培养骨骼肌细胞生长状态良好。与对照组相比,新生和12周龄IUGR组仔鼠中骨骼肌细胞中,SOCS-1、SOCS-3的mRNA和蛋白表达水平均增加(P均0.05),而IRS-1的mRNA和蛋白表达水平均下降(P均0.05),IRS-1mRNA和蛋白表达水平均与SOCS-1、SOCS-3mRNA和蛋白表达水平呈负相关。结论原代组织块翻转干涸培养法可简单有效的体外培养大鼠骨骼肌细胞,可为后续研究稳定连续提供骨骼肌细胞。宫内发育迟缓大鼠子代骨骼肌细胞SOCS-1、SOCS-3表达增加,通过负性调节使得IRS-1表达降低,可能是骨骼肌胰岛素抵抗和成年代谢综合征发生的机制之一,提示SOCS-1和SOCS-3可以作为2型糖尿病和其他胰岛素抵抗的治疗靶点。目的构建针对大鼠细胞信号转导抑制分子-1(SOCS-1)和-3(SOCS-3)基因的短发夹RNA(shRNA)表达载体,并选取最有效shRNA模板序列进行后续研究。方法设计并合成编码的shRNA的两条寡核苷酸序列,经退火成互补双链,再克隆至pGPU6/GFP/Neo中构建重组表达载体,转化DH5α菌株,进行序列测定,转染至骨骼肌细胞中。Real- time PCR和Western blot检测各组SOCS-1和SOCS-3的表达情况。结果测序证实重组质粒构建成功。四对shRNA模板序列对SOCS-1和SOCS-3的表达抑制与空白及阴性对照相比均有统计学意义(P0.05),且Real-time PCR和Western blot结果均一致的显示SOCS-1-shRNAb和SOCS-3-shRNAa分别干预效果最佳。结论SOCS-1和SOCS-3特异性shRNA重组表达载体构建成功,能有效抑制大鼠骨骼肌肌细胞SOCS-1和SOCS-3的表达,为后续研究及代谢综合征的基因治疗奠定了基础。目的探讨体外干预对宫内发育迟缓大鼠骨骼肌细胞在基础状态下及不同胰岛素浓度刺激下的葡萄糖摄取的影响及机制。方法采用母孕期饥饿法建立IUGR大鼠模型,组织块法原代培养大鼠骨骼肌细胞,利用脂质体将SOCS-1和SOCS-3特异性shRNA重组质粒转染三月龄宫内发育迟缓大鼠骨骼肌细胞,应用激光共聚焦法观察转染后骨骼肌细胞在基础状态下和不同胰岛素浓度刺激下的葡萄糖摄取变化。通过Western blot检测骨骼肌细胞中葡萄糖转运体4(GLUT4)蛋白水平表达变化和细胞信号作用通路分子Akt的磷酸化水平。结果pGPU6/GFP/Neo-SOCS-1-shRNA、pGPU6/GFP/Neo-SOCS-3-shRNA重组质粒成功构建并转染12周龄宫内发育迟缓大鼠骨骼肌细胞及对照组后,细胞共聚焦显微镜观察骨骼肌细胞在基础状态下及不同胰岛素浓度(10-11、10-9、10-7mol/l)刺激下的GLUT4的转位较空白组均升高,且程度与胰岛素浓度成正比。Western blot检测骨骼肌细胞膜上葡萄糖转运体4(GLUT4)蛋白表达水平显示各组骨骼肌细胞在10-7mol/l胰岛素刺激下较基础状态增加,且SOCS-1-shRNA和SOCS-3-shRNA干预组较空白组GLUT4表达均增加。各组GLUT4表达水平与Akt磷酸化水平呈正相关。结论SOCS-1-shRNA和SOCS-3-shRNA均能成功下调靶基因的表达,并能改善胰岛素抵抗状态下骨骼肌细胞的葡萄糖摄取,是潜在的治疗代谢综合征新方法。
[Abstract]:Primary culture and identification of intrauterine growth retardation rats skeletal muscle cells, the insulin signaling pathway of insulin receptor substrate -1 (IRS-1), cell signal transduction inhibitor -1 (SOCS-1) and -3 (SOCS-3) expression and preliminary study on the mechanism of the occurrence and development of insulin resistance.
Methods the IUGR rat model was established by maternal starvation. In normal rats as control method in primary cultured rat skeletal muscle cells in tissue culture by using dry turning, SABC method for actin immunohistochemical staining of skeletal muscle cells were identified by Real-time. PCR and Western blot detection of newborn and skeletal muscle cells 12 week old IUGR rats IRS-1, SOCS-1, SOCS-3mRNA and protein levels.
The body weight of IUGR rats was born, were significantly less than the control group (P < 0.05). Tissue culture of skeletal muscle cell growth in good condition. Compared with the control group, SOCS-1 of skeletal muscle cells of newborn and 12 week old rats in group IUGR, SOCS-3, mRNA and protein expression levels were increased (P 0.05), while the expression of mRNA and protein levels of IRS-1 were decreased (P 0.05), and the expression level of IRS-1mRNA protein and SOCS-1 expression of SOCS-3mRNA and protein level was negatively correlated.
Cultured rat skeletal muscle cells. Conclusion primary tissue culture method is simple and effective dry turning in vitro, for follow-up study provides stable and continuous skeletal muscle cells. IUGR offspring rats skeletal muscle cells SOCS-1, SOCS-3 expression increased by negatively regulating the expression of IRS-1 was reduced, may be one of the mechanisms of skeletal muscle insulin resistance and adult metabolic syndrome, suggesting that SOCS-1 and SOCS-3 can be used as a therapeutic target in the treatment of type 2 diabetes and other insulin resistance.
Objective to construct short hairpin RNA (shRNA) expression vector targeting -1 (SOCS-1) and -3 (SOCS-3) gene of rat cell signal transduction, and select the most effective shRNA template sequence for subsequent research.
Two oligonucleotide sequences were designed and synthesized. The shRNA encoding method, obtained by annealing complementary strands, then cloned into pGPU6/GFP/Neo to construct the recombinant expression vector, transformation of DH5 alpha strains were sequenced and transfected into skeletal muscle cells in.Real- time PCR and Western blot to detect the expressions of SOCS-1 and SOCS-3 in each group.
The results of sequencing confirmed that the recombinant plasmids were successfully constructed. Four inhibition of shRNA template sequences of SOCS-1 and SOCS-3 expression with blank and negative contrast were statistically significant (P0.05), and Real-time PCR and Western blot results were consistent with SOCS-1-shRNAb and SOCS-3-shRNAa respectively, the intervention effect is the best.
Conclusion the construction of SOCS-1 and SOCS-3 specific shRNA recombinant vector can effectively inhibit the expression of SOCS-1 and SOCS-3 in skeletal muscle cells, and lay a foundation for subsequent research and gene therapy of metabolic syndrome.
Objective to investigate the effect and mechanism of in vitro intervention on glucose uptake in skeletal muscle cells of rats with intrauterine growth retardation in basal state and different insulin concentrations.
Methods the IUGR rat model was established by maternal starvation, tissue cultured rat skeletal muscle cells by liposome, SOCS-1 and SOCS-3 specific shRNA recombinant plasmid was transfected into 3 month old intrauterine growth retardation rats skeletal muscle cells, using confocal laser scanning was observed after transfection of skeletal muscle cells in basal condition and different the concentration of insulin stimulated glucose uptake by skeletal muscle cells. Changes of glucose transporter 4 in the detection of Western blot (GLUT4) and the changes of cell signal pathway molecule Akt phosphorylation level of protein expression.
The results of pGPU6/GFP/Neo-SOCS-1-shRNA, the recombinant plasmid pGPU6/GFP/Neo-SOCS-3-shRNA was successfully constructed and transfected into 12 weeks of intrauterine growth retardation rats skeletal muscle cells and the control group, the observation of skeletal muscle cells in basal condition and different insulin concentration cell confocal microscope (10-11,10-9,10-7mol/l) stimulated the translocation of GLUT4 compared with the blank group were increased, and the degree of concentration of insulin and bone the muscle cell membrane..Western blot detection of glucose transporter 4 (GLUT4) protein expression in skeletal muscle cells were increased compared with that of in 10-7mol/l induced by insulin, and SOCS-1-shRNA and SOCS-3-shRNA group compared with control group, the expression of GLUT4 was significantly increased. The expression level of GLUT4 in each group of Akt phosphorylation levels were positively correlated.
Conclusion both SOCS-1-shRNA and SOCS-3-shRNA can successfully reduce the expression of target genes and improve glucose uptake in skeletal muscle cells under insulin resistance, which is a potential new method for the treatment of metabolic syndrome.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R714.5

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