本文關(guān)鍵詞： 宮內(nèi)發(fā)育遲緩 胰島素受體底物-1 細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)抑制因子 胰島素抵抗 細(xì)胞因子信號(hào)轉(zhuǎn)導(dǎo)抑制因子 短發(fā)夾RNA RNA干擾 宮內(nèi)發(fā)育遲緩 細(xì)胞因子信號(hào)轉(zhuǎn)導(dǎo)抑制因子 短發(fā)夾RNA RNA干擾 葡萄糖轉(zhuǎn)運(yùn)體4　出處：《華中科技大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：目的宮內(nèi)發(fā)育遲緩大鼠骨骼肌細(xì)胞的原代培養(yǎng)及鑒定,研究其胰島素信號(hào)通路中胰島素受體底物-1(IRS-1),細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)抑制分子-1(SOCS-1)和-3(SOCS-3)的表達(dá)變化,初步探討胰島素抵抗發(fā)生發(fā)展的機(jī)制。 方法采用母孕期饑餓法建立IUGR大鼠模型。以正常新生鼠作為對(duì)照,組織塊翻轉(zhuǎn)干涸培養(yǎng)法體外原代培養(yǎng)大鼠骨骼肌細(xì)胞,運(yùn)用運(yùn)用SABC法對(duì)骨骼肌細(xì)胞作肌動(dòng)蛋白免疫組織化學(xué)染色進(jìn)行鑒定。通過Real-time PCR和Western blot檢測新生和12周齡IUGR大鼠骨骼肌細(xì)胞IRS-1、SOCS-1、SOCS-3mRNA和蛋白水平表達(dá)變化。 結(jié)果IUGR大鼠出生時(shí)體質(zhì)量、身長顯著小于對(duì)照組(P均0.05)。組織塊培養(yǎng)法培養(yǎng)骨骼肌細(xì)胞生長狀態(tài)良好。與對(duì)照組相比,新生和12周齡IUGR組仔鼠中骨骼肌細(xì)胞中,SOCS-1、SOCS-3的mRNA和蛋白表達(dá)水平均增加(P均0.05),而IRS-1的mRNA和蛋白表達(dá)水平均下降(P均0.05),IRS-1mRNA和蛋白表達(dá)水平均與SOCS-1、SOCS-3mRNA和蛋白表達(dá)水平呈負(fù)相關(guān)。 結(jié)論原代組織塊翻轉(zhuǎn)干涸培養(yǎng)法可簡單有效的體外培養(yǎng)大鼠骨骼肌細(xì)胞,可為后續(xù)研究穩(wěn)定連續(xù)提供骨骼肌細(xì)胞。宮內(nèi)發(fā)育遲緩大鼠子代骨骼肌細(xì)胞SOCS-1、SOCS-3表達(dá)增加,通過負(fù)性調(diào)節(jié)使得IRS-1表達(dá)降低,可能是骨骼肌胰島素抵抗和成年代謝綜合征發(fā)生的機(jī)制之一,提示SOCS-1和SOCS-3可以作為2型糖尿病和其他胰島素抵抗的治療靶點(diǎn)。 目的構(gòu)建針對(duì)大鼠細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)抑制分子-1(SOCS-1)和-3(SOCS-3)基因的短發(fā)夾RNA(shRNA)表達(dá)載體,并選取最有效shRNA模板序列進(jìn)行后續(xù)研究。 方法設(shè)計(jì)并合成編碼的shRNA的兩條寡核苷酸序列,經(jīng)退火成互補(bǔ)雙鏈,再克隆至pGPU6/GFP/Neo中構(gòu)建重組表達(dá)載體,轉(zhuǎn)化DH5α菌株,進(jìn)行序列測定,轉(zhuǎn)染至骨骼肌細(xì)胞中。Real- time PCR和Western blot檢測各組SOCS-1和SOCS-3的表達(dá)情況。 結(jié)果測序證實(shí)重組質(zhì)粒構(gòu)建成功。四對(duì)shRNA模板序列對(duì)SOCS-1和SOCS-3的表達(dá)抑制與空白及陰性對(duì)照相比均有統(tǒng)計(jì)學(xué)意義(P0.05),且Real-time PCR和Western blot結(jié)果均一致的顯示SOCS-1-shRNAb和SOCS-3-shRNAa分別干預(yù)效果最佳。 結(jié)論SOCS-1和SOCS-3特異性shRNA重組表達(dá)載體構(gòu)建成功,能有效抑制大鼠骨骼肌肌細(xì)胞SOCS-1和SOCS-3的表達(dá),為后續(xù)研究及代謝綜合征的基因治療奠定了基礎(chǔ)。 目的探討體外干預(yù)對(duì)宮內(nèi)發(fā)育遲緩大鼠骨骼肌細(xì)胞在基礎(chǔ)狀態(tài)下及不同胰島素濃度刺激下的葡萄糖攝取的影響及機(jī)制。 方法采用母孕期饑餓法建立IUGR大鼠模型,組織塊法原代培養(yǎng)大鼠骨骼肌細(xì)胞,利用脂質(zhì)體將SOCS-1和SOCS-3特異性shRNA重組質(zhì)粒轉(zhuǎn)染三月齡宮內(nèi)發(fā)育遲緩大鼠骨骼肌細(xì)胞,應(yīng)用激光共聚焦法觀察轉(zhuǎn)染后骨骼肌細(xì)胞在基礎(chǔ)狀態(tài)下和不同胰島素濃度刺激下的葡萄糖攝取變化。通過Western blot檢測骨骼肌細(xì)胞中葡萄糖轉(zhuǎn)運(yùn)體4(GLUT4)蛋白水平表達(dá)變化和細(xì)胞信號(hào)作用通路分子Akt的磷酸化水平。 結(jié)果pGPU6/GFP/Neo-SOCS-1-shRNA、pGPU6/GFP/Neo-SOCS-3-shRNA重組質(zhì)粒成功構(gòu)建并轉(zhuǎn)染12周齡宮內(nèi)發(fā)育遲緩大鼠骨骼肌細(xì)胞及對(duì)照組后,細(xì)胞共聚焦顯微鏡觀察骨骼肌細(xì)胞在基礎(chǔ)狀態(tài)下及不同胰島素濃度(10-11、10-9、10-7mol/l)刺激下的GLUT4的轉(zhuǎn)位較空白組均升高,且程度與胰島素濃度成正比。Western blot檢測骨骼肌細(xì)胞膜上葡萄糖轉(zhuǎn)運(yùn)體4(GLUT4)蛋白表達(dá)水平顯示各組骨骼肌細(xì)胞在10-7mol/l胰島素刺激下較基礎(chǔ)狀態(tài)增加,且SOCS-1-shRNA和SOCS-3-shRNA干預(yù)組較空白組GLUT4表達(dá)均增加。各組GLUT4表達(dá)水平與Akt磷酸化水平呈正相關(guān)。 結(jié)論SOCS-1-shRNA和SOCS-3-shRNA均能成功下調(diào)靶基因的表達(dá),并能改善胰島素抵抗?fàn)顟B(tài)下骨骼肌細(xì)胞的葡萄糖攝取,是潛在的治療代謝綜合征新方法。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R714.5

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