發(fā)布時(shí)間：2018-03-19 08:27

  本文選題：miR-26b　切入點(diǎn)：脂肪細(xì)胞　出處：《蚌埠醫(yī)學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:闡明mi R-26b過(guò)表達(dá)對(duì)脂肪細(xì)胞胰島素敏感性調(diào)控的分子機(jī)制。方法:1.高脂飲食喂養(yǎng)SD大鼠,建立飲食誘導(dǎo)肥胖(Diet-induced obesity,DIO)大鼠模型。高糖、高胰島素環(huán)境下誘導(dǎo)建立3T3-L1/HPA-v胰島素抵抗脂肪細(xì)胞模型;2.采用Real time PCR技術(shù),檢測(cè)動(dòng)物及細(xì)胞模型中mi R-26b的表達(dá);3.胰島素(Insulin)、3-異丁基-1-甲基黃嘌呤(3-isobutyl-1-methyxanthine,MIX)、地塞米松(Dexamethasone,Dex)混合誘導(dǎo)劑誘導(dǎo)3T3-L1前體脂肪細(xì)胞分化為成熟脂肪細(xì)胞。胰島素、3-異丁基-1-甲基黃嘌呤、地塞米松和羅格列酮(rosiglitazone)混合誘導(dǎo)劑誘導(dǎo)HPA-v前體脂肪細(xì)胞分化為成熟脂肪細(xì)胞;4.構(gòu)建mi R-26b過(guò)表達(dá)慢病毒表達(dá)載體,感染人成熟脂肪細(xì)胞;48h后在熒光顯微鏡下,判斷病毒感染的效率;5.采用同位素標(biāo)記葡萄糖進(jìn)行糖攝取實(shí)驗(yàn),檢測(cè)胰島素刺激下脂肪細(xì)胞對(duì)葡萄糖攝取量的變化;6.Western Blot技術(shù),檢測(cè)PI3K信號(hào)通路中關(guān)鍵蛋白分子胰島素受體(insulin recepor,IR)、胰島素受體底物(insulin receptor substrate,IRS)、AKT、p-AKT、葡萄糖轉(zhuǎn)運(yùn)體-4(Glucose transporter type 4,GLUT4)的表達(dá)及GLUT4膜蛋白(m GLUT4)的含量;7.生物信息學(xué)預(yù)測(cè)mi R-26b的靶標(biāo),采用熒光素酶報(bào)告實(shí)驗(yàn)進(jìn)行靶標(biāo)驗(yàn)證。結(jié)果:1.高脂飲食誘導(dǎo)后SD大鼠出現(xiàn)肥胖及代謝紊亂的特征,成功構(gòu)建DIO大鼠模型。DIO大鼠腸系膜脂肪組織中mi R-26b的表達(dá)水平下降,且與應(yīng)用穩(wěn)態(tài)模型評(píng)估的胰島素抵抗指數(shù)(Homeostasis model assessment of insulin resistance,HOMA-IR)呈顯著負(fù)相關(guān);2.胰島素抵抗細(xì)胞模型中mi R-26b的表達(dá)水平顯著降低;3.mi R-26b過(guò)表達(dá)促進(jìn)脂肪細(xì)胞胰島素刺激下的葡萄糖攝取;4.mi R-26b過(guò)表達(dá)可以顯著上調(diào)脂肪細(xì)胞胰島素刺激下的m GLUT4含量;5.mi R-26b過(guò)表達(dá)可以顯著提高磷脂酰肌醇激酶3(phosphatidylinositol3-kinase,PI3K)胰島素信號(hào)通路中p-AKT水平;6.熒光素酶實(shí)驗(yàn)的結(jié)果表明,第10號(hào)染色體缺失的磷酸酶及張力蛋白同源基因(phosphatase and tensin homolog deleted on chromosome 10,PTEN)為mi R-26b的直接靶基因。結(jié)論:mi R-26b可能通過(guò)調(diào)控PTEN的表達(dá),影響PI3K胰島素信號(hào)通路的正常傳導(dǎo),參與肥胖相關(guān)脂肪細(xì)胞胰島素抵抗發(fā)生的分子機(jī)制。
[Abstract]:Objective: to elucidate the molecular mechanism of the overexpression of mi R-26b on insulin sensitivity of adipocytes. Methods: High-fat diet was fed to SD rats to establish Diet-induced obesity-induced diol (Dio) rat model. A 3T3-L1 / HPA-v insulin resistant adipocyte model was established under hyperinsulin-induced hyperinsulinemia. Real time PCR technique was used. The expression of mi R-26b was detected in animal and cell models. Insulin insulinator 3isobutyl-1-methyxanthine 3-isobutyl-1-methyxanthine (MIXX) and dexamethasone Dexen (Dexx) were used to induce the differentiation of 3T3-L1 precursor adipocytes into mature adipocytes. Insulin 3-isobutyl-1-methylxanthine was induced to induce the differentiation of 3T3-L1 precursor adipocytes into mature adipocytes. The mixture of dexamethasone and rosiglitazone induced HPA-v precursor adipocytes to differentiate into mature adipocytes. The vector of mi R-26b overexpression lentivirus was constructed and infected with human mature adipocytes for 48 h. To determine the efficiency of virus infection. Glucose uptake assay was performed with isotopic labeled glucose to detect the glucose uptake of adipocytes stimulated by insulin. 6. Western Blot technique was used to detect glucose uptake by adipocytes. To detect the expression of insulin receptor (insulin receptor), insulin receptor receptor (insulin receptor), insulin receptor substratesil, glucose transporter (-4Glucose transporter type 4GUT4) and the content of GLUT4 membrane protein (GLUT4) in PI3K signaling pathway. Bioinformatics was used to predict the target of mi R-26b. The target was verified by luciferase report experiment. Results: 1. The expression of miR-26b in mesenteric adipose tissue of DIO rats was successfully established, which was characterized by obesity and metabolic disorder in SD rats induced by high-fat diet, and the expression level of mi R-26b was decreased in mesenteric adipose tissue of DIO rats. There was a significant negative correlation between homeostasis model assessment of insulin resistance and HOMA-IR2.The expression level of mi R-26b in insulin resistance cell model was significantly lower than that in adipocytes stimulated by insulin. Glucose uptake 4.mi R-26b overexpression could significantly up-regulate the content of m GLUT4 in adipocytes stimulated by insulin. 5.mi R-26b overexpression could significantly increase the level of p-AKT in phosphatidylinositol3-kinase- PI3K) insulin signaling pathway. The results of luciferase assay showed that, Phosphatase and tensin homolog deleted on chromosome 10 PTENs are the direct target genes of mi R-26b. Conclusion the normal transduction of PI3K insulin signaling pathway may be affected by regulating PTEN expression. It is involved in the molecular mechanism of insulin resistance in adipocytes associated with obesity.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R589.2

【共引文獻(xiàn)】

相關(guān)期刊論文 前7條

1 譚新睿;肖延風(fēng);;CIDEC與胰島素抵抗相關(guān)研究進(jìn)展[J];中國(guó)當(dāng)代兒科雜志;2013年11期

2 李文娟;韓翰;孫丹;沈洪星;李友軍;張進(jìn)祥;;microRNA-215異常激活抑制Dicer1促進(jìn)肝癌細(xì)胞遷移和轉(zhuǎn)化[J];中國(guó)科學(xué):生命科學(xué);2015年02期

3 Megha Haridas Upadya;Jude Juventus Aweya;Yee-Joo Tan;;Understanding the interaction of hepatitis C virus with host DEAD-box RNA helicases[J];World Journal of Gastroenterology;2014年11期

4 王舒;張振海;顧冬飛;周建平;呂慧俠;;外排體作為新型給藥系統(tǒng)的研究進(jìn)展[J];中國(guó)藥科大學(xué)學(xué)報(bào);2014年02期

5 Sumadi Lukman Anwar;Ulrich Lehmann;;DNA methylation,microRNAs,and their crosstalk as potential biomarkers in hepatocellular carcinoma[J];World Journal of Gastroenterology;2014年24期

6 張乃鍵;趙超;趙亞萍;;肥胖相關(guān)微RNA的研究進(jìn)展[J];醫(yī)學(xué)綜述;2015年09期

7 Xu-Feng Lu;Yong-Jie Zhou;Lei Zhang;Hong-Jie Ji;Li Li;Yu-Jun Shi;Hong Bu;;Loss of Dicer1 impairs hepatocyte survival and leads to chronic inflammation and progenitor cell activation[J];World Journal of Gastroenterology;2015年21期

相關(guān)博士學(xué)位論文 前1條

1 鮑美華;let-7家族在ox-LDL所致人血管內(nèi)皮損傷中的作用與機(jī)制研究[D];中南大學(xué);2014年

相關(guān)碩士學(xué)位論文 前7條

1 田捧;DGCR8對(duì)卵巢癌發(fā)生發(fā)展的體內(nèi)外實(shí)驗(yàn)研究[D];鄭州大學(xué);2013年

2 韓冰;相關(guān)高血壓性腦出血miRNAs基因標(biāo)記物的研究[D];河北大學(xué);2013年

3 李興;檳榔堿對(duì)3T3-L1前脂肪細(xì)胞分化的影響[D];南華大學(xué);2013年

4 楊靜;沉默Dicer基因?qū)θ松圜[癌細(xì)胞株Tca-8113生物學(xué)行為的影響[D];南方醫(yī)科大學(xué);2013年

5 李金朋;PDCD5在膽道閉鎖患兒膽管組織中的表達(dá)及意義[D];華中科技大學(xué);2013年

6 余波;MiR-18a靶向Dicer1抑制肝癌細(xì)胞的侵襲遷移[D];蘇州大學(xué);2014年

7 趙鍇;miR-10a抑制肝癌細(xì)胞生長(zhǎng)及其機(jī)制探討[D];北京協(xié)和醫(yī)學(xué)院;2014年

，

本文編號(hào)：1633452