【作者】 陈琛；

【导师】 莫朝晖；

【作者基本信息】 中南大学 ， 内分泌， 2009， 硕士

【摘要】 目的观察胰岛素诱导基因2(insulin-induced gene2,insig2)在不同糖脂水平诱导3T3-L1细胞分化中的作用,探讨insig2在脂肪细胞分化中的作用。方法体外培养3T3-L1细胞(Tc)、转染pcDNA3.1(+)的3T3-L1细胞(Kc)及转染pcDNA3.1(+)-insig2的3T3-L1(Ic)细胞,分别置于高糖高脂(SF)、单纯高糖对照(S)、低糖高脂(F)、单纯低糖对照(D)的环境中分化培养12天,油红O染色后计算染色阳性细胞面积,RT-PCR法测定insig2、脂肪细胞型脂肪酸结合蛋白2(adipocyte fattyacid-binding protein2,aP2)、固醇调节元件结合蛋白(sterol regulatoryelement-binding protein,SREBP)mRNA的表达。结果同种细胞在高糖的分化环境中,高脂与单纯高糖对照处理后相比较,染色阳性细胞面积、aP2、insig2、SREBP mRNA表达均无显著差别(P＞0.05);同种细胞在低糖的分化环境中,高脂与单纯低糖对照处理后相比较,前者的染色阳性细胞面积、aP2、insig2、SREBPmRNA表达均较后者显著升高(P＜0.05);在四种分化环境中,分化前后转染pcDNA3.1(+)-insig2的3T3-L1细胞中insig2基因持续高表达,且诱导分化第12天染色阳性细胞面积、aP2、SREBP mRNA表达均较对照组3T3-L1细胞及转染pcDNA3.1(+)的3T3-L1细胞显著下调(P＜0.05);3T3-L1细胞组与转染pcDNA3.1(+)的3T3-L1细胞组比较,分化前后染色阳性细胞面积、aP2、insig2、SREBP mRNA表达均无显著差别(P＞0.05)。结论1.高糖和/或高脂即可促进脂肪细胞分化,而低糖低脂不利于脂肪细胞的分化2.insig2在细胞内的表达水平与脂肪细胞成熟及分化程度相关,与分化环境中糖脂浓度有一定关系;3.3T3-L1细胞在任何糖脂水平的分化环境中,过表达insig2均能显著抑制而非完全阻断其分化成熟与脂质合成。更多还原

【Abstract】 Objective:To observe the influence of insulin-induced gene2 (insig2) on the differentiation of 3T3-L1 in different concentration of glucose and free fatty acid,and to explore the effect of insig2 in the differentiation and formation of adipocytes and lipogenesis.Method:The 3T3-L1 cells and the pcDNA3.1(+) cells and the pcDNA3.1(+)-insig2 cells were induced to differentiate in high glucose concentration(25 mmol/L GS)(with or without oleic acid(0.5 mmol/L OA)) and low glucose concentration(5.6 mmol/L GS)(with or without oleic acid(0.5 mmol/L OA)),respectively.After differentiation,the differentiation of three kinds of cells was examined by Oil Red O straining, and the expression of insig2 mRNA and adipocyte fatty acid-binding protein2(aP2) and sterol regulatory element-binding protein(SREBP) mRNA was examined by RT-PCR.Result:After differentiating in the high concertration of glucose,it was observed that there were no significant difference in the Oil Red O straining and the expression of aP2,insig2 and SREBP mRNA of the conspecific cells(P＞0.05),no matter the culture medium was add in oleate or not;After differentiating in the low concertration of glucose,it was observed that the Oil Red O straining and the expression of aP2 or insig2 or SREBP mRNA were significant different in the conspecific cells, depend on the culture medium was add in oleate or not(P＜0.05);the Oil Red O straining and the expression of aP2 or insig2 or SREBP mRNA of those cultured in oleate-free culture medium were significant decreased (P＜0.05),compared to another.The insig2 was consistently high expressd during all the differentiation and the expression of the Oil Red O straining and the expression of aP2 or insig2 or SREBP mRNA were significant decreased in the pcDNA3.1(+)-insig2 cells(P＜0.05),compared to the contrast two kinds of cells.And there was no significant difference between the contrast two kinds of cells in the Oil Red O straining and the expression of aP2 or insig2 or SREBP mRNA(P＞0.05).Conclusion:Both high concertration of glucose and free fatty acid can increase the differention of 3T3-L1,while low concertration of glucose or free fatty acid is not benefit to the differention of 3T3-L1;the expression of insig2 in 3T3-L1 is connect with the degree of the differention of 3T3-L1 and the concertration of glucose or free fatty acid in the inducing surroundings;overpressed insig2 may have a depressant effect on adipocyte differentiation in any of the four inducing surroundings,but not block the lipid synthesis completely.更多还原