【作者】 解芸菲；

【导师】 孙超；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2011， 硕士

【摘要】 脂肪细胞不仅是机体能量的主要贮存和释放场所,它还分泌多种细胞因子对其自身和其他细胞发挥重要的调节作用。TNF-α作为其中最早发现的分泌产物之一,它对脂肪细胞可产生多种效应,如抑制脂肪细胞生长,诱导前体脂肪细胞和脂肪细胞凋亡。但由于脂肪细胞自身的特点,TNF-α诱导脂肪细胞凋亡鲜有研究和报道。SOCS家族是JAK/STAT信号通路的负调控因子。JAK2/STAT3通路是多种细胞生长、存活、分化及凋亡功能发挥过程中重要的细胞内信号传导途径。本试验以3T3-L1前体脂肪细胞和小鼠前体脂肪细胞为研究对象,应用siRNA沉默SOCS3基因,研究其对TNF-α诱导的前体脂肪细胞凋亡的影响,主要研究结果如下:1.以20、40、60、80、100、150、200 ng/mL TNF-α处理3T3-L1前体脂肪细胞24 h,光学显微镜观察细胞的形态学变化。结果表明,TNF-α诱导3T3-L1前体脂肪细胞凋亡呈剂量依赖性。100 ng/mL TNF-α处理后,3T3-L1前体脂肪细胞出现明显的细胞凋亡,并伴随出现细胞体积缩小,染色质凝集,核质固缩等特征。2.采用siRNA干扰技术,成功干扰SOCS3基因的表达,其中SOCS3 siRNA1基因沉默效果最明显,在3T3-L1前体脂肪细胞和小鼠前体脂肪细胞中SOCS3 mRNA表达分别下降了57%和50%,为后续以SOCS3基因为靶点的研究工作奠定基础。3. siRNA沉默3T3-L1前体脂肪细胞中SOCS3基因后,100 ng/mL TNF-α处理24 h,采用Hoechst 33258和PI染色剂染色,荧光显微镜观察细胞的形态学变化。RT-PCR和Western Blotting方法检测凋亡相关基因c-myc、survivin、mcl-1、bcl-2、bax、NF-κB和JAK2/STAT3通路关键基因SOCS1、SOCS2、JAK2、STAT3的mRNA和蛋白的表达变化。结果表明,与对照组相比,沉默SOCS3表达后细胞凋亡数量明显减少;survivin和NF-κB mRNA表达显著上升(P<0.05),c-myc和bcl-2 mRNA表达极显著上升(P<0.01),bax和SOCS1 mRNA表达显著下降(P<0.05),而mcl-1 mRNA表达无显著变化(P>0.05);Western blotting检测发现,p-STAT3蛋白表达显著上升(P<0.05),Bcl-2和NF-κB蛋白表达极显著上升(P<0.01),Bax蛋白表达显著下降(P<0.05)。而脂质体组和阴性对照组中各基因、蛋白的表达与空白对照组之间均无显著变化。结果表明,沉默SOCS3通过调控JAK2/STAT3通路关键基因和凋亡相关基因的表达,显著抑制了TNF-α诱导的3T3-L1前体脂肪细胞凋亡。4. siRNA沉默小鼠前体脂肪细胞中SOCS3基因后,100 ng/mL TNF-α处理24 h,Hoechst 33258和PI染色发现,沉默SOCS3表达后细胞凋亡数量明显减少;RT-PCR检测证明,NF-κB mRNA表达显著上升(P<0.05),mcl-1和bcl-2 mRNA表达极显著上升(P<0.01),SOCS1 mRNA表达极显著下降(P<0.01),而bax、c-myc和survivin mRNA表达无显著变化(P>0.05);Western blotting检测发现,Bcl-2、NF-κB和p-STAT3蛋白表达极显著上升(P<0.01),Bax蛋白表达显著下降(P<0.05)。结果表明,沉默SOCS3通过JAK2/STAT3通路调控了凋亡相关基因的表达,从而显著抑制了TNF-α诱导的小鼠前体脂肪细胞凋亡。更多还原

【Abstract】 Adipocyte can not only reserve or release energy, but also secrete many cytokines to play an important role in regulating itself and other cells. TNF-αis one of the cytokines, it can produce a variety of effects to adipocyte, such as inhibit the growth of adipocyte, inducing the apoptosis of preadipocytes and adipocytes. However, owing to the evidently ability of adipocyte to resist apoptosis, TNF-αinduced adipocyte apoptosis was not well studied and poorly understood. SOCS is a negative feedback regulator of the JAK/STAT signaling pathway. JAK2/STAT3 signaling pathway participate in many signal transduction systems, influencing cell growth, survival, differentiation and apoptosis functions.In this study, we used siRNA interfere technique to silence SOCS3 gene in 3T3-L1 preadipocytes and mouse preadipocytes. Study the effect of SOCS3 siRNA on TNF-αinduced apoptosis in both cells. The main results were summarized as following:1. 3T3-L1 preadipocytes were cultured in vitro and treated with TNF-αat the concentrations of 20、40、60、80、100、150、200 ng/mL for 24 h, respectively. Optical microscope to observe morphological changes during apoptosis. TNF-αinduced 3T3-L1 preadipocytes apoptosis showed a dose dependent manner. After treated with 100 ng/mL TNF-αfor 24h, primary preadipocyte apoptosis was apparent, accompanied by reduced cell volume, chromatin condensation, and nuclear shrinkage.2. Using siRNA interfere technique, this study successfully interfere the expression of SOCS3 gene. The gene silencing effect of SOCS3 siRNA1 is the most obvious. SOCS3 mRNA expression were suppressed by 57% and 50% by SOCS3 siRNA1 in 3T3-L1 preadipocytes and mouse preadipocytes, it may lay solid foundation for the further research on the function of SOCS3.3. After SOCS3 expression was inhibited by SOCS3 siRNA infection, 3T3-L1 preadipocytes were treated with TNF-αat 100 ng/mL for 24 h. Morphological changes during apoptosis were observed by fluorescence microscope after staining by Hoechst 33258 and PI fluorescent dyes respectively. RT-PCR and Western blotting to measure the expression of apoptosis-associated gene c-myc、survivin、mcl-1、bcl-2、bax、NF-κB, and JAK2/STAT3 pathway key gene SOCS1, SOCS2, JAK2, STAT3. Compared with control group, in SOCS3 siRNA group the number of cells apoptosis was decreased remarkably; the expression level of survivin and NF-κB mRNA increased significantly (P<0.05), the expression level of c-myc and bcl-2 mRNA increased extremely significantly (P<0.01), the expression level of bax and SOCS1 mRNA decreased significantly (P<0.05), no changes were found for the expression of mcl-1 (P>0.05); Western blotting analysis showed that inhibition of SOCS3 also upregulated the expression of Bcl-2、NF-κB and p-STAT3 (P<0.05), but downgulated bax expression (P<0.05). Here, no distinct change was detected in LipofectineTM2000 group or negative control group. Taken together, our data established that knocking down SOCS3 can regulate the expression of apoptosis-associated gene by JAK2/STAT3 pathway, then effectively inhibit TNF-αinduced apoptosis in 3T3-L1 preadipocytes.4. After SOCS3 expression was inhibited by SOCS3 siRNA infection, mouse preadipocytes were treated with TNF-αat 100 ng/mL for 24 h. Hoechst 33258 and PI staining showed that the number of cells apoptosis was decreased remarkably; RT-PCR analysis showed that, the expression level of NF-κB mRNA increased significantly (P<0.05), the expression level of mcl-1 and bcl-2 mRNA increased extremely significantly (P<0.01), the expression level of bax and SOCS1 mRNA decreased extremely significantly (P<0.01), no changes were found for the expression of bax、c-myc and survivin (P>0.05); Western blotting analysis showed that inhibition of SOCS3 also upregulated the expression of p-STAT3、Bcl-2 and NF-κB (P<0.01), but downgulated bax expression (P<0.05). Our data established that knocking down SOCS3 can regulate the expression of apoptosis-associated gene by JAK2/STAT3 pathway, then effectively inhibit TNF-αinduced apoptosis in mouse preadipocytes.更多还原