| 摘要: |
| 【目的】探讨SOCS3对3T3-L1细胞凋亡的影响。【方法】以小鼠脂肪组织提取的RNA为模板，用RT-PCR扩增SOCS3基因，将其克隆至pMD18-T Simple，构建pMD18-T SOCS3重组质粒，经测序验证后，以pMD18-T SOCS3为模板扩增SOCS3基因，将其克隆至pEGFP-N1，构建真核重组质粒pEGFP-N1 SOCS3，测序正确后，将重组质粒pEGFP-N1 SOCS3转染3T3-L1细胞，荧光下观察GFP的表达，RT-PCR和Western blotting检测细胞内SOCS3 mRNA和蛋白的表达，观察细胞凋亡的变化。【结果】测序表明，pMD18-T SOCS3和pEGFP-N1 SOCS3的核苷酸序列正确率为100%；在荧光显微镜下发现，脂质体组和SOCS3组均有GFP的表达；RT-PCR和Western blotting检测表明,3T3-L1细胞中SOCS3 mRNA表达显著提高（P<0.05）；Hoechst 33258染色发现，SOCS3组的细胞凋亡显著；Bax和c-myc基因的mRNA表达水平显著升高（P<0.05），bcl-2和mcl-1基因表达水平显著降低（P<0.05）。【结论】SOCS3对脂肪细胞凋亡的发生有促进作用。 |
| 关键词: 细胞凋亡 SOCS3 pEGFP-N1 |
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| 基金项目:国家自然科学基金项目(30871785)；教育部新世纪优秀人才计划项目(NCET-06-0865) |
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| Construction of mice SOCS3 eukaryotic vector and its effects on 3T3-L1 cells apoptosis |
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| Abstract: |
| 【Objective】The research is aimed at discussing the effects of SOCS3 on 3T3-L1 Lipoapoptosis.【Method】With RNA extracted from mice adipose tissue as a template,SOCS3 was amplified to pMD18-T by RT-PCR and recombinant plasmid pMD18-T SOCS3 was constructed.After sequencing,SOCS3 was amplified to pEGFP-N1 with pMD18-T SOCS3 as a template and the eukaryotic recombinant plasmid pEGFP-N1 SOCS3 was contructed,after sequencing the pEGFP-N1 SOCS3.GFP under the fluorescence microscope is expressed and the expressions of SOCS3 mRNA and protein by RT-PCR and Western blotting was used to detect the change of apoptosis.【Result】The recombinant plasmid pMD18-T SOCS3 and pEGFP-N1 SOCS3 are qualified to be right by sequence identification in genbank;Lipofectamine group and SOCS3 group show that GFP is expressed under the fluorescence microscopy;SOCS3 mRNA and protein in the cells detected by RT-PCR and Western blotting express significantly(P<0.05).The apoptosis of SOCS3 group increased significant after Hoechst 33258 staining;Compared with the control group,SOCS3 group significantly increased apoptosis,the mRNA expression level of Bax and c-myc gene was significantly increased(P<0.05),while bcl-2 and mcl-1 was significantly decreased(P<0.05).【Conclusion】SOCS3 can promote fat cell apoptosis. |
| Key words: apoptosis SOCS3 pEGFP-N1 |
