【作者】 张焕莉；

【导师】 肖新华；

【作者基本信息】 南华大学 ， 内科学， 2011， 硕士

【摘要】 目的:构建重组小鼠锌α2糖蛋白(Zinc-α2-glycoprotein )真核表达载体,并稳定转染3T3-L1细胞,观察3T3-L1细胞过表达ZAG后对线粒体生物合成相关因子的影响。方法:提取正常小鼠肝脏组织总RNA,通过RT-RCR方法扩增出ZAG序列,克隆入经XbaⅠ和HindШ双酶切后的pcDNA3.1(-),重组小鼠ZAG真核表达质粒(pcDNA3.1(-)-mZAG)经转化大肠杆菌JM109,筛选阳性克隆,酶切和核苷酸测序鉴定后经脂质体转染3T3-L1细胞,G418筛选阳性克隆扩增,RT-PCR法鉴定ZAG阳性细胞株并检测过氧化物酶增殖型受体γ辅助活化因子1α(PGC-1α)、核呼吸因子1/2(NRF-1/2)、线粒体转录因子A(mtTFA)mRNA表达,Western Blot检测PGC-1α、NRF1/2、mtTFA蛋白的表达。结果:成功克隆小鼠ZAG cDNA全序列,并将其与pcDNA3.1(-)载体片段连接,构建成pcDNA3.1(-)-mZAG表达质粒。重组真核表达质粒经限制性核酸内切酶XbaⅠ和HindШ酶切后获得5.4kb和924bp两个片段,大小与理论值一致,并由核苷酸序列测定证实。pcDNA3.1(-)-mZAG成功转染3T3-L1细胞,在转染的3T3-L1细胞中ZAG基因的转录明显上调,过表达ZAG后,PGC-1α、NRF-1/2、mtTFA表达均上调。结论:成功构建ZAG基因的真核表达质粒pcDNA3.1(-)-mZAG,构建ZAG稳定过表达细胞系,在稳定转染的3T3-L1细胞中,ZAGmRNA表达水平与线粒体生物合成有关,ZAG对线粒体生物合成相关因子PGC-1α、NRF-1/2、mtTFA的表达有促进作用。目的:探讨锌α2糖蛋白(Zinc-α2-glycoprotein, ZAG)促进3T3-L1细胞线粒体生物合成相关因子的PKA和p38MAPK信号通路机制。方法:体外培养3T3-L1细胞,分别加入ZAG、PKA抑制剂(H89)、H89+ZAG、p38MAPK抑制剂(SB203580)和SB203580+ZAG。应用RT-PCR法检测过氧化物酶增殖型受体γ辅助活化因子1α(PGC-1α)、核呼吸因子1/2(NRF-1/2)、线粒体转录因子A(mtTFA)的表达水平。结果: RT-PCR法显示ZAG促进线粒体生物合成相关因子的表达,PKA特异性抑制剂H89和p38MAPK特异性抑制剂SB203580抑制线粒体生物合成相关因子的表达,而H89+ZAG和SB203580+ZAG处理细胞则抑制ZAG对线粒体生物合成相关因子的促进作用。结论:ZAG能够促进线粒体生物合成相关因子的表达,这种作用可能是通过PKA和p38MAPK信号通路介导。更多还原

【Abstract】 Objective: To construct pcDNA3.1(-)-mZAG recombinant mammalian expression vector , which was transfected into 3T3-L1 cell and observe its effects on relevant factors of mitochondria biogenesis after overexpression of ZAG.Methods: The total RNA was extracted from mouse liver cell, and the sequence of ZAG was amplified with RT-PCR.The PCR product was then cloned into the prokaryotic expression vector pcDNA3.1(-) after enzyme by XbaⅠand HindШ. After identification by double restriction enzyme digestion and DNA sequencing, the recombinant eukaryotic expression plasmid pcDNA3.1(-)-mZAG was transfected into 3T3-L1 cell by lipofectamine 2000. The stable transfected 3T3-L1 cell line was established after selection with G418. The expression of ZAG mRNA and PGC-1α、NRF-1/2、mtTFA mRNA were detected by RT-PCR. The expression of PGC-1α、NRF-1/2、mtTFA protein were detected by Western Blot.Results: Mouse ZAG coding sequence was amplified successfully and ligated with vector fragments by T4 ligase. The construction of pcDNA3.1(-)-mZAG plasmid containing mouse ZAG coding sequence of 5.4kb fragment and another 924bp fragment when digested by XbaⅠand HindШ. PcDNA3.1(-)-ZAG was transfected into the 3T3-L1 cell successfully, the expression of PGC-1α、NRF-1/2、mtTFA and ZAG mRNA were up-regulated in the transfected 3T3-L1 cell.Conclusion: The recombinant eukaryotic expression vector pcDNA3.1(-)-ZAG was constructed successfully. The stable 3T3-L1 cell line over-expression of ZAG was established. In the stable transfected 3T3-L1 cell, the expression of ZAG mRNA is correlated with mitochondria generation, which have promote effects on expression of relevant factors of mitochondria biogenesis. Objective: To explore whether PKA and p38MAPK could mediate the effects of ZAG on relevant factors of mitochondria biogenesis.Methods: 3T3-L1 cells were treated with ZAG、PKA inhibitor H89、H89+ZAG、p38MAPK inhibitor SB203580 and SB203580+ZAG. The expression of PGC-1α、NRF-1/2、mtTFA mRNA and protein were detected by the method of RT-PCR and Western Blot.Results: ZAG promoted the expression of relevant factors of mitochondria biogenesis, PKA inhibitor H89、p38MAPK inhibitor SB203580 inhibited the expression of relevant factors of mitochondria biogenesis, H89+ZAG、SB203580+ZAG decreased the promote effects of ZAG on mitochondria biogenesis.Conclusion: ZAG have a promote effect on relevant factors of mitochondria biogenesis, PKA and p38MAPK signal pathway can mediate this effect.更多还原