人遗传印记基因PEG10重组真核表达质粒的构建及其在转染细胞株中的表达  

Construction of recombinant plasmid human imprinted gene PEG10 and the primary functional identification in transfected cell lines

作　　者：张琼[1] 谢娜[1] 王晓燕[1] 常莹[1] 林园园[1] 林菊生[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院消化内科,武汉430030

出　　处：《中华肝脏病杂志》2007年第4期287-290,共4页Chinese Journal of Hepatology

基　　金：国家自然科学基金（30471983）

摘　　要：目的构建人遗传印记基因PEG10的全长表达基因质粒,观察PEG10的过表达对人正常肝细胞及其非肝脏来源的对照细胞的作用。方法将PEG10基因全长cDNA直接连入真核细胞表达质粒pcDNA3．1hisC中,以脂质体介导的方法将该表达质粒pcDNA3．1hisC-PEG10转染入人正常肝细胞及其对照细胞,采用RT-PCR、Western blot、MTT、TUNEL等方法分析PEG10基因在正常人肝细胞及其对照细胞中的表达和功能。结果限制性酶切及DNA测序结果均说明所构建质粒为PEG 10全长表达质粒。该质粒经过稳定筛选后稳定表达于细胞内,促进人肝细胞的生长,抑制其凋亡,但对非肝脏来源的对照组细胞没有明显影响。结论PEG10全长表达质粒的构建为进一步研究PEG10基因的效应和机制提供了有利的工具,研究结果显示PEG10基因可以促进人正常胚肝细胞增殖并抑制其凋亡,但对非肝脏来源的对照组细胞人胚肾细胞株293细胞没有明显效应。Objectives To construct a plasmid expressing human imprinted gene PEG10 and to study the effect of overexpression of PEG10 in a stable transfected human normal liver cell line L02 and in nonliver derived cell line 293. Methods Full length cDNA of PEG10 open reading frame 1 was amplified and subcloned into a mammalian expression vector pcDNA3. I hisC. Recombinant plasmid was stably transfected into L02 cells and control cells via Lipofectamine 2000. The expression and the function of PEG10 in L02 cells and control group cells were examined using RT-PCR, Western blot, MTT and TUN-EL. Results Recombinant plasmid was successfully constructed and confirmed through DNA sequencing and restriction digesting. PEG10 gene accelerated the growth of L02 cells and inhibited their apoptosis but it had no conspicuous effect on the non-liver derived cells. Conclusion The constructed expressing vector pcDNA3. 1 hisC-PEG 10 provides a useful tool for further study on the effects and mechanisms of PEG10. Over-expression of PEG10 may promote L02 cells＇ proliferation and inhibit their apoptosis, but not in the non-liver derived cell line 293.

关 键 词：转染 质粒 PEGIO 

分 类 号：R686[医药卫生—骨科学]