人前脑啡肽原基因在体细胞系细胞的表达  

Expression of human preproenkephalin gene in somatic cell line cells

作　　者：杨茂元[1] 郑从义[2] 毕好生[1] 屈三甫[2] 张咸伟[1] 张传汉[1] 

机构地区：[1]同济医科大学附属同济医院麻醉学教研室,武汉市430030 [2]武汉大学中国典型培养物保藏中心

出　　处：《中华麻醉学杂志》2000年第4期226-228,共3页Chinese Journal of Anesthesiology

基　　金：国家自然科学研究基金!资助项目 ( 3 990 0 13 9) ;卫生部科研基金资助项目 !( 98 1 12 9)

摘　　要：目的　检测人前脑啡肽原基因 (hppe)鼠、犬、人体细胞系细胞的表达水平 ,评估转脑啡肽基因细胞系用于细胞镇痛的可行性。方法　采用磷酸钙共沉淀法或脂质体包裹法 ,将重组质粒pCMVhPPE(pE)单独或与pRSVneo(pN)共转染导入三种哺乳动物体细胞系细胞 (NIH 3T3,MDCK ,WISH)中。用G418培养液筛选阳性共转染细胞 ,扩增培养。以放射免疫法测定细胞培养上清液中脑啡肽 (ENK)的含量。结果　双质粒共转染加G418筛选所获转化细胞的ENK分泌量 10～ 2 0ng 10 5 个细胞明显高于 pE质粒单转染者的 4～ 6ng 10 5 个细胞 (P <0 0 1)。两者的基因瞬时表达水平无显著性差异 (P >0 0 5 )。双质粒共转染时 ,脂质体包裹法和磷酸钙共沉淀法的基因转染率相仿 ,前者的转染率和表达水平为 0 1%和 13～ 2 0ng 10 5 个细胞 ,后者为 0 0 8%和 6～ 10ng 10 5 个细胞 ,两者无显著性差异 (P >0 0 5 )。结论　hppe基因能在体细胞系细胞中良好表达 ,转化细胞的ENK分泌量(10 7～ 10 9个细胞 )可以满足大鼠脊髓镇痛的需要。Objective To assay the expression of exogenic enkephalin gene in mammalian somatic cell line cells and evaluate the feasibility of the gene modified cells in cell therapy for chronic pain Methods Recombinant plasmid pCMVhPPE (pE) lonely or combined with plasmid pRSVneo (pN) was transfected into somatic cell line(NIH/3T3, MDCK or WISH) cells of three kinds of mammalian using the calcium phosphate precipitation method or lipofectamine method for gene transfer The positive cotransfected cells were selected with G418 culture medium and were subcultured The concentration of enkephalin (ENK) in cell culture medium was measured with radioimmunoassay Results ENK concentration secreted by cotransfected cells (10 20ng/10 5 cells) was markedly higher than that by the cells transfected with plasmid pE (4 6ng/10 5 cells) (P<0 01) The both instant expressions were at same level (P>0 05) The rate of transfection and expression level with both plasmids by lipofectamine method were similar to those by calcium phosphate precipitation method (0 1% and 13 20ng/10 5 cells vs 0 08% and 6 10ng/10 5 cells) (P>0 05) Conclusions The human preproenkephalin gene can express well in somatic cell line cells, and the transfected cells (10 7 10 9 cells) can secret ENK enough to produce analgesic effect

关 键 词：基因表达 前脑啡肽原基因 细胞系 镇痛 

分 类 号：R742.5[医药卫生—神经病学与精神病学]