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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 detect the expression of human enkephalin gene (hppe) mouse, canine and human cell lines and to evaluate the feasibility of using enkephalin gene cell lines for cell analgesia. Methods The recombinant plasmid pCMVhPPE (pE) was co-transfected into three mammalian somatic cell lines (NIH 3T3, MDCK, WISH) alone or with pRSVneo (pN) using calcium phosphate co-precipitation or liposome encapsulation. Screening positive co-transfected cells with G418 culture medium, expansion and culture. The content of enkephalin (ENK) in cell culture supernatant was determined by radioimmunoassay. Results The amount of ENK secreted by the double-plasmid co-transfected cells plus G418 was 10 ~ 20 ng 10 5 cells higher than that of the pE plasmid transfected cells by 4 ~ 6 ng 10 5 cells (P <0.01). There was no significant difference in transient expression between the two genes (P> 0.05). When the two plasmids were co-transfected, the transfection rates and expression levels of the former were 0 1% and 13 ~ 20 ng 10 5 cells, and the latter was 0 0 8% and 6 ~ 10ng 10 5 cells, no significant difference between the two (P> 0 05). Conclusion hppe gene can be well expressed in somatic cell lines, ENK secretion of transformed cells (10 7 ~ 10 9 cells) can meet the needs of spinal cord pain in rats.