高水平稳定表达人睫状神经营养因子细胞株的建立

The establishment and identincation of cell lines with stable expressionof human ciliary neurotrophic factor

目的 建立能稳定且高水平表达人睫状神经营养因子(CNTF)的永久细胞株。方法 采用阳离子脂质体介导CNTF表达质粒转染C2C12、NIH3T3、HLF、CRL－2302细胞,经氨甲喋呤筛选出抗性克隆后,分别采用原位杂交检测细胞中CNTF mRNA的表达,采用免疫组织化学染色、Western blot方法检测细胞浆中CNTF蛋白质的表达,采用ELISA法检测培养液中CNTF的分泌水平。结果 不同的克隆产生和分泌CNTF的能力不同,但其中产生和分泌CNTF能力最强的克隆其培养液中CNTF的质量浓度分别是C2C12－CNTF 114OOpg/ml、NIH3T3－CNTF 9069.071pg/ml、HLF-CNTF 91046.15pg/ml及CRL-2302－CNTF 77 578.4 pg/ml。 结论 构建的多株细胞株均能持续、稳定、高水平表达和分泌CNTF。

Objective To establish cell lines which can stably and highly express human ciliary neurotrophic factor. Methods Plasmid encoding human CNTF was transfected into C2C12, NIH3T3. HLF,CRL-2302 cell line,and the stably transfected clones were selected with Amethopterin. CNTF mRNA expression of transfected clones was examined by in situ hybridization, CNTK protein expression was detected by immunohistochemistry and Western blot assay and the ability of the stably transfeeted clones to secrete CNTK was measured with a sandwich enzyme-linked immunosorbent assay ( ELISA ). Results Although examined clones showed varying ability to express and secrete CNTF,the content of CNTF in medium per 2 × 10~4 cells over a 24-hr ineubation period reachedll 400 pg/ml ( C2C12-CNTF) , 9 069. 071pg/ml ( NIH3T3-CNTF). 1 046. 15 pg/ml ( HLF-CNTF) , 77 578. 4pg/ml ( CKI.-2302-CNTF ). Conclusion The selected clones could stably and highly express and secrete human eiliary neurolrophic factor.