稳定表达小鼠白介素12的间充质干细胞株的筛选与鉴定  

Screening and Identification of Mesenchymal Stem Cell Strains to Secret Mouse Interleukin-12 Mediated with Lenti-viral Vector

作　　者：徐健蓉[1,2,3] 李红霞[2] 王国庆[2] 杜小波[2] 魏于全[2] 赵菊梅[2] 

机构地区：[1]四川大学生命科学学院,成都610064 [2]四川大学华西医院生物治疗国家重点实验室 [3]西南科技大学

出　　处：《四川大学学报（医学版）》2009年第4期584-587,共4页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金创新研究群体科学基金(No.30221001)资助

摘　　要：目的筛选获得慢病毒介导的分泌表达小鼠白介素12(mIL-12)的间充质干细胞株。方法采用PCR反应从pORF-mIL-12中扩增得到mIL-12基因的完整序列,克隆入pENTRTM11质粒,构建含小鼠IL-12基因的重组质粒pENTR-mIL-12,与pLenti6/V5-Dest质粒进行细胞外重组,得到pLenti6/V5-mIL-12质粒,经PCR、多酶切鉴定正确后送Invitrogen公司上海分公司测序;用293FT细胞进行病毒包装,得到具有感染性的病毒;分离小鼠间充质干细胞,用杀稻瘟素筛选Lenti-mIL-12-MSC的稳定表达株,并鉴定。结果构建了重组质粒pENTR-mIL-12和pLenti6/V5-mIL-12,测序结果与GenBank收录序列一致,未发生突变;包装得到Lenti6/V5-mIL-12病毒;筛选得到稳定表达的Lenti-mIL-12-MSC细胞株,并用RT-PCR方法及酶联免疫吸附实验(ELISA)检测验证了mIL-12体外表达。结论筛选得到稳定表达mIL-12的间充质干细胞株。Objective To screen the stable expression cell strains of mouse interleukin-12 （raiL-12） from mouse Mesenchymal Stem Cells （mMSCs） transfeeted with lenti-mIL-12 virus. Methods The raiL-12 cDNA was amplified from plasmid pORF-mIL-12 （Invivogen） by PCR. The cDNA was subcloned into pENTR.TM 11 to generate recombinant plasmid pENTR-mIL-12. Then, pENTR-mIL-12 was homologously reeombinated with pLenti6/V5- Dest. The recombinant was named as pLenti6/VS-mIL-12 and confirmed by PCR and DNA sequencing. The Lenti6/VS-mIL-12 virus was packaged using 293FT cells. The Lenti-mIL-12-MSC monoclone was picked from the mMSCs infected by the Lenti6/V5-mIL-12 virus using Blasticidia and verified by RT-PCR and ELISA. Results The recombinant pLenti6/VS-mIL-12 was constructed, The sequence of amplified mIL-12 gene was consistent with that reported in GenBank. By RT-PCR and ELISA, it was confirmed that the mIL-12 protein could be expressed and secreted into the supernatant of MSC strain culture. Conclusion The recombinant mMSC strains lentivirally engineered to secret raiL-12 were obtained.

关 键 词：白细胞介素12 间充质干细胞 慢病毒载体 

分 类 号：R73-36[医药卫生—肿瘤]