人脐血间充质干细胞携带人肿瘤坏死因子α基因逆转绒癌耐药体外研究

In vitro study on reversing the drug resistance of choriocarcinoma cell line JEG-3/VP16 by the expression of adenovirus-mediated hTNFα gene in human umbilical cord blood-derived mesenchymal stem cells

目的观察人脐血间充质干细胞携带人肿瘤坏死因子α(TNF-α)基因对绒癌耐药细胞株耐药指数的影响。方法应用AdMax系统构建携带TNF-α基因的腺病毒载体。鉴定、分离、培养、扩增、纯化人脐血间充质干细胞(hUCB-MSCs)。携带TNF-α基因的重组腺病毒感染hUCB-MSCs后,与绒癌耐药细胞株JEG3/VP16共培养,空载腺病毒(Ad-EGFP)感染的hUBC-MSCs做为对照,MTT法测定共培养48h后JEG-3/VP16对依托泊苷(VP16)、氟尿嘧啶(5-FU)、甲氨蝶呤(MTX)耐药情况。结果(1)酶切、聚合酶链式反应(PCR)鉴定构建的重组腺病毒携带TNF-α基因。(2)重组腺病毒可高效感染hUCB-MSCs。(3)JEG-3/VP16和转染TNFa基因的hUCB-MSCs共培养,JEG-3/VP16对VP16、5-FU、MTX的IC50分别下降至未共培养前IC50的1/12.8,1/6.75和1/10.1。结论携带hTNFα基因的hUCB-MSCs与绒癌耐药细胞株JEG-3/VP16共培养后,逆转了JEG-3/VP16的耐药性。

Objective To construct the replication-deficient recombinant adenoviruses vectors contained human TNFα gene and to investigate TNFα expression in human umbilical cord blood-derived mesenchymal stem cells（hUCB-MSCs）. And to observe the drug-resistance changes of JEG-3/VP16 after co-culture with hUCB-MSCs transfected with adenovirus-mediated hTNFα gene. Method Human TNF-α gene was amplified by PCR method from plasmid pCMV-SPORT6-TNF and cloned into shuttle vector pDC316-IRES-EGFP which was carried into 293 cells together with backbone plasmid pBHGlox_E1,3Cre by lipofectamine 2000 to obtain packed recombinant adenovirus Ad-TNFα. hUCB-MSCs were isolated,cultivated and identified in vitro. The resistance index of JEG-3/VP16 was determined by MTT test after co-culture with hUCB-MSCs transfected with hTNFα gene. Result （1）The packed recombinant adenovirus Ad-TNFα was identified using PCR and enzyme digestion.（2）The adenovirus vector was constructed successfully and high efficient expression of TNFα in hUCB-MSCs was detected. （3）MTT test showed that the resistance index of JEG-3/VP16 decreased after co-culture with hUCB-MSCs transfected huTNF-α gene. Conclusion The drug resistance of JEG-3/VP16 can be reversed after co cultured with hUCB-MSCs transfected with hTNFα gene.