| Objective The drug resistance is a major obstacle to successful treatment of choriocarcinoma. Transfecting huTNFa gene into a drug-resistance choriocarcinoma cell line was able to reverse its drug resistance at both mRNA level and protein level, increase its sensitivity to cytotoxic drugs and reduce its tumorigenesity. However, TNFa administered by the conventional intravenous method can cause severe side effects. Furthermore, clinical trials have shown limited antitumor efficacy due to low concentration reached to tumor sites during systemic administration. It is important to find an appropriate vehicle for delivery huTNFa gene and generate tumor necrosis factor protein in local site. Mesenchymal stem cells (MSCs) possess key functional vector attributes for cancer gene therapy such as the capabilities of self renewal and long term in vivo repopulation. We investigated the possibility of using MSCs as huTNFa gene carriers based on the documented tumor-homing abilities of this cell population. This study is designed to observe both the drug-resistance changes of JEG-3/VP16 after co-culture with human MSCs from umbilical cord blood(huUCB-MSCs) transfected with adenovirus-mediated huTNFa gene and the migratory capacity of transgene-expressing huUCB-MSCsMethod huUCB-MSCs were isolated,cultivated and identified in vitro. Construct recombinant adenovirus vector containing huTNFa gene and transfect into huUCB-MSCs. Elisa method was applied to assay the TNFa in culture medium. And the activity of TNFa in supernatant was measured by crystal violet staining. The expression of huTNFa was detected using RT-PCR and Western blot, the expression of enhanced green fluorescent protein was observed under fluorescent microscope. We have established drug-resistance cell line JEG-3/VP16 by pulse exposure choriocarcinoma cell line JEG-3 to VP16 and demonstrated drug-resistance of JEG-3/VP16 using MTT. The resistance index of JEG-3/VP16 was determined by MTT test after co-culture with huUCB-MSCs transfected with huTNFa gene. Transwell migration assay in vitro was used to analyze the migration ability of huUCB-MSCs transfected with huTNFa gene towards tumor cell.Result When cultured in suitable medium in vitro, huUCB-MSCs displayed a fibroblast-like morphology and expressed several MSCs-related antigens CD29,CD44,CD90 but not hematopoietic stem cells (HSCs) antigens CD34,CD45. It can be affirmed as the MSCs which characterized with multi-differentiating potentiality after lipogenic and osteoblastic differentiation. Human TNF-a 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_El,3Cre by lipofectamine 2000 to obtain packed recombinant adenovirus. Virus titer could reach as high as 5.2×10~8PFU/ml. We observed the expressions of huTNFa gene with flurescent microscope 48 hours after the huUCB-MSCs were infected by the viruses. RT-PCR revealed that huTNFa gene was transcripted and expressed in transgene huUCB-MSCs. The TNF-a concentration in the supernatant fluids was measured by ELISA at the different time points, which increased gradually during the period of 16h-48h after transfection and continued for 10 days. The TNFa activity in the supernatant fluids was determined. Establish drug-resistance choriocarcinoma cell line JEG-3/VP16 successfully. MTT test showed that the resistance index of JEG-3/VP16 decreased after co-culture with huUCB-MSCs transfected huTNF-a gene. The number of huUCB-MSCs that migrated across the filter in the JEG-3/VP16 group was significantly larger than those of fibroblast group and blank control group.Conclusion It is possible to obtain single cell-derived, clonally expanded MSCs from UCB with remarkable potential to differentiate into multiple lineages, which could be used as huTNFa gene vectors in vitro study. The TNFa gene can be transfected into huUCB-MSCs efficiently and safely by adenovirus vector, and the tumor necrosis factor protein can be secreted into the supernatant liquid in a relatively high level during a relatively long period. The drug resistance of JEG-3/VP16 can be reversed after co cultured with huUCB-MSCs transfected with huTNFa gene. We demonstrated that huUCB-MSCs transfected with huTNF-a gene, when cultured in vitro, migrate targeting tumor cell JEG-3/VP16, while continuing to express a foreign gene. huUCB-MSCs transfected with huTNFa gene might be an alternative for the treatment of drug-resistance choriocarcinoma. |
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