[Recombinant human insulin gene lentivirus transfecting human umbilical cord mesenchymal stem cells in vitro].
OBJECTIVE
To construct the lentiviral vector to co-express enhanced green fluorescent protein (EGFP) gene and human insulin (insulin) gene, and to explore the condition to transfect human umbilical cord mesenchymal stem cells (hUCMSCs) so as to lay a foundation for tissue engineered adipose reconstruction and transplantation in vivo in future.


METHODS
The insulin gene was cloned to lentiviral expression vector with EGFP [pLenti6.3-internal ribosome entry site (IRES)-EGFP] by recombinant DNA technology, the positive clones were screened, and lentiviral packaged systems and target gene plasmid were co-transfected to package virus in 293T cells by lipofectin. The reporter gene expression was observed by fluorescent inverted phase contrast microscope, virus supernatant was collected, purified and concentrated, and the titer of recombinant viruses was determined, hUCMSCs from umbilical cord tissue of mature neonates were isolated and cultured by different multiple of infection (MOI, 0, 1, 3, 5, 7, 10, 15, and 20). By recombinant lentiviral infected hUCMSCs with reporter gene green fluorescent protein expression, the best MOI was screened; recombinant lentiviral infected hUCMSCs at the best MOI, then real-time PCR and Western blot methods were applied to detect insulin gene and insulin protein expression levels in cells.


RESULTS
The recombinant lentiviral vector of co-expressing insulin gene and EGFP gene (pLenti6.3-insulin-IRES EGFP) was successfully constructed. Virus could be packaged, purified and concentrated successfully. The virus titer was 1.3 x 10(8) TU/mL. The best MOI was 10 and the transfer efficiency was up to 90% in the same time. Real-time PCR results showed that insulin gene expression of transfected group was positive and non-transfected group was negative; Western blot detection confirmed that insulin protein expression of transfected group was positive in cells and supernatant, but that of non-transfected group was both negative.


CONCLUSIONS
Lentiviral vector pLenti6.3-insulin-IRES-EGFP carrying recombinant insulin gene could effectively transfect hUCMSCs and express insulin protein.
Liu
Yi
Y
Center for Military Burns and Plastic Surgery, Lanzhou General Hospital, Lanzhou Command of Chinese PLA, Lanzhou Gansu, 730050, P R China. liuzhih20002003@yahoo.com.cn
Xue
Meisi
M
D002478:Cells, Cultured; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D023822:Tissue Engineering / Q000379:methods; D014162:Transfection; D014470:Umbilical Cord / Q000166:cytology*
D007328:Insulin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins