PMID- 20695379
OWN - NLM
STAT- MEDLINE
DCOM- 20110111
LR  - 20181201
IS  - 1002-1892 (Print)
IS  - 1002-1892 (Linking)
VI  - 24
IP  - 7
DP  - 2010 Jul
TI  - [Recombinant human insulin gene lentivirus transfecting human umbilical cord 
      mesenchymal stem cells in vitro].
PG  - 822-7
AB  - 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. CONCLUSION: Lentiviral vector pLenti6.3-insulin-IRES-EGFP carrying 
      recombinant insulin gene could effectively transfect hUCMSCs and express insulin 
      protein.
FAU - Liu, Yi
AU  - Liu Y
AD  - 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
FAU - Xue, Meisi
AU  - Xue M
LA  - chi
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - China
TA  - Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi
JT  - Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = 
      Chinese journal of reparative and reconstructive surgery
JID - 9425194
RN  - 0 (Insulin)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 147336-22-9 (Green Fluorescent Proteins)
SB  - IM
MH  - Cells, Cultured
MH  - Gene Expression
MH  - Genetic Vectors
MH  - Green Fluorescent Proteins/genetics
MH  - Humans
MH  - Insulin/*genetics
MH  - Lentivirus/genetics
MH  - Mesenchymal Stem Cells/*cytology
MH  - Recombinant Fusion Proteins/genetics
MH  - Tissue Engineering/methods
MH  - Transfection
MH  - Umbilical Cord/*cytology
EDAT- 2010/08/11 06:00
MHDA- 2011/01/12 06:00
CRDT- 2010/08/11 06:00
PHST- 2010/08/11 06:00 [entrez]
PHST- 2010/08/11 06:00 [pubmed]
PHST- 2011/01/12 06:00 [medline]
PST - ppublish
SO  - Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 Jul;24(7):822-7.