Osteogenic Activity of MSC Infected by Recombinant Adenovirus Vector Ad-LMP-1
- VernacularTitle:重组腺病毒载体Ad-LMP-1的构建及其转染MSC后成骨活性
- Author:
Zhian CHENG
;
Dongbin LIU
;
Yanfeng WU
;
Lin HUANG
;
Huiyong SHEN
;
Shangli LIU
- Publication Type:Journal Article
- Keywords:
LIM mineralization protein-1;
mesenchymal stem cells;
osteogenic activity;
gene transfect
- From:
Journal of Sun Yat-sen University(Medical Sciences)
2010;31(2):199-206
- CountryChina
- Language:Chinese
-
Abstract:
[Objective]This study was designed to construct a recombinant adenovirus vector contains LMP-1 gene,and investigate the osteoinductive activity of MSC which were transfected recombinated adenoviral vector carrying LMP-1 gene.[Methods]Total RNA was extracted from mt osteoblast and the LMP-1 gene was acquired by RT-PCR,the LMP-1 gene and entry vector pENTR/D-TOPO were used to create the entry clone with the directional TOPO clone technology,then the entry clone and the expression vector were used to create the expression clone throush the LR recombination reaction.The adenovirus expression clone was linearized by PacI and transfected to the 293A cell line to harvest a high titer.Ad-LMP-1 was infected into the 3rd passage MSC,the expression of LMP-1 was detected by Western blot.The osteogenic activity of MSC was evaluated by the expression of collagen Ⅰ,ALP,osteocalcin and the formation of bone nodule.[Result]The LMP-1 gene was successfully acquired and confirmed,the entry clone and the expression clone were both verified by enzymes digestion,and the expression clone was further confirmed by sequenced.The expression of LMP-1 was detected successfully in MSC.The increasing expression of collagen Ⅰ,osteocalcin.ALP and bone nodule were observed by comparing to the control group.[Conclusion]Gateway technology not only make construction of the pAd-LMP-1 recombination adenovirus vector simple and fast,but also get a high transfection efficacy in MSC.LMP-1 gene can induce the osteoblast differention of MSCs,and improve its osteogenic activity.The adenovirus vector is reliable to be used in further gene therapy research.
