Experimental study of the effects of marrow mesenchymal stem cells transfected with hypoxia-inducible factor-1alpha gene.
OBJECTIVE
To construct the eukaryotic expression vector hypoxia-inducible factor 1alpha-pcDNA(3.1) and to investigate its transfective efficiency into mesenchymal stem cells (MSCs) in vitro and the expression of HIF-1alpha gene in MSCs.


METHODS
mRNA of Wistar Rats' myocardial cells was extracted, and cDNA was synthesized with Reverse Transcription Kit, HIF-1alpha was amplified by polymerase chain reaction (PCR), and constructed into pcDNA(3.1). Transfected HIF-1alpha-pcDNA(3.1) into MSCs by liposome mediated method. The expression of HIF-1alpha in the cells was detected by Western Blot Analysis and ELISA.


RESULTS
Eukaryotic expression vector HIF-1alpha-pcDNA(3.1) was constructed successfully. Analyzed by flow cytometer, The MSCs' surfaces mark were CD44+, SH3(CD73)+, CD34-, CD45- and the CD44+ cells and SH3(CD73)+ cells were 94.7% and 97.3%, respectively, showing the high purity of the cultured MSCs. After inducing, the cultured MSCs can differentiate into osteoblasts and adipocytes successfully. In HIF-1alpha gene transfected MSCs, the expression of HIF-1alpha mRNA and HIF-1alpha protein were both increased obviously.


CONCLUSIONS
HIF-1alpha was cloned successfully. HIF-1alpha-pcDNA(3.1) can be transfected into MSCs by liposome-mediated method effectively and which resulting stable expression of HIF-1alpha in transfected MSCs.
Yang
Jinfu
J
Department of Cardiothoracic Surgery, Second Xiangya Hospital, Central South University, Hunan, Changsha 410011, China.
Tang
Tao
T
Li
Feng
F
Zhou
Wenwu
W
Liu
Jian
J
Tan
Zhiping
Z
Zheng
Wei
W
Yang
Yifeng
Y
Zhou
Xinmin
X
Hu
Jianguo
J
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D015687:Cell Hypoxia / Q000235:genetics; D048430:Cell Shape / Q000235:genetics; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods*
D015703:Antigens, CD; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D002118:Calcium