Experimental Study of the Effects of Marrow Mesenchymal Stem Cells Transfected with Hypoxia-InducMe Factor-lalpha Gene

摘要

Objective. To construct the eukaryotic expression vector hypoxia-inducible factor la-pcDNA_(3.1) and to investigate its transfective efficiency into mesenchymal stem cells (MSCs) in vitro and the expression of HIF-la gene in MSCs. Methods. mRNA of Wistar Rats' myocardial cells was extracted, and cDNA was synthesized with Reverse Transcription Kit, HIF-la was amplified by polymerase chain reaction (PCR), and constructed into pcDNA_(3.1). Transfected HIF-la-pcDNA_(3.1) into MSCs by liposome mediated method. The expression of HIF-la in the cells was detected by Western Blot Analysis and ELISA. Results. Eukaryotic expression vector HIF-lalpha-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-la gene transfected MSCs, the expression of HIF-la mRNA and HIF-la protein were both increased obviously. Conclusion. HIF-la was cloned successfully. HIF-lalpha-pcDNA_(3.1) can be transfected into MSCs by liposome-mediated method effectively and which resulting stable expression of HIF-la in transfected MSCs.