The Protective Effect Of HO-1 Gene Transfection On Mesenchymal Stem Cell Injury Caused By Analogous Ischemia In Vitro

Objective To survey the protective effect of HO-1 gene transfection on mesenchymal stemcells (MSCs) injury caused by analogous ischemia in vitro, for the evidences of HO-1 gene-modified MSCs transplantation for treatment of myocardial ischemia.Methods Density gradient centrifugalization combined with adherence method were used to segregate and cultivate swine MSCs. Cultivated MSCs were characterized using immunofluorescence cytochemistry technology. Lentivirus tranfection method was used to transfect MSCs with swine HO-1 gene and the transfected MSCs were detected by fluorescence ion, growth curves was made as well as. Polymerase chain reaction(PCR) was used to examine the expression of HO-1mRNA. Serum and oxygen deprivation (SOD)was used to cause analogous ischemia injury of MSCs in vitro. Flow cytometry, Western blotting analysis and RT-PCR were used to evaluate apoptosis, survival of MSCs and measure HO-1mRNA and HO-1protein expression on different time points 3h, 6h, and 9h after SOD, respectively.Results Density gradient centrifugalization combined with adherence method could segregate and purify swine MSCs effectively. 48 hours after transferring gene into MSCs, strongfluorescence was detected. The results of immunofluorescence cytochemistry showed that CD44 and CD105 expression were positive for MSCs. Growth curves showed that there were no significant differences between genetically modified MSCs(HO-1-MSCs) and not genetically modified MSCs(MSCs); The results of flow cytometry showed that the number of apoptosis cell of HO-1-MSCs was less than that of MSCs after serum and oxygen deprivation (P<0.01) on different time points, respectively. The expression of HO-1mRNA and protein of HO-1-MSCs were higher than that of nontransfected MSCs (P<0.01) on different time points, respectively.Conclusions1. According to the adhesion properties, porcine bone marrow MSCs could be successfully isolated and cultured.2. At MOI = 20, lentiviral packaging system could make HO-1 gene transfer successful, having no significant effect on the proliferation of MSCs .3. The transfection of HO-1 gene, with its stable protein expression could increase the HO-1-MSCs'stability of hypoxia, ischemia against ischemic injury in vitro, and antagonize HO-1-MSCs'apoptosis thus had protective effect.