Effects Of Transplantation With Survivin Modified Bone Marrow-derived Mesenchymal Stem Cells On Cerebral Infarction In Rats

Objective :Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) has been proposed as a potential therapeutic strategy for cerebral ischemia .However, marginal recovery of neurological function has been resulted from considerable cells death and apoptosis after transplantation into the infarct brain. Here, we genetically modified BMSCs with anti-apoptotic and pro-angiogenesis Survivin (SVV) gene and evaluated cell survival, the lever of neurotrophic factor and functional improvement in the brains post focal cerebral ischemia in rats.Methods: 1. BMSCs were prepared from the bone marrow of rats based on their adherence to tissue culture surface. P3 BMSCs were identified by fluorescence-activated cell sorter (FACS) for CD14, CD29, CD45, CD90 , CD106 antibodies.2. P1 BMSCs were infected by SVV recombinant virus or Mock-virus, which was named as SVV/GFP+BMSCs or GFP+BMSCs, respectively, and after they were cultured for 96 hours, the GFP-positive cells were quantified by FACS. When they were passaged to P3, FACS was used to detect the effect of infect, immunofluorescent staining was used to detect the expression of SVV in the BMSCs ,3. 48 male adult Sprague-dawley rats were randomly divided into sham group,PBS treated group (vechile group), GFP+BMSCs treated group and SVV/GFP+BMSCs treated group, Rat's focal cerebral ischemia was induced by right middle cerebral arteryocclusion (MCAO) with modified Longa's methods.SVV/GFP+BMSCs were injected after 24 hours of MCAO. the neurological functions were tested by modified neurological severity scores(mNSS), The surviving cells were tracked by immunofluorescent staining after transplantation. Expression of VEGF ,bFGF, IGF-1 in the infarct zone were assay by reverse transcript (RT)-PCR and immunostaining, cell apoptosis in situ were assay by TUNEL staining.Results: 1. P3 BMSCs were CD29, CD90, CD106-positive and CD14, CD45-negtive.2. P1 BMSCs were GFP-positive after infected 96 hours.There are also more than 90% of successively BMSCs were GFP-positive then passaged to P3. SVV/GFP+BMSCs group were survivin-positive and GFP+BMSCs group were survivin-negtive. 3. The rat's neurological functions in SVV/GFP+BMSCs group at 7d and 14d after MCAOwere better than that in GFP+BMSCs and vechile group.Transplantation with SVV/GFP+BMSCs increased about 1.2-fold,2-fold of the cellular survival at 4days and 14 days respectively when compared with GFP+BMSCs group (P﹤0.01). Expression of VEGF, bFGF, IGF-1 in the infarct zone was higher in GFP+BMSCs group only at 4days after MCAO than in vechile group. Expression of these growth factors in SVV/GFP+BMSCs was not only higher than vechile group but also than in GFP+BMSCs group(P﹤0.05or P﹤0.01 ) at 4days and 14 days after MCAO. the cell apoptosis at the border of infarct core in SVV/GFP+BMSCs group was significantly lower than in GFP+BMSCs group (P﹤0.05)and vechile group(P﹤0.01).Conclusion: SVV gene modification enhanced survival of BMSCs under ischemic condition, and transplantation with SVV engineered BMSCs may mediate substantial functional recovery after cerebral infarction.