Experimental Study On The Therapeutic Benefit Of Transplantation Of VEGF Gene-modified Mesenchymal Stem Cells After Cerebral Infarction In Rats

Objective: To investigate the efficiency of Mesenchymal stem cells (MSCs) transfection after constructing VEGF gene's recombinant eukaryotic expression vector pIRES2-EGFP-VEGFA and then transferring it to Mesenchymal stem cells of rat and detect the expression level of VEGF gene in Mesenchymal stem cells after transfecting; To survey the therapeutic benefit of intravenous transplantation of VEGF gene-modified Mesenchymal stem cells (MSCs) in rats after Cerebral Infarction.Methods: With the technology of gene rearrangement, we digested the plasmid with EcoR I to obtained the target sequence of VEGF gene, inserted the gene fragment into vector pIRES2-EGFP and got the recombinant eukaryotic expression vector pIRES2-EGFP-VEGFA. The recombinant vector was identified by restriction enzyme analysis, sequenced, and transfected MSCs with lipofectamine 2000. We used flow cytometry and fluorescence microscope to detect the efficiency of transfection and detect the expression of VEGF protein in MSCs by immunobloting. The rat model of focal cerebral ischemia was established with the left middle cerebral artery suture occlusion (MCAO) method and reperfused with suture extracted after MCAO 90 mins. Rats were randomly divided into VEGF-MSCs group,MSCs group,PBS group and Model group; 1ml VEGF gene-modified MSCs, MSCs and PBS were intravenous injected respectively through tail vein on the next day after ischemia. Then 7d after transplantation, cerebral infarction volume of rats was detected through tetrazolium chloride (TTC) staining; and the expression of Ang-2, CD34 in cortex around cerebral infarction zone was detected through immunohistochemistry. Neurological function was evaluated with modified Neurological Severity Scores (NSS) on 1d and 7d after transplantation.Results: (1) The plasmid pIRES2-EGFP-VEGFA was successfully constructed, containing 656bp fragments of VEGFA cDNA, certified by restriction enzyme analysis and sequencing, many green fluorocytes were shown under fluorescence microscope and the efficiency of transfection was (34±3.6) %. We also used flow cytometry to further detect the efficiency of transfection (37±2.8) %. The protein level of VEGF in tansfected cells was detected by Western. (2) NSS: 1day after transplantation, every group have no statistical significance (P>0.05). By 7 day after transplantation, compared with PBS group and Model group, NSS score reduced in VEGF-MSCs group and MSCs group (P<0.05), VEGF-MSCs group was much lower than MSCs group (P<0.05). PBS group and Model group have no statistical significance(P>0.05) (3) TTC: Compared with PBS group and Model group, the percentage of cerebral infarction volume of VEGF-MSCs group, MSCs group were lower (p<0.05), and the group of VEGF-MSCs was much lower than MSCs group (p<0.05) ; PBS group and Model group have no statistical significance(P>0.05). (4) The expression of Ang-2 and CD34 in cortex around cerebral infarction was detected through immunohistochemistry after transfecting. Compared with PBS group and Model group, the expression of VEGF-MSCs group and MSCs group were higher (p<0.05), and the group of VEGF-MSCs was much higher than the MSCs group (p<0.05); PBS group and Model group have no statistical significance(P>0.05).Conclusion: (1) pIRES2-EGFP-VEGFA of the eukaryotic expression vector can be constructed successfully as well as transfected effectively into Mesenchymal stem cells with lipofectamine 2000, the study provided an experimental basis for gene modified Mesenchymal stem cells transplantation in the therapy of stroke. (2) Intravenous transplantation of VEGF gene-modified MSCs in rats after Cerebral Infarction can ameliorate neurological deficit and diminish the percentage of cerebral infarction volume of rats as well as facilitate angiogenesis.