| PARTⅠVitro experiments of the therapeautic effects of VEGF gene transfected MSCs on rat model of cerebral infarctionObjective:Transfect recombinant eukaryotic expression vector pIRES2-EGFP-VEGFA into bone marrow mesenchymal stem cells.Investigate and detect the VEGF gene-modified bone marrow mesenchymal stem cells whether have the ability to enhanced the secretion of VEGF in the ischemic environment. In next, explored the mechanism of MSCs in the ischemic reperfusion injury, to provid a reliable basis for clinical application.Methods:Isolation MSCs and cell culture. The recombinant vector was transfected into MSCs with lipofectamine2000. We used Optical density and flow cytometry to detect the efficiency of transfection and detect the expression of VEGF protein in MSCs by Western blot. The rat model of focal cerebral ischemia was established with the left middle cerebral artery suture occlusion (MCAO) method. VEGF gene-modified MSCs were intravenous injected respectively through tail vein after ischemia. The level of VEGF was detected through Enzyme-linked immunosorbent assay (ELISA)in rar’s brain and in cultured medium of MSC.Results:Many green fluorocytes were shown under fluorescence microscope and the efficiency of transfection was (28±3.4)%. We also used flow cytometry to further detect the efficiency of transfection(32±2.8)%. The protein level of VEGF in tansfected-cells was higher than untansfected-cells,which was detected by Western blot. The level of VEGF in the brain tissue of the rats transplantated with MSCs was Significantly higher than that of the rats untransplanted. The results in vitro experiments show that:The VEGF level of the MSCs culture which was added with th ischemic brain extract was Significantly higher than that was not done,.Which could be enhanced after MSCs modified by VEGFConclusion:Our experiment results shows that ischemic environment can enhance the ability of MSCs to secrete VEGF, and this ability is significantly increased after the VEGF gene-modified MSCs PART ⅡAngiogenesis of VEGF gene transfected-MSCs on rat model of cerebral infarctionObjective:This part of the experiment is designed to confirme the angiogenesis of VEGF gene-modified MSCs transplantation in the rat tMCAO/R model,and to explore the mechanism of angiogenesis after cerebral infarction.Methods:SD rats were randomly divided into sham operation group, model group, MSCs group and VEGF-MSCs group.Suture method was used to produce tMCAO/R model, MSCs group rats wered injected2×106MSCs via the tail vein one day after cerebral infarction.VEGF-MSCs group rats wered injected2×106MSCs transfered VEGF gene via the tail vein one day after cerebral infarction. ELISA was adopted to measure VEGF level expression of every groups in rat brain tissue.Immune staining was used to observe the Ang-2and CD34expression around the infarct zone. Western-blot was used to observe the Ang-2expression around the infarct zone Transmission electron microscopy was adopted to observe the cerebral cortex vascular clearance and edema after cerebral infarction.Results:ELISA results showed that the model rats infarction brain tissue VEGF expression levels reached a peak in about4d, come down to the sham group (normal) level or even lower after7～10d;Compared with model group. VEGF levels in VEGF-of MSCs group and MSCs group are significantly higher than those of the model group at each time point.(P<0.05),and VEGF levels in VEGF-MSCs group was higher than that in the MSCs group (P<0.05), Remained at high level in the10d.The Immunohistochemical results suggest that the Ang-2-positive cell count and CD34expression around infarct area in VEGF-of MSCs group and MSCs group were significantly more than those of the model group after infarct10days (P<0.05),.,and Vthe Ang-2-positive cell count and CD34expression around infarct area in VEGF-MSCs group was higher than that in the MSCs group (P<0.05),Western-blot results also show the same trendConclusions:Compared with MSCs transplantation group,VEGF gene transfection MSCs transplantation tMCAO model in rats group.Infarction side of the brain tissue concentration of VEGF can improve cerebral infarction, the increase of Ang-2level increase in the peripheral capillary density of infarcted area. We speculate that the synergistic effection of VEGF and Ang-2, may be one of the mechanisms of angiogenesis after cerebral ischemia. part ⅢNeuroprotective of VEGF gene transfected-MSCs on rat model of cerebral infarctionObjective:This study was designed to detect the different changes of cerebral infarction、MSCs、MSCs transplantation after VEGF gene transfection on tMCAO/R model rat model of cerebral infarction and to confirm whether MSCs transplantation after VEGF gene transfection play a neuroprotective role on tMCAO/R model rat model of cerebral infarction.Methods:SD rats were randomly divided into sham group, model group, MSCs group and VEGF-MSCs group. NSS scores was used to assess the behavioral score of each group at each time point. TTC was used to measure the infarct volume of rats in each group. Cell apoptosis of infarcted area in each group was detected by TUNEL in situ. TEM was used to observe the cortex mitochondria and synapses changes in the infarction neurons. Western blot analysis was used to measure Caspase-3protein expression levels of each group. Real-time quantitative PCR was applied to analysis the Bcl-2mRNA transcription level of each group in each time point.Results:the NSS score of the sham group (normal) was o, there was no cerebral infarction, and occasionally physiological apoptotic cells (data not shown). VEGF-MSCs group and MSCs group after4d,7d,10d, the NSS score was significantly lower than model group (P<0.05).compared with MSCs group, VEGF-of MSCs group was lower (P<0.05).TTC results showed that after10d of infarction, the brain volume of VEGF-MSCs group and MSCs group were significantly lower than model group (P<0.05). Compared with MSCs group, VEGF-of MSCs group was lower (P<0.05).And the TUNEL assay results also showed that the same trend. TEM results showed that mitochondrial was swell after cerebral infarction, compared to VEGF-MSCs group and MSCs group. However, changes of VEGF-of MSCs group and MSCs group were improved.The result of Immunohistochemistry and Western blot results showed that caspase-3positive cells and caspase-3protein expression level in VEGF-MSCs group and MSCs group were significantly lower than the model group(P<0.05) and the VEGF-MSCs group was lower than the MSCs group (P<0.05).The result of Real-time quantitative PCR show that the level of mRNA transcription of Bcl-2increased on rat model of cerebral infarction, especially VEGF-MSCs group and MSCs group (P<0.05),.Besides, VEGF-of MSCs group was higher than MSCs group (P<0.05).Conclusion:MSCs transplantation after VEGF gene transfection can reduce infarct volume,the number of neuronal apoptosis and improve neurological function. The mechanism of VEGF play a direct neuroprotective role on the basis of VEGF can promote angiogenesis and improve the microenvironment of focal cerebral ischemia in the brain tissue and up-regulate Bcl-2gene expression, inhibit caspase-3expression. |
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