| Objective:Ischemic stroke is a disease which threatens the human's life and health seriously. Following a stroke, neural cell necrosis, astrocyte proliferation and scar formation frequently lead to permanent brain damage and the loss of neurological function. At present, there are no effective treatments that could facilitate the repair of the injured nervous tissue and the functional recovery. The discovery of the neural stem cells (NSC) in adult central nervous system (CNS) offers a series of novel strategies for treatment of stroke. Recently, most studies have shown that bone-derived mesenchymal stem cells (BMSC) transplantation could promote the proliferation of endogenous NSC and improve animal neurological functional recovery after stroke. However, the mechanisms underlying the therapeutic benefits were not elucidated. Many studies have demonstrated that Notch signaling pathway plays an important role in the control of the proliferation and differentiation of NSC in the embryonic and adult CNS. Previously, our teams have shown that BMSC can promote the proliferation of NSC through increasing the activation of the Notch signaling pathway ex vivo. Therefore, we inferred that BMSC transplantation after stroke may affect the activation of the Notch signaling pathway, and then promote the proliferation of endogenous NSC. This study based on our previous results will further investigate the effect of BMSC transplant after ischemic stroke on the proliferation of NSC and the expression of the Notch signaling molecules (notch1, hes1, mash1) in the subventricular zone (SVZ) of ischemic hemisphere, then explore the role of Notch signaling pathway in the proliferation of NSC after stroke.Methods1. BMSC from SD rats were isolated, cultured and purified by adherence screening method, and the BMSC of forth passage were collected for transplantation experiments after identification by immunocytofluorescence staining of CD34 and CD29.2. The right middle cerebral artery occlusion (MCAO) model of SD rats was produced by obstructing middle cerebral artery using a single strand nylon thread. At 24h after MCAO, the brain tissue were extracted and stained by TTC. The rats were randomly divided into BMSC transplantation group, ischemic control group and normal group. BMSC were administrated at 24 hours after MCAO in transplantation group, while the same amout of PBS was injected in ischemic control group. The rats were killed at 1, 3, and 7 days after MCAO, and the brain tissues were isolated.3. The transplanted BMSC were identified and counted in the ischemic and contralateral zone under the fluorescence microscopy in each group.4. The Ki-67-, Nestin- and Notch1-positive cells were identified and counted in the SVZ of ischemic hemisphere under the fluorescence microscopy in each group.5. The expressions of Notch1 and Hes1 in the SVZ of ischemic hemisphere were detected by RT-PCR method in each group.Results1. After cultivation for 72 hours, the primary cells showed a fibroblast-like appearance and form some small cell colonies. After the flask is confluent for 10~14d cultivation, the cells were passaged. The fourth passage cells were identified by the immunocytofluorescence staining, and the results showed that CD29 was positively expressed and CD34 was negatively expressed in the cells, which were the characteristics of BMSC.2. At 24h after MCAO, the brain tissues were stained by TTC. The color of the infarct cortex and striatum is white, and the non-infarct region is pink.3. The number of BMSC in ischemic area were significantly higher than that in the contralateral area at the 1st and 7th day after BMSC transplantation. It suggested that the transplanted BMSC survived and preferentially localized to the ipsilateral hemisphere.4. The number of ki-67 positive cells in the SVZ of the ischemic hemisphere in the BMSC transplantation group were significantly increased compared with the ischemic control group (13.28±1.501 vs. 8.32±0.610 at the 1st day, 25.28±1.418 vs. 14.32±1.425at the 3rd day, 31.4±1.960vs. 27.12±1.724 at the 7th day, P<0.05, n = 5, respectively). The number of ki-67-positive cells in the SVZ of the sham-operated group is 5.56±0.713. These results indicated that BMSC transplantation after stroke can promote the cell proliferation in the SVZ of the ischemic hemisphere.5. The number of nestin-positive cells in the SVZ of the ischemic hemisphere in the BMSC transplant group were significantly increased than the ischemic control group (11.52±0.879 vs. 7.36±0.699 at the 1st day, 23.68±1.197 vs. 13.68±1.137 at the 3rd day, 28.56±1.466 vs. 21.68±1.308 at the 7th day, P<0.05, n = 5, respectively). The number of nestin-positive cells in the SVZ of the sham-operated group is 4.08±0.303. These results indicated that BMSC transplant after stroke can promote the NSC proliferation in the SVZ of the ischemic hemisphere.6. The number of notch1 positive cells in the SVZ of the ischemic hemisphere in the BMSC transplant group were significantly increased than the ischemic control group (12.04±1.763 vs. 8.72±0.482 at the 1st day, 28.6±1.086 vs. 15.76±1.090 at the 3rd day, 32.2±3.181 vs. 27.34±1.760 at the 7th day, P<0.05, n = 5, respectively). The number of notch1-positive cells in the SVZ of the sham-operated group is 7.32±0.415. These results indicated that BMSC transplantation after stroke can promote the expression of notch1 protein in the SVZ of the ischemic hemisphere.7. The expression of Notch1 mRNA in BMSC transplantation group was significantly higher than that both in ischemic control group (p<0.05) and in sham-operation group (p<0.05). The expression of Notch1 mRNA in ischemic control group was significantly lower than in sham-operation group (p<0.05) at the 1st and 3rd day. However, the expression of Notch1 mRNA in ischemic control group was significantly higher than that in sham-operation group (p<0.05) at 7th day. These results indicated that BMSC transplantation could promote the expression of the Notch1 mRNA in the SVZ of the ischemic hemisphere.8. The expression of Hes1 mRNA in sham-operation group is low, and it is significantly higher than in ischemic control group (p<0.05). Furthermore, the expression of Hes1 mRNA in BMSC transplantation group was significantly upregulated compared with ischemic control group (p<0.05). These results indicated that BMSC transplantation could promote the expression of the Hes1 mRNA in the SVZ of the ischemic hemisphere.9. The expression of Mash1 mRNA in sham-operation group is low, and it is significantly upregulation in ischemic control group (p<0.05). The Mash1 mRNA expression in BMSC transplantation group was significantly higher than in sham-operation group (p<0.05) and in the ischemic control group (p<0.05) at the 1st and 3rd day. The expression of Mash1 mRNA in the BMSC transplantation group was significantly higher than in the ischemic control group (p<0.05) at 7th day. These results indicated that BMSC transplantation could affect the expression of the Mash1 mRNA in the SVZ of the ischemic hemisphere.Conclusion:The expression of Notch1 protein and Notch1, Hes1 and Mash1 mRNA in the SVZ of the ischemic hemisphere in BMSC transplantation group were higher than those in ischemic control group and sham-operated group. These results indicated that BMSC transplantation could activate Notch signaling pathway, and then promote the proliferation of endogenous NSC after stroke.
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