| Cerebrovascular disorders belong to the category of "wind stroke" in traditional Chinese medicine (TCM), which accounts for70%to80%among all the cerebrovascular patients and brings great torture and burden to the patients, their families and the society, resulting in high rates of morbility, mortality and disability. Hence, how to effectively prevent and treat the nerve injuries following cerebral ischemia and promote the nerve regeneration becomes the key issue. In recent years, with the further investigation of mesenchymal stem cell (MSCs) transplantation, it is found that it could improve the neurological functions for the cerebral ischemia, which has attractive prospects for the clinical applications. However, the specific mechanisms are still uncertain. The transplanted MSCs could promote the neurological outcome maybe via transforming into neuron-like cells, accelerating angiogenesis, alleviating local inflammation and edema and inhibiting cellular apoptosis. Conception and Governor Vessel acupuncture has certain clinical basis in treatment of cerebral ischemic apoplexy. Previous studies indicated that electro-acupuncture (EA) at Conception and Governor Vessels continuously promotes the proliferation and differentiation of endogenous neural stem cells (NSCs) in the cerebral ischemia/reperfusion rats. Meanwhile, EA at Conception Vessel could obviously up-regulate the expressions of growth factors and EA at Governor Vessel could inhibit the cellular apoptosis, which could promote the neurological function recovery following cerebral ischemia. Hence, both mechanisms of EA and MSCs transplantation in treatment of cerebral ischemia are similar. On the basis of the researches above, we put forward the following hypothesis, that is, compared with the simple MSCs intracerebral transplantation, acupuncture at Conception and Governor Vessels plus MSCs transplantation has greater impacts on the nerve protection following cerebral ischemia. Based on the hypothesis and the literatures referred, this dissertation takes the human umbilical cord blood-derived MSCs (HUCB-MSCs) transformation plus EA as the breakthrough point. By collecting and culturing the HUCB-MSCs and establishing the cerebral ischemia models, we explore the possible pathways and mechanisms of EA at Conception and Governor Vessels plus HUCB-MSCs transplantation in treatment of cerebral ischemia from the perspectives of literature and experimental studies.1. Objective:By collection and culture of the HUCB-MSCs, transplantation of the cells into the brains of cerebral ischemia rats and EA at Conception and Governor Vessels, the cellular structures, the vascular endothelial growth factor (VEGF) expression, the cellular apoptosis situation around the transplantation site and the neurological deficit scores were observed, which could reveal the possible pathways and mechanisms of Conception and Governor Vessels EA plus MSCs transplantation in treatment of cerebral ischemia and provides the theoretical and experimental basis for the future integrative acupuncture and stem cell transplantation treatment of cerebral ischemia.2. Methods:2.1HUCB-MSCs in vitro culture and biological studyWith the informed written maternal consent to the delivery women and their families, during childbearing, seventeen bags of umbilical cord blood of the delivery women from the Maternal Department, Affiliated Shenzhen Traditional Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, were collected. With the centrifugation in a ficoll-Hypaque gradient, the mononuclear cells were isolated and suspended in Dulbecco’s Minimum Essential Medium/F12(DMEM/F12), containing10%fetal bovine serum (FBS), glutamine, human epidermal growth factor (EGF), recombinant human fibroblast growth factor-basic (bFGF) and penicillin-streptomycin, and seeded at a concentration of1×106cells/cm2. Non-adherent cells were taken out by changing the medium after one week. Cultures were maintained at37℃in a humidified atmosphere containing5%CO2and passaged at80%to90%confluency for three passages. Before transplantation, the cultured cells were washed with phosphate buffered saline (PBS), with the concentration as1×105cells/u1. For surface phenotype analysis, some cells of primary passage and passage 3were stained with monoclonal antibodies like CD90, CD44, CD73, CD45, CD29, CD34, CD19, CD105, CD14and HLA-DR and analyzed by flow cytometry with a FACS Diva cytometer. Some cells of passage3were also collected for the cellular growth test.2.2Study of Conception and Governor vessel EA plus HUCB-MSCs transplantation on nerve protection of cerebral ischemia ratsA total of one-hundred and twenty specific-pathogen free (SPF) male adult Sprague Dawley(SD) rats were randomized into the following five groups, namely, sham surgery group (Group S), PBS transplantation group (Group P), HUCB-MSCs transplantation group (Group B), HUCB-MSCs transplantation plus Conception and Governor Vessel EA group (Group CG), HUCB-MSCs transplantation plus Governor Vessel EA group (Group G). In each group, all the rats again were randomized into seven-day group, fourteen-day group and twenty-eight-day group, with8rats in each sub-group. With the thread artery-insertion method, the cerebral ischemia/reperfusion models were established. After24hours, the HUCB-MSCs were transplanted into the right striatum and subcortex of the rats in the Group B, Group CG and Group G, and the PBS of same volume was transplanted into the rats in the Group P.24hours following transplantation, rats in the Group CG and Group G were applied with EA, with the sparse-dense wave and the frequency of6to15volts. The acupoints applied include Chengjiang (CV24), Guanyuan (CV4) and Qihai (CV6) on the Conception Vessel and Dazhui (GV14), Baihui (GV20) and Shuigou (GV26) on the Governor Vessel, and the rats were sacrificed on the time-point days and the brains were collected. The pathomorphism change of the focus around the transplantation site was detected with HE staining; the VEGF-positive cells around the transplantation site were observed with immunohistochemical staining; the apoptotic cells around the transplantation site were observed with TUNEL method; and the neurological function was evaluated with the modified neurological severity scores (mNSS). The positive cell images were analyzed by the analytical system of LeiCa DC Software (Germany), counted at light microscope of200X and compared the differences with SPSS15.0software.3. Results:3.1A total of seventeen bags of umbilical cord blood were isolated and4bags of blood were finally cultured into HUCB-MSCs, with the successful rate as23.5%. After added with the culture medium and the growth factors, after3 to7days, some adherent cells with diverse morphology could be observed with the microscope and there were a large number of floating cells. After two to five changes of the medium, the cells got over80%confluence, and the cellular colonies with fibroblastic morphology formed, which presented a radiating appearance. After subcultivation, the cells became adherent soon, with the day four and day five growing most. It took fifteen to thirty days during the primary passage cultivation, and about four or five days during the following passages. Flow cytometric analysis revealed that HUCB-MSCs were positive for mesenchymal antigens, including CD90, CD73, CD105, and adhesion molecules CD44and CD29; but negative for CD45, HLA-DR, CD14, CD34and CD19.3.2Compared with the Group S, after surgeries, rats in other groups appeared the neurological deficit symptoms such as contralateral hemiplegia (limb disturbance such as flexion and adduction) and contralateral circling while walking. With the prolonging of the reperfusion periods, all the mNSS had the tendency of decreasing, with the7-day highest,14-day decreased and28-day lowest, with significant difference (P<0.01). After seven days and fourteen days, the mNSS in the Group B were lower than that in the Group P (P<0.05), and the mNSS in Group G and Group CG were lower than that in the Group B respectively (P<0.01). However, there was no significant difference of the mNSS between the Group G and Group CG (P>0.05).3.3After cerebral ischemia/reperfusion injuries, there was obvious edema and necrosis around the focus, which implied that the model establishment was successful. Compared with the simple HUCB-MSCs transplantation, EA plus MSCs transplantation could better alleviate the cerebral ischemia injuries.3.4Results of the immunohistochemistry showed that after ischemia/reperfusion injuries, the VEGF protein expression was up-regulated. However, the up-regulation of VEGF expression was time-constrained. With the prolonging of the reperfusion period, it had the tendency of decreasing. Compared with simple HUCB-MSCs transplantation, EA plus MSCs transplantation could up-regulate VEGF protein expression at least after14days following reperfuion, with the effects in Group CV better than that in Group G.3.5Results of the TUNEL indicated that after ischemia/reperfusion injuries, diverse TUNEL-positive cells exist around the cerebral focus. With the prolonging of the reperfusion period, it had the tendency of decreasing. Compared with simple HUCB-MSCs transplantation, EA plus MSCs transplantation could down-regulate TUNEL-positive cells numbers at least after7days following reperfuion, with the effects in Group CV better than that in Group G.4. Conclusions:There are actually MSCs in the HUCB, which highly express the MSCs-specif ic surface antigen markers. Compared with simple MSCs transplantation, EA plus HUCB-MSCs transplantation could better up-regulate VEGF protein expression and down-regulate the apoptotic cells numbers around the cerebral focus, promote the neurological function recovery and alleviate the local pathological lesions hence better protect the nerve and restore the injuries. |
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