补脑Ⅰ号对阿尔茨海默病模型小鼠脑内移植骨髓间充质干细胞后海马VEGF、BDNF表达及超微结构的影响

Effect of Bunao No.1(补脑Ⅰ号)on the Expression of VEGF and BDNF and Ultrastructure of Hippocampus after Transplantation of Bone Marrow Mesenchymal Stem Cells in the Brain of Alzheimer's Disease Model Mice

目的探讨补脑Ⅰ号治疗阿尔茨海默病(AD)的可能机制。方法分离C57BL/6小鼠股骨骨髓细胞,传代培养获得第三代骨髓间充质干细胞(BMSCs)。32只APP/PS1双转基因小鼠随机分为模型组、BMSCs组、补脑Ⅰ号组、补脑Ⅰ号+BMSCs组,每组8只。同系种C57BL/6小鼠8只设为正常组。BMSCs组、补脑Ⅰ号+BMSCs组将第三代BMSCs移植入侧脑室内,移植2h后补脑Ⅰ号组、补脑Ⅰ号+BMSCs组予补脑Ⅰ号方26g/(kg·d)灌胃,BMSCs组、正常组、模型组予0.2ml生理盐水灌胃,每天1次,连续灌胃4周。Western blot法检测海马血管内皮生长因子(VEGF)、脑源性神经营养因子(BDNF)蛋白表达,实时荧光定量(qRT-PCR)检测VEGF、碱性成纤维细胞生长因子(bFGF)及BDNF mRNA表达,透射电镜观察海马区微血管形态、神经细胞、神经纤维及突触结构的改变。结果与正常组比较,模型组BDNF、VEGF蛋白表达,BDNF、VEGF、bFGF mRNA表达均显著降低(P<0.01);与模型组比较,BMSCs组、补脑Ⅰ号组、补脑Ⅰ号+BMSCs组VEGF蛋白表达,VEGF、BDNF、bFGF mRNA表达均升高(P<0.05或P<0.01),补脑Ⅰ号+BMSCs组BDNF蛋白表达升高(P<0.05);与BMSCs组、补脑Ⅰ号组比较,补脑Ⅰ号+BMSCs组VEGF蛋白表达,VEGF、BDNF、bFGF mRNA表达升高(P<0.05或P<0.01)。补脑Ⅰ号组、BMSCs组、补脑Ⅰ号+BMSCs组均能够改善海马受损的血管、神经元、有髓神经纤维和突触的超微结构,以补脑Ⅰ号+BMSCs组更明显。结论补脑Ⅰ号可促进AD模型小鼠脑内移植BMSCs后海马区血管、神经及突触结构的改善,其机制可能与提高海马区BDNF、VEGF、bFGF的表达,促进神经血管再生与突触重塑有关。

Objective To explore the effect and possible mechanism of Bunao No.1(补脑Ⅰ号) on Alzheimer′s disease(AD). Methods C57 BL/6 mouse femur bone marrow cells were isolated and subcultured to produce the third generation of bone marrow mesenchymal stem cells(BMSCs). Thirty-two APP/PS1 double transgenic mice were randomly divided into model group, BMSCs group, Bunao No.1 group and the combination(Bunao No.1 plus BMSCs) group, with eight mice in each group. Eight C57 BL/6 mice of the same strain were set as the normal group. In the BMSCs group and the combination group, the third generation of BMSCs was transplanted into the lateral ventricle;two hours after the transplantation, 26 g/(kg·d) of Bunao No.1 was given by gavage to the BMSCs group, Bunao No.1 group and the combination group, while the other groups were administered with 0.2 ml normal saline by gavage once daily, for 4 weeks. Western blotting was used to detect the protein expression of hippocampal vascular endothelial growth factor(VEGF) and brain-derived neurotrophic factor(BDNF);real-time fluorescent quantitative polymerase chain reaction(qRT-PCR) test was used to detect mRNA expression of VEGF, basic fibroblast growth factor(bFGF) and BDNF;transmission electron microscope was used to assess hippocampal microvascular morphology, nerve cells, nerve fibers and synaptic structure changes. Results Compared to the normal group, the model group had significantly lower protein expression of BDNF and VEGF, as well as lower mRNA expression of BDNF, VEGF and bFGF(P<0.01);compared to the model group, the BMSCs group, Bunao No.1 group and the combination group had significantly increased VEGF protein expression and VEGF, BDNF, bFGF mRNA expression(P<0.05 or P<0.01);the combination group also had increased BDNF protein expression(P<0.05). Compared to BMSCs group and Bunao No.1 group, the combination group had higher expression of VEGF protein as well as VEGF, BDNF and bFGF mRNA(P<0.05 or P<0.01). The Bunao No.1 group, BMSCs group and the combinaion group showed improvement of the ultrastructure of damaged blood vessels, neurons, myelinated nerve fibers and synapses in the hippocampus, and the combination group had most obvious effect. Conclusion Bunao No.1 can promote the improvement of the structure of blood vessels, nerves and synapses in the hippocampus after BMSCs transplantation in the brain of AD model mice. Its mechanism may be related to increasing the expression of BDNF, VEGF and bFGF in the hippocampus, and promoting neurovascular regeneration and synapses remodeling.