电针对移植的BMSCs在脑出血大鼠脑内分化影响的研究
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摘要
目的：观察电针对脑出血(intracerebral hemorrhage,ICH)后移植的外源性骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BMSCs)分化的影响。方法：通过脑立体定位仪向成年SD大鼠脑内尾壳核(Caudate-Putamen)注射胶原酶和肝素钠建立ICH动物模型,在模型建立后的第3天,选择神经功能评分(Neurological Severity Score,NSS)超过9分的SD大鼠并随机分为生理盐水对照组(NS组)、BMSCs移植组(BMSCs组)和BMSCs移植结合电针刺激治疗组(EABMSCs组)三组。然后把生理盐水、等量BMSCs分别移植到NS组、BMSCs组和EA BMSCs组SD大鼠的ICH部位；移植2h后在EABMSCs组SD大鼠的百会穴、大椎穴给与电针刺激,以后每天一次,直至SD大鼠被处死为止。按照注射生理盐水或进行细胞移植后各组SD大鼠的再喂养时间不同(1d、3d、5d、7d、14d),把每组的SD大鼠都分成五个亚组,每个亚组5只SD大鼠。在动物模型建立2h后开始对各实验组的SD大鼠进行NSS评分并记录,以后每天一次直至处死。然后灌注取脑、冰冻切片及免疫组化染色,最后利用荧光显微镜观察外源性BMSCs在ICH的SD大鼠脑内的分布与分化情况。
结果：在建模后移植前同一时间点各实验组(NS组、BMSCs组、EABMSCs组)之间,各SD大鼠的NSS评分差异无统计学意义。在移植后同一时间点(1d、3d除外)BMSCs组SD大鼠的NSS评分低于生理盐水组SD大鼠的NSS评分,差异具有统计学意义,p<0.05；同一时间点(1d除外)EA BMSCs组SD大鼠的NSS评分低于BMSCs组SD大鼠的NSS评分,差异具有统计学意义,p<0.05；同一实验组后一时间点SD大鼠的NSS评分均低于前一时间点SD大鼠的NSS评分,差异具有统计学意义,p<0.05。外源性BMSCs移植进入到ICH大鼠尾壳核处后,随着时间的推移,在纹状体、胼胝体、背侧丘脑以及大脑皮层都可见被绿色荧光蛋白(Green Fluorescent Protein, GFP)标记的外源性BMSCs分化而来的表达胶质细胞原纤维酸性蛋白(Glial Fibrillary Acidic Protein, GFAP)的星形胶质细胞和表达神经丝蛋白-200(Neurofilament- 200, NF-200)的成熟的神经元样细胞。
结论：外源性BMSCs移植联合电针刺激较单纯BMSCs移植更能改善ICH后SD大鼠神经功能缺失症状,其机制可能是电针有助于促进外源性BMSCs向神经元样细胞、星形胶质细胞的分化,替代原来受损的神经细胞,完成一定的神经组织结构修复和环境重建。
Abstract:Objective:Observe the impact on electroacupuncture to the Bone marrow mesenchymal stem cells (BMSCs) transplanted in the rat brain of intracerebral hemorrhage(ICH). Methods:The animal model of intracerebral hemorrhage is established by injecting collagenase and heparin into the Caudate-Putamen in brain of the adμlt SD rats through the brain stereotaxic apparatus. In the third day after the model established, the SD rats selected NSS score more than 9 points were randomly divided into 3 groups:normal saline control group (NS group), BMSCs transplanting group (BMSCs group), BMSCs transplanting combined with electroacupuncture treatment group (EA BMSCs group). Then, the Normal saline (NS group)、equivalent BMSCs (BMSCs group and EA BMSCs group) were transplanted into the plance of ICH; The rats of EA BMSCs group were treated by electroacupuncture after 2h cells transplanted and one time a day until to the death of the SD rats.According to different feeding times (1d,3d, 5d,7d,14d) in NS group、BMSCs group、EA BMSCs group), they were divided into five subgroups (5 rats in subgroups). After 2h of animal model established, we make a Neurological Severity Score (NSS) and record it, then record it one time a day until to death of SD rats. Then making the perfusion of brain, taking out of the brain. freezing sections and making immunohistochemical staining. At last, the distribution and differentiation of BMSCs in the brain is observed by fluorescence microscopy. Results:NSS score difference at the same time points before transplantation was not statistically significant between every experimental group (NS group, BMSCs group, EA BMSCs group). BMSCs group's NSS score lower than the score of NS group at the same time point after transplantation, the difference was statistically significant, p<0.05 (1d,3d excluded). The EA BMSCs group's NSS score lower than that of BMSCs group at the same time point after transplantation, the difference was statistically significant, p<0.05 (1d excluded). The NSS score at the time lower than that of next time point in the same experimental group, it was statistically significant,p<0.05 The exogenous BMSCs transplanted into the ICH rat's Caudate-Putamen nucleus are visible in the corpus callosum, striatum, dorsal thalamus and the cerebral cortex, and the differentiation nerve cells and the astrocytes were discovered in these places. Conclusion:Electroacupuncture combined with BMSCs transplantation can improve neurological deficits of the ICH SD rats more than those of a simple of BMSCs transplantation. The mechanism may be that Electroacupuncture combined with exogenous BMSCs contributes differentiating to the nerve cells, astrocyte cells which replace the original nerve cells of necrosis and change the cytoarchitecture of the brain.
结果：在建模后移植前同一时间点各实验组(NS组、BMSCs组、EABMSCs组)之间,各SD大鼠的NSS评分差异无统计学意义。在移植后同一时间点(1d、3d除外)BMSCs组SD大鼠的NSS评分低于生理盐水组SD大鼠的NSS评分,差异具有统计学意义,p<0.05；同一时间点(1d除外)EA BMSCs组SD大鼠的NSS评分低于BMSCs组SD大鼠的NSS评分,差异具有统计学意义,p<0.05；同一实验组后一时间点SD大鼠的NSS评分均低于前一时间点SD大鼠的NSS评分,差异具有统计学意义,p<0.05。外源性BMSCs移植进入到ICH大鼠尾壳核处后,随着时间的推移,在纹状体、胼胝体、背侧丘脑以及大脑皮层都可见被绿色荧光蛋白(Green Fluorescent Protein, GFP)标记的外源性BMSCs分化而来的表达胶质细胞原纤维酸性蛋白(Glial Fibrillary Acidic Protein, GFAP)的星形胶质细胞和表达神经丝蛋白-200(Neurofilament- 200, NF-200)的成熟的神经元样细胞。
结论：外源性BMSCs移植联合电针刺激较单纯BMSCs移植更能改善ICH后SD大鼠神经功能缺失症状,其机制可能是电针有助于促进外源性BMSCs向神经元样细胞、星形胶质细胞的分化,替代原来受损的神经细胞,完成一定的神经组织结构修复和环境重建。
Abstract:Objective:Observe the impact on electroacupuncture to the Bone marrow mesenchymal stem cells (BMSCs) transplanted in the rat brain of intracerebral hemorrhage(ICH). Methods:The animal model of intracerebral hemorrhage is established by injecting collagenase and heparin into the Caudate-Putamen in brain of the adμlt SD rats through the brain stereotaxic apparatus. In the third day after the model established, the SD rats selected NSS score more than 9 points were randomly divided into 3 groups:normal saline control group (NS group), BMSCs transplanting group (BMSCs group), BMSCs transplanting combined with electroacupuncture treatment group (EA BMSCs group). Then, the Normal saline (NS group)、equivalent BMSCs (BMSCs group and EA BMSCs group) were transplanted into the plance of ICH; The rats of EA BMSCs group were treated by electroacupuncture after 2h cells transplanted and one time a day until to the death of the SD rats.According to different feeding times (1d,3d, 5d,7d,14d) in NS group、BMSCs group、EA BMSCs group), they were divided into five subgroups (5 rats in subgroups). After 2h of animal model established, we make a Neurological Severity Score (NSS) and record it, then record it one time a day until to death of SD rats. Then making the perfusion of brain, taking out of the brain. freezing sections and making immunohistochemical staining. At last, the distribution and differentiation of BMSCs in the brain is observed by fluorescence microscopy. Results:NSS score difference at the same time points before transplantation was not statistically significant between every experimental group (NS group, BMSCs group, EA BMSCs group). BMSCs group's NSS score lower than the score of NS group at the same time point after transplantation, the difference was statistically significant, p<0.05 (1d,3d excluded). The EA BMSCs group's NSS score lower than that of BMSCs group at the same time point after transplantation, the difference was statistically significant, p<0.05 (1d excluded). The NSS score at the time lower than that of next time point in the same experimental group, it was statistically significant,p<0.05 The exogenous BMSCs transplanted into the ICH rat's Caudate-Putamen nucleus are visible in the corpus callosum, striatum, dorsal thalamus and the cerebral cortex, and the differentiation nerve cells and the astrocytes were discovered in these places. Conclusion:Electroacupuncture combined with BMSCs transplantation can improve neurological deficits of the ICH SD rats more than those of a simple of BMSCs transplantation. The mechanism may be that Electroacupuncture combined with exogenous BMSCs contributes differentiating to the nerve cells, astrocyte cells which replace the original nerve cells of necrosis and change the cytoarchitecture of the brain.
引文
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