骨髓间充质干细胞移植对实验性大鼠缺血性脑梗死免疫炎性反应的影响研究

目的：探讨骨髓间充质干细胞移植对实验性大鼠缺血性脑梗死免疫炎性反应的影响，为脑梗死治疗提供方法。方法：①实验性大鼠缺血性脑梗死模型建立。选择2015年3月~2017年5月实验室进行实验的SD大鼠60例，采用改良Zealonga线栓法完成大鼠缺血性脑梗死模型建立。②干细胞的获取及培养。另取大鼠5只，利用DMEM培养基冲洗骨髓腔，收集骨髓细胞，采用贴壁法完成大鼠骨髓间充质干细胞分离制备。③模型大鼠处理。将建模成功后的大鼠60只，随机分为模型组、对照组和实验组。模型组建模后仅进行血管结扎，不插入线栓；对照组在病灶部位注入等体积PBS；实验组在病灶部位注入10 μl第三代骨髓间充质干细胞，测定三组大鼠免疫水平、炎性因子水平，评估骨髓间充质干细胞在实验性大鼠缺血性脑梗死大鼠中的应用效果。结果：①倒置显微镜下第3天第一代骨髓间充质干细胞体积增大，呈多角形或长梭形，细胞开始聚集，第3代细胞迅速增殖，排列具有一定的方向性，呈旋涡状生长，并且具有多层，界限不清；②荧光显微镜观察结果表明：获得细胞中99%被荧光标记，获得的细胞为骨髓间充质干细胞；③对照组与模型组治疗后1个月、2个月及3个月神经功能评分比较，差异无统计学意义（P>0.05）；实验组治疗后1个月、2个月及3个月神经功能评分，低于对照组与模型组（P<0.05）；④实验组治疗后3个月IL-6、IFN-γ水平，高于对照组（P<0.05）；实验组治疗后3个月IL-10及TGF-β水平，低于对照组（P<0.05）；⑤实验组大鼠治疗后1个月、2个月及3个月CD4⁺CD25⁺Tregs细胞占CD4⁺T细胞的百分比，高于对照组与模型组（P<0.05）。结论：移植骨髓间充质干细胞有助于调节大鼠免疫，降低炎症因子水平，能促进神经功能早期恢复。

Effect of bone marrow mesenchymal stem cells transplantation on immune inflammatory response in experimental ischemic cerebral infarction in rats

Objective:To investigate the effect of bone marrow mesenchymal stem cell transplantation on immunoinflammatory response in experimental cerebral infarction rats,and to provide a method for the treatment of cerebral infarction.Methods:①Experimental rat model of ischemic cerebral infarction was established.Sixty SD rats in the lab from March 2015 to May 2017 were selected to complete the ischemic cerebral infarction model established by modified Zea-longa suture method.②Acquisition and culture of stem cells.In addition,5 rats were used to wash the bone marrow cavity with DMEM medium to collect bone marrow cells and the rat bone marrow mesenchymal stem cells could be isolated and prepared by adherent method.③Treatment of model rats.After successful modeling,60 rats were randomly divided into model group,control group and experimental group.After modeling,the model group only performed vascular ligation and did not insert the line plug;the control group injected equal volume of PBS at the lesion site;the experimental group injected 10 μl of third generation bone marrow mesenchymal stem cells at the lesion site and measured the immune levels of the three groups of rats.To evaluate the effect of bone marrow mesenchymal stem cells in experimental rats with ischemic cerebral infarction.Results:①In the inverted microscope,the volume of the first generation of MSCs became polygonal or fusiform on the third day,and the cells started to aggregate.The third generation of cells proliferated rapidly and arranged in a certain direction with swirling growth,and had multiple layers,the boundaries are unclear;②Fluorescence microscopy showed that:99% of cells obtained by fluorescence labeling,the obtained cells were bone marrow mesenchymal stem cells;③Control group and model group 1 month after treatment,there was no significant difference in neurological score between 2 months and 3 months(P> 0.05).Neurological scores at 1,2 and 3 months after treatment in the observation group were lower than those in the control group and the model group(P<0.05)；④The levels of IL-6 and IFN-γ in the observation group were higher than those in the control group at 3 months after treatment(P<0.05).The levels of IL-10 and TGF-β in the observation group at 3 months after treatment were lower than those in the control group(P<0.05)；⑤The percentage of CD4⁺CD25⁺Tregs in CD4⁺ T cells at 1 month,2 months and 3 months after treatment in observation group was higher than that in control group and model group(P<0.05).Conclusion:Transplantation of bone marrow mesenchymal stem cells can help regulate rat immunity,reduce the level of inflammatory cytokines and promote the early recovery of neurological function.