自分化法培养大鼠骨髓间充质干细胞移植治疗大鼠帕金森病的研究

Therapy on rat hemiparkinsonian models by transplantation of the self-differentiation rat bone marrow mesenchymal stem cells

目的观察自分化法培养的大鼠骨髓间充质干细胞(SDrBMSCs)向大鼠纹状体内移植大鼠帕金森病(PD)模型的治疗作用。方法 SDrBMSCs以不换液、不外加任何诱导物质培养,用免疫细胞化学染色鉴定SDrBMSCs及用普通法培养大鼠骨髓间充质干细胞(rBMSCs)的分化情况,制作大鼠PD模型,设SDrBMSCs移植组、rBMSCs移植组(PD大鼠12只/组),向PD大鼠纹状体内植入SDrBMSCs或rBMSCs(1×106个/只),以免疫荧光组织化学染色鉴定其在纹状体部细胞体内存活迁移能力,高效液相色谱电化学方法(HPLC)检测纹状体内多巴胺(DA)含量,同时设PBS模型对照组(PD大鼠12只/组)做比较。结果 SDrBMSCs培养至第4代21 d移植入PD大鼠纹状体内,在无外源干预的情况下,表达nestin、β-tublinⅢ、DCX和MAP2,而同样条件移植入rBMSCs的PD大鼠仅表达少量的nestin;SDrBMSCs或rBMSCs移植入大鼠纹状体内后均可在体内存活、迁移并与周围组织整合。SDrBMSCs移植组PD大鼠在移植后28 d能表达GFAP,并有分化为TH阳性神经元的现象,使纹状体内DA含量提高,但与移植后14 d比较无明显变化(P>0.05);PD大鼠的旋转行为以移植后56 d时改善最为明显(P<0.05),旋转降至移植前的55.02%。rBMSCs移植组PD大鼠在移植后28 d可表达GFAP,也有改善PD大鼠旋转行为的效果(P<0.05),但纹状体内DA含量明显低于植入SDrBMSCs的效果(P<0.05)。结论 SDrBMSCs脑内移植后主要分化为星形胶质细胞,极少分化为TH阳性神经元,与rBMSCs相比对PD大鼠模型有明显治疗作用。

Objective To explore the therapeutic role of self-differentiation rat bone marrow mesenchymal stem cells （SDrMSCs） for Parkinson＇s disease （PD） by transplanting SDrMSCs into the striatum of rat hemiparkinsonian models. Methods The SDrMSCs were first cultured without changing culture medium andadding inducing substances, and then were identified by immunocytochemistry. Next, rat bone marrow mesenchymal cells （rBMSCs） were cultured and self-differentiated. Two experimental groups were created： SDrMSCs transplantation and＇ rBMSCs transplantation （12 PD models per group）. Either SDrMSCs or rBMSCs was transplanted into the striatum in each rat model （1×10^6 per rat）. After grafting,im- munofluorescent staining was used in the striatum to determine the cells＇ ability of survival and migration. High performance liquid chromatography （HPLC） coupled with electrochemical detection were employed next to determine the concentration of dopamine at the striatal level. These experimental values were then compared with the those of the rBMSCs cultured by basal medium and the PBS control group （ 12 PD models per group）. Results After self-differentiating for 21 days until the fourth generation,SDrBMSCs were transplanted into the striatum of PD rat models. In the absence of exogenous interference, the SDrBMSCs expressed nestin ,β-tublin IU, DCX and MAP2 ;under the same condition,the rBMSCs could only express nestin. It was clearly seen that the grafted ceils survived very well in vivo and migrated extensively in the host striatum. At 28 d, SDrBMSCs began to express GFAP in the striatum. Among these cells, few of the SDrBMSCs were converted into TH-positive ceils. The DA concentration at the striatal level increased in the PD rats that received grafting of SDrBMSCs, but exhibited no significant？ differences with the DA concentration at 14 d （P 〉 0. 05）. Moreover, those animals grafted with SDrBMSCs were shown to improve rotational behavior significantly at 56 d （P 〈0. 05 ）, as manifested by a decrease in asymmetric rotation to 55.02%. The rBMSCs could express GFAP in the striatum at 28d and could also improve rotational behavior （P 〈 0. 05 ）, but the DA concentration at the striatal levels was lower than that with the grafting of SDrBMSCs （ P 〈 0. 05 ）. Conclusion SDrBMSCs grafted in the striatum of PD rat models differentiate into astrocytes ,with only few were converted into TH- positive cells. Compared with rBMSCs, the SDrMSCs had significant therapeutic effects.