CAJ | 학술논문

目的体外将骨髓间充质干细胞(BMSCs)诱导分化为多巴胺能神经元,观察分化过程中细胞形态的改变,检测细胞膜特异性抗原的阳性表达率,并对诱导后细胞间所形成的连接进行形态和功能的检测;探讨体外诱导BMSCs为特殊组织学类型神经元的可行性.方法将大鼠BMSCs进行分组诱导:实验对照组Ⅰ(加入bFGF+GDNF诱导);实验组Ⅱ(加入bFGF+GDNF+WHI-P131 +Shh诱导);空白对照组Ⅲ(不加入诱导剂).观察细胞形态学改变;检测胞神经元特异性表面抗原阳性率及细胞上清中多巴胺含量;采用细胞免疫组化法显示突触后致密蛋白95(PSD-95)的表达,观察各组细胞是否有成熟突触结构的形成;利用荧光染料FM1-43进行突触小泡的染色,间接观察分化后各组细胞突触循环的效能.结果实验组Ⅱ的细胞被证实高表达多巴胺能神经元特异性表面抗原多巴胺转运蛋白(DAT)和酪氨酸羟化酶(TH),且于诱导后细胞培养上清液中检测到多巴胺的分泌.并且,BMSCs诱导为多巴胺能神经元样细胞后细胞突起数量、长度以及PSD-95的免疫组化阳性率和FM1-43染色后荧光密度均高于实验对照组I.结论 BMSCs诱导为多巴胺能神经元样细胞样细胞后,在神经元特异性表面标志物阳性率、诱导分化率及细胞存活率上无明显改变;在细胞突起数量、长度、多巴胺分泌量、多巴胺神经元特异性表面标志物(DAT、TH)阳性率以及突触功能指标(PSD-95阳性率、突触泡荧光密度)等方面高于未诱导为多巴胺能神经元样细胞组.
목적 체외장골수간충질간세포(BMSCs)유도분화위다파알능신경원,관찰분화과정중세포형태적개변,검측세포막특이성항원적양성표체솔,병대유도후세포간소형성적련접진행형태화공능적검측;탐토체외유도BMSCs위특수조직학류형신경원적가행성.방법 장대서BMSCs진행분조유도:실험대조조Ⅰ(가입bFGF+GDNF유도);실험조Ⅱ(가입bFGF+GDNF+WHI-P131 +Shh유도);공백대조조Ⅲ(불가입유도제).관찰세포형태학개변;검측포신경원특이성표면항원양성솔급세포상청중다파알함량;채용세포면역조화법현시돌촉후치밀단백95(PSD-95)적표체,관찰각조세포시부유성숙돌촉결구적형성;이용형광염료FM1-43진행돌촉소포적염색,간접관찰분화후각조세포돌촉순배적효능.결과 실험조Ⅱ적세포피증실고표체다파알능신경원특이성표면항원다파알전운단백(DAT)화락안산간화매(TH),차우유도후세포배양상청액중검측도다파알적분비.병차,BMSCs유도위다파알능신경원양세포후세포돌기수량、장도이급PSD-95적면역조화양성솔화FM1-43염색후형광밀도균고우실험대조조I.결론 BMSCs유도위다파알능신경원양세포양세포후,재신경원특이성표면표지물양성솔、유도분화솔급세포존활솔상무명현개변;재세포돌기수량、장도、다파알분비량、다파알신경원특이성표면표지물(DAT、TH)양성솔이급돌촉공능지표(PSD-95양성솔、돌촉포형광밀도)등방면고우미유도위다파알능신경원양세포조.
Objective Bone marrow mesenchymal stem cells (BMSCs) were induced to differentiate to the special histological types of neurons in vitro.The morphological change of cells and positive expression of specific antigen on membrane were studied,and the function of connection between the induced BMSCs was also detected.The feasibility of BMSCs differentiate to the special histological types of neurons was investigated.Methods BMSCs were divided into group Ⅰ (induced with bFGF+GDNF),group Ⅱ (induced with bFGF+GDNF+WHI-P131 +Shh),and control group (no revulsive).The morphologic change of cells was observed,and the positive rate of neuron specific surface antigen and the content of dopamine were detected.Formation of mature synaptic structure was detected by immunohistochemical assay of postsynaptic density protein 95 (PSD-95) expression,and synaptic loop was shown by FM1-43 stain synaptic vesicles.Results By immunohistochemical staining,the positive rates of dopamine transporter (DAT) and tyrosine hydroxylase (TH) in group Ⅱ were significantly higher than those in group Ⅰ,and dopamine can been detected in cell culture supematant of group Ⅱ.After BMSCs was induced into dopamine neuron-like cells,number and length of cell protrusions,positive rate of PSD-95 and fluorescence intensity of FM1-43 in group Ⅱ were significantly higher than those of group Ⅰ.Conclusions There were no significant change in positive rate of neuron-specific surface markers,rate of cell survival and differentiation rate after BMSCs differentiated to dopaminergic neuron-like cells.The number and length of cell protrusions,content of dopamine in cell culture supematant,positive rate of dopaminergic neuron-specific surface antigen (DAT and TH),synaptic function index (positive rate of PSD-95 and fluorescence intensity of synaptic loop) of group Ⅱ were all significantly higher than that of group Ⅰ.