成人外周血多能干细胞对帕金森病大鼠的治疗作用

The therapeutic effects of adult human peripheral blood derived multipotent stem cells on rat models of Parkinson disease

目的研究成人外周血胰岛素释放细胞（PB-IPC）对帕金森病大鼠的治疗作用。方法分离、培养PB—IPC8d后，观察干细胞的形态，RT—PCR检测PB—IPC分化相关的基因表达。将Brdu标记的PB-IPC在脑立体定位仪下注射到帕金森病大鼠的黑质致密部和纹状体。观察大鼠旋转行为学的变化，免疫荧光双标法检测PB-IPC酪氨酸羟化酶、多巴胺转运体表达情况。结果（1）PB—IPC表现出类似脐带血多能干细胞的形态和生长特点：圆形或椭圆形，贴壁生长、扩增；（2）PB-IPC表达胚胎干细胞多向分化潜能相关的转录因子c-Myc、Klfd、Nanog、Sox2，以及促神经元分化的转录因子Ngn1、Ngn2和Mashl；（3）术后第2、4、6、8周计算PB．IPC移植组帕金森病大鼠在阿扑吗啡诱导后30min内的旋转圈数，较术前分别减少了（26±2）％、（34±4）％、（39±7）％和（37±5）％，而对照组无明显改变，各观察点结果同对照组相比差异均有统计学意义（P〈0．05）；（4）免疫荧光显示大部分移植的PB—IPC的酪氨酸羟化酶、多巴胺转运体染色阳性。结论PB—IPC具有类似胚胎干细胞的自我更新和多向分化潜能，将其移植入帕金森病大鼠，能发挥治疗作用。

Objective To study the therapeutic effects of peripheral blood insulin-producing cells （PB-IPC） on rat model of Parkinson＇s disease （PD）. Methods PB-IPC,isolated and cultured from healthy adult human donors, were quantified mRNA expression for genes associated with plasticity of stem cell and neurogenesis through Quantitative real-time PCR test. After labeled by bromodeoxyuridine （Brdu）, PB-IPC were injected into the right of the substantia nigra compact （SNC） and candate putamen （CPU） of PD rats by using the stereotaxic instrument. The behavioral changes of the rats were observed after transplantation; brain frozen sections were carried out immunofluorescene through double staining. Results （1） We observed that PB-IPC displayed morphological and growing characteristics similar with CB-SCs （Cord blood-derived multipotent stem cells） ： round or oval and adhering to the bottom of Petri dish tightly; （2） It has been established that PB-IPC make an apparent expression of Sox2, Nanog, c-Myc, Klf4, Ngnl, Ngn2 and Mashl;（3 ）The rotation scores of PD rats in the Transplantation group by the apomorphine irritation were reduced consistently after transplantation （26± 2） % at the 2th week, （34 ± 4） % at the 4th week, （39 ±7 ） % at the 6th week, （ 37 ± 5 ） % at the 8th week; on the contrary ,the rotation scores of PD rats in the Control group kept stable at these observational points, which showed a significant statistical differences between Transplantation and Control group （ P 〈 0. 05 ） ; （4） Histochemical analyses showed that most of grafted cells made a positive expression of special markers of dopamine neuron in the treated rats： tyrosine hydroxylase and dopamine transporter. Conclusions These findings demonstrated that PB-IPC shared with embryonic stem cells （ESCs） some characteristics including selfrenewal and multidifferentiation potential. Transplantation of PB-IPC could make an significant therapeutic effect on PD rats.