【摘要】 目的观察不同途径移植人羊膜间充质干细胞(human amniotic mesenchymal stem cells,h AMSCs)对帕金森病(Parkinson’s disease,PD)模型大鼠的生物学效应及其在体内的分化。方法采用胰蛋白酶-胶原酶消化法分离hAMSCs,流式细胞术分析表型。40只雌性Wistar大鼠随机分为假手术组、模型组、hAMSCs静脉移植组和原位移植组。采用单侧前脑内侧束(MFB)注射6-羟基多巴胺建立PD大鼠模型。通过舌下静脉或于MFB原位移植3×105个hAMSCs。腹腔注射阿朴吗啡诱导旋转观察大鼠的行为变化,免疫荧光染色法检测人细胞核抗原及神经元微管结合蛋白(microtubule-associated protein 2,MAP-2)的表达,免疫组化染色法检测酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达。结果与模型组比较,hAMSCs静脉移植组和原位移植组大鼠旋转次数均明显减少(均P<0.05),前者行为学改善可持续至移植后6 w,后者则至8 w;免疫荧光染色显示,hAMSCs在原位移植区可存活至少12 w,并表达MAP-2;免疫组化染色显示静脉和原位移植hAMSCs均可上调PD模型大鼠黑质TH表达,但后者强于前者。结论 hAMSCs能改善PD模型大鼠的运动行为,原位移植优于静脉移植,其机制可能与上调黑质TH表达有关。hAMSCs可在原位移植部位分化为多巴胺能神经元样细胞。更多还原

【Abstract】 Objective To compare the treatment effects of human amniotic mesenchymal stem cells( h AMSCs) in a rat model of Parkinson’s Disease( PD) by different transplantation approaches. Methods h AMSCs derived from human amnion were isolated by trypsin and collagenase digestion method. Immunohistochemical staining and FCM were employed to identify h AMSCs. Forty date-mated female Wistar rats were randomly divided into four groups,including sham operation group( n = 10),control group( n = 10),in situ transplantation group( n = 10) and sublingual vein transplantation group( n= 10). PD model was built by injection 6-OHDA into single medial forebrain bundle( MFB) position of hemisphere. 3 ×105cells were transplanted by sublingual vein and in situ respectively. Rotations were induced by apomorphine( APO,0. 5mg / kg) injection after cell transplantation weekly. Survival and differentiation of donor cells were investigated by immunofluorescence staining and immunohistochemistry staining were employed to detect the expression of human microtubule associated protein-2( MAP-2) and tyrosine hydroxylase( TH) respectively. Results Compared with model group,the rotation frequenciesy of rats in h AMSCs vein transplantation group and in situ transplantation group both were decreased significantly( all P < 0. 05). The former was sustainable to the 6th week,the latter lasted to the 8th weeks after transplantation. Survival of donor cells,and the expression of MAP-2 were found in brain tissue of PD rats at 2 weeks and 12 weeks after in suit transplantation. Moreover,compared with control group that expression of TH was negative,which the expression lever of TH in h AMSCs in situ transplantation group and sublingual vein transplantation group was positive,but the former was stronger.Conclusion h AMSCs can improve the movement behavior of PD model rats,and in situ transplantation is superior to the vein transplantation. Its mechanism may be related to raise TH expression in the substantia nigra. Donor cells can differentiate into dopaminergic-like neurons after in situ transplantation.更多还原