含NURR1基因腺病毒修饰神经干细胞移植治疗帕金森病的试验

Transplantation of neural stem cell modified by adenovirus with NURR1 gene in the treatment of Parkinson disease

目的:观察含NURR1基因腺病毒修饰的C17.2神经干细胞移植治疗对帕金森病模型症状和组织学的改善。方法:实验于2004-03/2005-01在中山大学附属第二医院林百欣实验中心完成。6-羟基多巴脑内立体定向及单侧纹状体两点定位注射制作SD大鼠帕金森病模型,1周后,行阿朴吗啡行为学检测,即阿朴吗啡(0.5mg/kg)腹腔内注射后30min内,计数大鼠平均每分钟身体旋转360°的次数,达到6圈/min及以上者为合格帕金森病模型,每周测1次,连续4周,获取稳定动物模型共31只,分为帕金森病模型对照组10只、帕金森病+C17.2组11只、帕金森病+C17.2+腺病毒组10只,无菌收集所需的C17.2神经干细胞、含NURR1基因腺病毒载体修饰C17.2神经干细胞,浓度为(1.0～4.0)×109L-1细胞,对入组的帕金森病模型进行无菌细胞注射移植,注射点与造模时注射点相同,每点约3μL,帕金森病模型对照组注射生理盐水。细胞移植后10d和4周进行阿朴吗啡诱发的帕金森病模型旋转试验,免疫组化检测帕金森病模型纹状体内酪氨酸羟化酶阳性神经元数量。结果:在实验过程中又有4只死亡,共有27只帕金森病模型鼠进入试验。①帕金森病模型旋转圈数:移植后10d和4周帕金森病+C17.2组和帕金森病+C17.2+腺病毒组均少于帕金森病模型对照组,帕金森病+C17.2+腺病毒组少于帕金森病+C17.2组(移植后10d:帕金森病模型对照组21.57±5.26,帕金森病+C17.2组3.90±0.90,帕金森病+C17.2+腺病毒组1.56±0.58;移植后4周:帕金森病模型对照组18.40±5.84,帕金森病+C17.2组3.60±0.67,帕金森病+C17.2+腺病毒组1.00±0.55,P<0.05)。②纹状体内酪氨酸羟化酶阳性细胞数:移植后10d帕金森病+C17.2组和帕金森病+C17.2+腺病毒组多于帕金森病模型对照组,帕金森病+C17.2+腺病毒组多于帕金森病+C17.2组(帕金森病模型对照组3.50±0.67,帕金森病+C17.2组13.17±1.89,帕金森病+C17.2+腺病毒组20.50±1.67,P<0.05)。结论:NURR1基因结合神经干细胞有效改善了帕金森病模型症状,提高移植后酪氨酸羟化酶阳性神经元细胞的数量。

AIM： To observe symptom and histologic amelioration of Parkinson disease models transplanted by C17.2 neural stem ceils （NSC） modified by adenovirus with NURR1 （Ad-NURR1） gene. METHODS： The experiment was performed at the Linbaixin Experimental Center, Second Affiliated Hospital, Sun Yat-sen University from March 2004 to January 2005. SD rat Parkinson disease models were induced by 6-hydroxyl-dopamine in two points injection of half-corpus striatum. One week later, emetomorphine praxiology examination was performed： emetomorphine （0.5 mg/kg） was treated by intraperitoneal injection before within 30 minutes mean cycles of body rotation 360° in a minute was counted. The successful PD models could rotate 6 cycles in a minute, tested once in a week for 4 weeks. Totally 31 stable animal models were obtained. Of them, there were 10 in Parkinson disease model control group, 11 in Parkinson disease plus C17.2 group and 10 in Parkinson disease plus C17.2 plus adenovirus group. C17.2 NSC, C17.2 NSC modified by Ad-NURR1 gene and （1.0-4.0）×10^9 L^-1 cells were collected sterilely. Parkinson disease models were transplanted with sterile cell injection. The injection point was the same as that during establishing models, about 3 μL. Parkinson disease model control group received saline. After cell transplantation for 10 days and 4 weeks, Parkinson disease model rotation trial induced by emetomorphine was performed. Number of tyrosine hydroxylase positive neurons in striatum of Parkinson disease models was measured with immunohistochemistry. RESULTS： Four rats died during the trial, totally 27 Parkinson disease model rats were involved in the trial. ①Cycles of rotation of Parkinson disease models： At day 10 and week 4 after transplantation, it was fewer in the Parkinson disease plus C17.2 group and Parkinson disease plus C17.2 plus adenovirus group than that in the Parkinson disease model control group; It was fewer in the Parkinson disease plus C17.2 plus adenovirus group than that in the Parkinson disease plus C17.2 group （10 days after transplantation： 21.57±5.26 in the Parkinson disease model control group, 3.90±0.90 in the Parkinson disease plus C17.2 group, 1.56±0.58 in the Parkinson disease plus C17,2 plus adenovirus group; 4 weeks after transplantation： 18.40±5,84 in the Parkinson disease model control group, 3.60±0.67 in the Parkinson disease plus C17.2 group, 1.00±0.55 in the Parkinson disease plus C17.2 plus adenovirus group,P 〈 0.05）. ②Number of tyrosine hydroxylase positive ceils in striatum： 10 days after transplantation it was more in the Parkinson disease plus C17.2 group and Parkinson disease plus C17.2 plus adenovirus group than that in the Parkinson disease model control group; It was more in the Parkinson disease plus C17.2 plus adenovirus group than that in the Parkinson disease plus C17.2 group （3.50±0.67 in the Parkinson disease model control group, 13.17±1.89 in the Parkinson disease plus C17.2 group, 20.50±1.67 in the Parkinson disease plus C17.2 plus adenovirus group,P 〈 0.05）. CONCLUSION： NURR1 gene combined with NSC can effectively ameliorate PD models symptoms and elevate the number of tyrosine hydroxylase positive neurons after transplantation.