基因修饰的骨髓基质细胞移植治疗帕金森病模型大鼠的研究

Study on transplantation of bone marrow derived stroma cells transfected by neurturin gene for treating rat model of Parkinson disease

目的研究转染neurturin基因（NTN）的骨髓基质细胞（BMSCs）移植治疗帕金森病模型大鼠，并探讨其治疗机制。方法将建模成功的帕金森病模型大鼠分为3组，将转染NTN基因的BMSCs（转基因组）、未转染NTN基因含空质粒的BMSCs（空质粒组）和生理盐水（生理盐水组）分别注入模型大鼠右侧的纹状体，在治疗后1～6个月分别观察大鼠阿朴吗啡诱导的旋转行为的变化。移植后第1个月分别进行大鼠脑冰冻切片荧光免疫组织化学鉴定[免疫荧光检测绿色荧光蛋白（GFP）分别与NTN（GFP／NTN）、胶质纤维酸性蛋白（GFAP）（GFP／GFAP）、神经元特异性烯醇化酶（NSE）（GFP／NSE）和酪氨酸羟化酶（TH）（GFP／TH）双标细胞]及检测1～6个月脑切片免疫组织化学TH阳性细胞并计数。原位杂交和蛋白印记法分别检测NTN在移植大鼠脑中mRNA和蛋白的表达。结果细胞移植后第1、2、3个月，转基因组大鼠的旋转次数[分别为（12．9±4．7）r／min、（9．4±3．3）r／min和（8．0±3．4）r／min]少于空质粒组[分别为（14．1±4．3）r／min、（12．5±4．8）r／min和（10．6±3．7）r／min]，而这两组在1～6个月的旋转次数均少与生理盐水组[分别为（15．1±4．2）r／min、（14．5±3．6）r／min、（13．8±3．7）r／min、（13．1±3．0）r／min、（12．9±2．8）r／min和（12．6±3．1）r／min3（P〈0．05）；在移植后第1个月转基因组大鼠移植区可见GFP／NTN、GFP／GFAP、6FP／NSE双标的细胞，未发现GFP／TH双标的细胞。结论转基因组治疗帕金森病模型大鼠的总体疗效好于空质粒组和生理盐水组。

Objective Study on the therapeutic effect of transplantation of bone marrow derived stroma cells（BMSCs） transfected with neurtuin（NTN） gene into Parkinson disease （PD） rat model. Methods BMSCs transfected by the adenoviral vector with NTN gene were transplanted into striatum of PD rat models. Those animals who showed more than 7 full body turns/min opposite side to the lesion were selected and allocated randomly into 3 groups. BMSCs with NTN gene （6 × 10^5 cells/each model）, BMSCs with mock gene （6 × 10^5 cells/each model） and physiological saline （12 μl） were injected into the right striatum of PD rat respectively. The rotation test was held from the first month to the sixth month after transplantation. Immunofluorescence double staining （GFP/NSE, GFP/GFAP, GFP/NTN and GFP/TH） were carried out one month after BMSCs transplantation. Meanwhile, immunohistochemical staining of the TH-immunoreactive（TH-IR） neurons and counting the number of TH-IR neurons were done from the first month to the sixth month after transplantation. NTN mRNA and protein expression were detected by insitu hybridization and Western blot. Results BMSCs with transfected NTN gene had better results of the treatment on PDmodel [（12.9＋4.7） r/min, （9.4＋3.3）r/min, （8.0＋3.4）r/min, respectively] than that of BMSCs without NTNgene 〈（14. 1＋4.3）r/min, （12.5＋4.8）r/min, （10.6＋3.7）r/min]during the first three months. BMSCs with transfected NTN gene and BMSCs with mock vector had better effect than that of physiological saline gruop during the six months[ （15.1＋ 4.2）r/min, （14. 5 ＋ 3.6）r/min,（13.8＋3.7）r/min, （13.1＋3.0）r/min,（12.9＋2.8）r/min, （12.6＋3.1 r/min） （all P〈0.05）]. Immunofluorescence double staining cells of GFP/NSE, GFP/GFAP and GFP/NTN were found in the striatum of PD rat alter one month of BMSCs transplantation. However, no immunofluorescence double staining cell of GFP/TH was found. NTN mRNA and protein were detected in the area of transplantation by insitu hybridization and Western blot. Conclusions BMSCs transplantation with transfected NTN gene has better therapeutic effect than that of BMSCs without NTN gene and physiological saline group. NTN might have a therapeutic value for the treatment of PD.