TH基因修饰细胞脑内移植治疗猴帕金森病的实验研究

Intracerebral transplantation of TH-transgene decorated cell in treating monkeys with Parkinson's disease

目的 评价包囊化酪氨酸羟化酶 (tyrosinehydroylase ,TH)基因修饰的基因工程细胞脑内移植治疗帕金森病的疗效。方法 将pcDNA3/hTH质粒转染人神经母细胞瘤细胞系SY5Y细胞 ,筛选出阳性克隆 ,微包囊化处理后的含有TH基因修饰细胞植入PD猴模型脑内 ,观察其行为、CSF中DA含量的变化 ,用免疫组化法检查移植细胞的存活情况。结果 (1)pcDNA3 /hTH基因经亚克隆 ,提取纯化的质粒 ,经ECORI酶切后产生 1 9Kb和 5 5Kb的片段。转基因后的SY5Y细胞免疫细胞化学染色显示TH染色强阳性 ;(2 )移植后PD猴症状明显改善 ,脑脊液中DA合量升高 ;(3)SABC免疫组化发现移植区存在大量TH阳 /性细胞。结论 构建的TH基因工程细胞体外和体内均表达人类TH基因 ;微包囊化处理后的基因工程细胞在PD猴脑内存活并发挥治疗作用。

Objective Transfecting recombined plasmid with human TH gene to neuroblastoma cell to construct cell lines expressing TH gene. After being capsulated, they were transplanted into brains of monkey models. By efficient and stable expression,TH deficiency of substansia negro was correctd, and DA concentration of striatum increased to a therapeutic level.Methods Cloned E. Coli with pcDNA3/hTH plasmids were transfected into human neuroblastoma line SY5Y. Positive clones selected by G418 were cultured.TH-decorated cells dealt by microcapsulating were then transplanted into PD monkey models.Behavior and DA concentration in CSF were observed. Transplanted cells were also examined by immunohistochemistry to determine their living status.Results 1.Subcloned and purified plasmids with pcDNA3/hTH gene were resolved into fragments of 1.9Kb and 5.5Kb by ECORI enzyme. It was in accordance with anticipated result. so recombined plasmids were tested to be right. Immunohistochemical staining of post-transgeneic SY5Y cells showed strong positive. 2.Symptoms of post-transplanted monkeys improved dramatically. DA concentration in CSF also increased. 3.Large numbers of transplanted TH-stained positive gliocytes were found in transplanting areas by SABC immunohistochemistry.Conclusions (1)Human neuroblastoma SY5Y cells constructed by gene transfection can exspress human TH gene in vitro and post-transplantation. (2)Microcapsulated transgeneic cells can live well in monkey's brain and perform treating effect.