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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 To evaluate the therapeutic effect of intracerebral transplantation of genetically engineered cells modified with tyrosine hydroxylase (TH) gene in the treatment of Parkinson’s disease. Methods The pcDNA3 / hTH plasmid was transfected into human neuroblastoma cell line SY5Y and the positive clones were screened. The microencapsulated TH gene-modified cells were implanted into the brain of the model of PD monkeys to observe its behavior, Content changes, using immunohistochemistry to check the survival of transplanted cells. Results (1) The pcDNA3 / hTH gene was subcloned and the purified plasmid was extracted and digested with ECORI to generate fragments of 1 9Kb and 5 5Kb. Immunocytochemical staining of SY5Y cells after transgene showed that TH staining was strongly positive; (2) The symptoms of PD monkeys were significantly improved after transplantation, and the amount of DA in cerebrospinal fluid increased; (3) SABC immunohistochemistry showed a large number of TH positive / cell. Conclusion The constructed TH gene engineering cells expressed human TH gene both in vitro and in vivo. The microencapsulated genetically engineered cells survived in the brain of PD monkey and played a therapeutic role.