重组hpAKP逆转录病毒载体的构建及其在新生小鼠脑内的表达

Construction of the Recombinant hpAKP Retrovirus Vector and its Expression in Newborn Mouse Brain

摘要:

目的： 探索逆转录病毒载体 (RV)作为新生儿神经系统疾病基因治疗载体的可行性及靶基因在其脑内表达特点。方法： 将从质粒DAP获得的报告基因人胎盘碱性磷酸酶 (hpAKP)克隆至RVpLXSN,用脂质体转染法导入包装细胞系PT67,获得高滴度的重组病毒。将其注射入生后1d的小鼠脑内,通过原位杂交、酶组化法,检测hpAKP在新生小鼠脑内的表达。通过免疫组化法,确定所转染细胞的种类。 结果： 注射后,新生鼠脑内hpAKP表达渐增高,但28d表达已较前减弱。RV转染的细胞位于皮层的Ⅱ～Ⅴ层锥体细胞、脑干神经核,小脑Purkenje细胞层,脑血管壁,脑膜及室管膜细胞等处。绝大多数细胞GFAP(+),少数GFAP(-)细胞也有转染,未见RV对神经系统的损害。结论： RV可高效广泛转染包括神经元在内的新生小鼠脑细胞,靶基因表达正常,RV适于作为新生动物神经系统疾病基因治疗的载体。

Abstract:

OBJECTIVE: To explore the possibility of using the retrovirus vector (RV) in the gene therapy of neonatal neuropathy and the expression properties of target genes in the brain. METHODS: We combined human placenta alkaline phosphatase (hpAKP) cDNA obtained from plasmid DAP with RV pLXSN and transferred recombinant plasmid into packed cell line PT67 by the liposome entrapment method. We injected high titer recombinant hpAKP RV into the 1 day old mouse brain, and then determined the hpAKP expression in the brain by hybridation in situ and the enzyme histological chemistry method as well as the type of expression cells by the immunological histological chemistry method. RESULTS: The infected cells lay in the Ⅱ Ⅴ stratum of the cerebral cortex, the nuclei of the brain stem, the Purkenje cell stratum of the cerebellum, the vascular wall, and the cerebral mater. The area of positive cells increased from the 1st to the 7th day after the injection, but was smaller on the 28th day than on the 7th day after the injection, part of those being GFAP(-). RV was not found harmful to the nervous system. CONCLUSIONS: RV can transfer target genes into the newborn mouse brain effectively, including neurons. Target genes express normally. It is likely that RV could be used in the gene therapy of neonatal neuropathy.