CONSTRUCTION OF RECOMBINANT HPAKP RETROVIRUS VECTOR AND ITS EXPRESSION IN NEWBORN MOUSE’S BRAIN AFTER HYPOXIA ISCHEMIA DAMAGE

Bo T., Mao J., Han YK

Department of Paediatrics, the Second Clinical Hospital, China Medical University, Shenyang, the People’s Republic of China

Objective: To explore the possibility of using retrovirus vector (RV) as vector on the gene therapy of newborn hypoxic ischemic brain damage (HIBD) and the expression properties of target gene in the brains after hypoxia ischemia (HI).

Methods: We combinated human placenta alkaline phosphatase (hpAKP) cDNA being obtained from plasmid DAP into RV pLXSN and transferred recombinant plasmid into packed cell line PT67 by liposome entrapment method. We replicated the newborn HIBD by ligation of the bilateral uteral arteries in pregnant full-term rats. Then we injected the high titer recombinant hpAKP-RV into the 1-day-old normal or HIBD mouse brain, then determined the expression on hpAKP in their brains by RT-PCR, hybridation in situ and enzyme histological chemistry method as well as the type of expression cells by the immunological histological chemistry method.

Results: In two groups, the infected cells lied in the Ⅱ-Ⅴ stratum of cerebral cortex, the nuclei of medulla, the Purkenje cell stratum of cerebellum, the vascular wall, and cerebral mater. By semi-quantitive RV-PCR analysis, the area of positive cells increased from the 1^st to the 7^th day after injection, and was less in the 28^th day than in the 7^th day after injection. But at the different time point, the hpAKP mRNA expression was lower in HIBD group than in normal group. Part of transfer positive cells. It was not found that RV was harmful to nervous system.

Conclusions: RV can transfer the target gene into the neurocytes and target gene can be expressed in normal newborn mouse’s as well as in newborn mouse’s brain damaged by HI. Target gene can expresses normally, but the express density in latter is lower than it in former,

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