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Immortalized Rat Astrocyte Strain Genetically Modified by Rat Preprogalanin Gene

Journal of Huazhong University of Science and Technology(Medical Sciences) 2005年第2期25卷 144-146,197页

作者：安珂田玉科杨辉高峰王鹏 Department of Anesthesiology Tongji Hospital Tongji Medical College Huazhong Universityof Science and Technology Wuhan 430030 China

Summary: To construct an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene (IAST/GAL) and detect its galanin (GAL) expression and secretion, a cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was inserted into eukaryotic expression vector pcDNA3.1(+) by DNA recombinant technology, then the restriction enzyme digestion and DNA sequencing were carried out to evaluate the recombinant. The pcDNA3.1(+)-GAL and pcDNA3.1(+) construct were transfected into immortalized rat astrocyte strain (IAST) by lipofectamine and the population of cells which stably integrated the construct was selected with 600 μg/mL G418. Individual clones were screened and expanded into clonal cell strains. Detection of Neo gene was used to validate the success of the transfection. Immunocytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion level of GAL. The recombinant had been successfully constructed by restriction enzyme digestion and DNA sequencing. Detection of Neo gene showed that the pcDNA3.1(+)-GAL and pcDNA3.1(+) have been successfully transfected into IAST. After selection by using G418, IAST/GAL and IAST/Neo cell strains were obtained. IAST/GAL, IAST/Neo and IAST were immunostained positively for GAL, but the GAL average optical density of IAST/GAL was significantly higher than that of IAST/Neo and IAST (P0.05). It was concluded that IAST/GAL strain was constructed successfully and it might provide a basis for the further study of pain therapy.

关键词： preprogalanin astrocyte immortalized cell strain biologic minipumps

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