构建大鼠前甘丙肽原基因修饰永生化星形胶质细胞株对镇痛研究的意义

Construction of immortalized rat astrocyte strain genetically modified by rat preprogalanin gene for the analgesia research

目的:转基因细胞移植镇痛是慢性疼痛治疗的新方向,构建大鼠前甘丙肽原基因修饰的永生化大鼠星形胶质细胞株,为转甘丙肽基因细胞移植镇痛的研究提供理论依据。方法:实验于2003-05/2004-10在华中科技大学同济医学院附属同济医院麻醉学研究室完成。采用DNA重组技术将质粒pBSKS(+)-前甘丙肽原中的大鼠前甘丙肽原基因克隆至真核表达载体pcDNA3.1(+)上,经酶切鉴定和测序分析后,采用脂质体介导法将重组质粒pcD-NA3.1(+)-前甘丙肽原和空质粒pcDNA3.1(+)分别转染永生化大鼠星形胶质细胞株,经G418(600mg/L)筛选,挑选阳性克隆并扩大培养。采用免疫细胞化学法、反转录-聚合酶链反应和放射免疫法检测转染细胞大鼠甘丙肽的表达及分泌,同时检测Neo基因的存在以证实转染成功。结果:重组质粒经酶切后分别出现5.4kb和521bp片段,测序分析与文献报道结果一致,表明重组质粒pcDNA3.1(+)-前甘丙肽原亚克隆成功。Neo基因检测显示重组质粒和空质粒已成功转染到星形胶质细胞株中,筛选获得星形胶质细胞株/前甘丙肽原和星形胶质细胞株/Neo两种细胞株。星形胶质细胞株,星形胶质细胞株/前甘丙肽原和星形胶质细胞株/Neo的前甘丙肽原免疫染色均阳性,平均光密度分析结果显示星形胶质细胞株/前甘丙肽原的前甘丙肽原表达明显高于星形胶质细?

AIM:Transgenic cell transplantation for analgesia is a new direction to treat chronic pain.This paper aims to construct an immortalized rat astrocyte strain(I AST) genetically modified by rat preprogalanin(GAL) gene in order to provide the oretical bases for analgesia by GAL genetic transplantation. METHODS:The experiment was completed in the Institute of Anesthesiology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology f rom May 2003 to October 2004.A cDNA fragment of rat GAL in plasmid of pBS KS(+) GAL was cloned into eukaryotic expression vector pcDNA3.1(+) by DNA recombina nt technology,and the restriction enzyme digestion and DNA sequencing were carri ed out to evaluate this recombinant plasmid.The pcDNA3.1(+) GAL and pcDNA3.1( +)construction were transfected into IAST by standard lipofectamine techniques respectively and the population of cells which stably integrated was selected wi th 600 mg/L G418.Then,individual clone was screened and expanded into clonal cel l strain.Immuncytochemical staining,RT PCR and radioimmunoassay were used to de tect the expression and secretion of GAL in the transfected rats.Detection of Ne o gene was used to validate successful transfection. RESULTS:After yhe recombinant plasmid constructed by restriction enzyme digest ion and DNA sequencing,5.4 kb and 521 bp fragments were found,and their sequenci ng analysis accorded with that reported in related literatures,indicating that r ecombinant plasmid pcDNA3.1(+) GAL was subcloned successfully.Detection of Neo gene showed that the pcDNA3.1(+) GAL and pcDNA3.1(+) have been transfected i nto IAST successfully.After selected by G418,IAST/GAL and IAST/Neo cell strains were obtained.IAST/GAL,IAST/Neo and IAST were immunohistochemically stained posi tive for GAL,but the GAL average absorbance value of IAST/GAL was significantly higher than that of IAST/Neo and IAST(P< 0.001).The level of GAL mRNA expression and the supernatant concentration of GAL secretion in cultured IAST/GAL were hi gher significantly than those of IAST and IAST/Neo(P< 0.001),but there was no si gnificant differences between the IAST and IAST/Neo(P >0.05). CONCLUSION:An immortalized rat astrocyte line modified genetically by rat prep rogalanin gene(IAST/GAL) is constructed successfully,which provides a research b asis for transgenic cell transplantation for analgesia.