人单纯疱疹病毒PDGF-BB转基因重组体的构建、转染及活性研究  

Construction of human herpes simplex virus carried PDGF-BB gene vector and its bioactivity evaluation

作　　者：陈晓雷 修光辉 李秀玲[2] 蒲倩 孙洁[1] 凌斌 CHEN Xiao-lei;XIU Guang-hui;LI Xiu-ling(Department of Intensive Care Unit, The No.4 Hospital Affiliated to Kunming Medical University(The Second People's Hospital of Yunnan Province), Kunming 650021, China)

机构地区：[1]云南大学附属医院(云南省第二人民医院,昆明医科大学第四附属医院)重症医学科,昆明650021 [2]云南省第一人民医院产科

出　　处：《临床神经外科杂志》2020年第6期634-637,644,共5页Journal of Clinical Neurosurgery

基　　金：国家自然科学基金面上项目(81901950);云南省科技厅-昆明医科大学联合专项(2015FB079);云南省基础研究计划项目(2019FB099);云南省高层次卫生计生技术人才培养项目(H-2017060)。

摘　　要：目的研究构建人单纯疱疹病毒(HSV)血小板源性生长因子-BB(PDGF-BB)转基因重组体(HSV-PDGF-BB),转染大鼠脑星型胶质细胞后其表达水平及生物活性。方法采用聚合酶链式反应合成大鼠PDGF-BB基因,克隆并构建HSV-PDGF-BB重组体,构建成功后转染大鼠脑星型胶质细胞,并进行筛选鉴定。用免疫组化、蛋白印迹反应检测星型胶质细胞中PDGF-BB的定位及表达水平,并观察转染后星型胶质细胞的增殖能力。结果成功构建了HSV-PDGF-BB重组体,转染后星形胶质细胞的PDGF-BB表达水平明显上调(P<0.05),且明显促进星形胶质细胞的增殖(P<0.05)。结论构建的HSV-PDGF-BB重组体能有效转染大鼠脑星型胶质细胞,并促进PDGF-BB的表达,增强转染后细胞的增殖能力。Objective To construct the recombinant vector of herpes simplex virus(HSV)carried platelet derived growth factor-BB(PDGF-BB)gene,detect the expression level and activity after transfectting astrocytes of rats.Methods Reverse transcription polymerase chain reactions synthesized and cloned PDGF-BB gene from rats,constructted the recombinant vector of HSV carried PDGF-BB(HSV-PDGF-BB)gene.Then the rat brain astrocytes were transfected.Successfully transfected cells would be chose and identified.The PDGF-BB level and subcellular localization were detected by western blotting and immunohistochemistry test.The proliferation of astrocytes after transfection was observed by cell counting kit-8 assay.Results The recombinant vector of HSV-PDGF-BB gene was successfully built.PDGF-BB level and proliferational activity of astrocytes were increased significantly in PDGF-BB transfectted group(P<0.05).Conclusions The recombinant vector of HSV-PDGF-BB gene was successfully constructed.It could effectively transfect rat astrocytes,then over-expressing PDGF-BB,and increased the ability of the proliferation of astrocytes.

关 键 词：血小板源性生长因子-BB 人单纯疱疹病毒 重组载体 星型胶质细胞 细胞增殖 

分 类 号：Q784[生物学—分子生物学] R739.41[医药卫生—肿瘤]