骨髓基质细胞中Von Hippel-Lindau基因的siRNA表达载体构建及其检测  

Construction and detection of gene expression vector of the Von Hippel-Lindau in bone marrow stromal cells

作　　者：邹多宏[1,2] 赵君[3] 夏伦果[3] 张秀丽[4] 蒋欣泉[4] 黄远亮[2] 

机构地区：[1]同济大学口腔医学院,上海200072 [2]同济大学附属东方医院口腔科,上海200120 [3]上海交通大学医学院附属第九人民医院.口腔医学院口腔颌面外科,上海200011 [4]上海交通大学医学院附属第九人民医院.口腔医学院口腔组织工程实验室,上海200011

出　　处：《上海口腔医学》2010年第4期403-409,共7页Shanghai Journal of Stomatology

基　　金：上海市科学技术委员会资助项目(9411954800);上海市口腔医学重点实验室开放基金(S30206)~~

摘　　要：目的:构建骨髓基质细胞(bone marrow stromal cells BMSCs)中Von Hippel-Lindau(VHL)基因的siRNA表达载体并检测其对BMSCs细胞中VHL基因的干扰作用。方法:根据犬的VHL基因序列设计并合成4对siRNA oligo,然后用载体构建试剂盒进行重组克隆,将4对双链的siRNA oligo分别插入到siRNA表达载体pcDNATM6.2-GW/EmGFPmiR中,构建4个siRNA表达质粒(SR144-1,SR144-2,SR144-3,SR144-4)。用载体通用引物进行菌落PCR筛选,筛选得到的阳性克隆进行测序,以验证重组克隆中插入片段序列是否与设计的oligo序列一致。干扰载体瞬时转染BMSCs,通过qPCR和Western印迹分别检测干扰载体对目的基因的沉默效果。为了提高转染效率及VHL基因的沉默效果,对SR144-4进行慢病毒干扰载体(pLenti-mi-VHL)构建。结果:通过对构建质粒克隆进行测序,证实重组克隆中插入片段序列与设计的oligo序列一致,转染BMSCs细胞24h和48h后分别用qPCR与Western印迹检测,显示SR144-4干扰效果最理想。通过测序结果比对,插入序列完全正确,pLenti-mi-VHL构建成功。结论:成功构建了BMSCs的siRNA-VHL表达载体,为进一步实验研究奠定了基础。PURPOSE： To construct siRNA-VHL expression vector and detect the effect of VHL gene interference on BMSCs.METHODS： According to the dog’s VHL gene sequences,four pairs of siRNA oligo were designed and synthesized.Using vector cloning kit reorganization,four pairs of double-stranded siRNA were inserted into the expression vector（pcDNATM 6.2-GW/EmGFPmiR） and 4 siRNA expression plasmids（SR144-1,SR144-2,SR144-3,SR144-4） were constructed.With the vector universal primers,colony PCR was screened.The positive clones were sequenced to verify whether the sequence of insert fragments in recombinant clones was consistent with oligo sequences designed or not.Interference vector transiently transfected the BMSCs.qPCR and Western blot were used to detect the gene silencing effect.In order to improve transfection efficiency of siRNA-VHL as well as the effect of the VHL gene silencing,pLenti-mi-VHL was constructed.RESULTS： Through sequencing the plasmids cloned,the fragment sequences inserted in recombinant clones were consistent with the designed oligo sequences.After 24h and 48h transfection of BMSCs cells by plasmids,SR144-4 showed the best effect of interference by qPCR and Western blot.Through comparing the sequencingresults,the inserted fragment sequences were completely correct and the pLenti-mi-VHL was successfully constructed.CONCLUSION： The siRNA-VHL expression vector for BMSCs is successfully constructed and applicable for further experiments.

关 键 词：VonHippel-Lindau基因 骨髓基质细胞 SIRNA干扰 低氧诱导因子 

分 类 号：R341[医药卫生—基础医学]