| 特异性针对大鼠甲状旁腺激素受体1基因SiRNA表达载体的构建、鉴定及对高糖状态下INS-1细胞周期的影响 |
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| 引用本文： | 梁华晟,薛耀明,钟宇华. 特异性针对大鼠甲状旁腺激素受体1基因SiRNA表达载体的构建、鉴定及对高糖状态下INS-1细胞周期的影响[J]. 南方医科大学学报, 2010, 30(9): 2093 |
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| 作者姓名： | 梁华晟 薛耀明 钟宇华 |
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| 作者单位： | 1. 广西医科大学第九附属医院内分泌科,广西,北海,536000
2. 南方医科大学南方医院内分泌科,广东,广州,510515 |
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| 基金项目： | 广西科技厅自然科学基金，广西北海市科学研究与技术开发计划项目 |
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| 摘 要： | 目的 构建并筛选携带针对大鼠甲状旁腺激素受体1基因(PTH1R)的pSUPERretro-GFP/Neo干扰逆转录病毒载体以及鉴定及观测高糖下对INS-1细胞周期的影响.方法 根据Genbank筛选PTH1R三个阳性克隆及一个阴性克隆序列,定向克隆入逆转录病毒载体pSUPERretro-GFP/Neo,并用RT-PCR和测序鉴定,将其转染至INS-1细胞中,Westernblotting观察PTH1R表达,鉴定其转染效率,筛选出最佳抑制基因后,应用流式细胞仪检测25mmol/LD-葡萄糖处理INS-1细胞周期.结果 菌落RT-PCR及基因测序鉴定结果表明序列正确.并能成功转染到INS-1细胞株中,Westernblot筛选最佳抑制基因.细胞周期检测提示PTHIR沉默后抑制INS-1细胞从G0/G1期进入S期.结论 成功构建并筛选出特异性沉默大鼠甲状旁腺激素受体1基因的pSUPERretro-GFP/Neo干扰逆转录病毒载体,高糖状态下PTH1R表达可能为INS-1细胞自我保护的一种作用.
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| 关 键 词： | 甲状旁腺激素受体1基因 小干扰RNA 小发卡结构RNA 转染 RNA干扰 细胞周期 |
Construction and identification of siRNA recombinant expression vector targeting parathyroid hormone 1 receptor gene and its effect on the cell cycle of INS-1 cells |
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| LIANG Hua-sheng,XUE Yao-ming,ZHONG Yu-hua. Construction and identification of siRNA recombinant expression vector targeting parathyroid hormone 1 receptor gene and its effect on the cell cycle of INS-1 cells[J]. Journal of Southern Medical University, 2010, 30(9): 2093 |
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| Authors: | LIANG Hua-sheng XUE Yao-ming ZHONG Yu-hua |
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| Affiliation: | LIANGHua-sheng1,XUEYao-ming2,ZHONGYu-hua1 1Department of Endocrinology,Ninth Affiliated Hospital of Guangxi Medical University,Beihai 536000,China,2Department of Endocrinology,Nanfang Hospital,SouthernMedical University,Guangzhou510515 |
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| Abstract: | Objective To constructthe expressionvectorof siRNA targeting parathyroid hormone 1 receptor(PTH1R) gene and evaluate its effectonthe cell cycle of INS-1 cells.Methods The sequences of PTH1Rgene was retrievedfromGenbank,and4 pairs of oligonucleotides were synthesizedandinsertedinto pSUPERretro RNAi,whichwas identifiedby RT-PCRandsequence analysis.The vectors were thentransfectedinto INS-1 cells,inwhichthe expressionof PTH1Rwas observedby Westernblotting to evaluate the transfection efficiency.The cell cycle ... |
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| Keywords: | parathyroid hormone 1 receptor small interfering RNA small hairpin RNA gene transfection RNA interference cell cycle |
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