CAJ | 학술논문

目的：探究慢病毒介导RNAi沉默SGMS2基因的单克隆细胞系构建中最佳感染复数（ multiplicity of infection，MOI）及BSD基因筛选抗生素（ blasticidin）浓度。方法荧光标记小鼠SGMS2干扰阴性对照慢病毒并按照MOI值0、10、30、60、120（ TU number／cell）分别侵染INS-1空白细胞，培养72 h后使用荧光显微镜拍照并计算细胞的荧光比率（％）及死亡率（％），以确定最佳MOI值。小鼠胰岛素瘤INS-1空白细胞中加入0、1、2、3μg／mL blasticidin，第7天时采用MTT法检测细胞的死亡率，以确定细胞抗生素敏感浓度。使用SGMS2干扰阴性对照慢病毒及SGMS2干扰慢病毒（病毒滴度：1×108 TU／mL）按照最佳MOI值侵染细胞，并用blasticidin敏感浓度进行阳性细胞筛选，获得混合系细胞。当细胞的荧光率达90％时，进行单克隆稳转细胞系的构建。结果最佳MOI值为60，此时细胞的荧光率达100％，但细胞的死亡率＜0.5％，细胞保持原有的形态。当blasticidin敏感浓度为2μg／mL，此时空白细胞失去原有的贴壁性，全部死亡。 INS-1-SEMS2细胞第2次检测的Ct值28.21大于第1次检测的Ct值27.58，且siRNA的干扰效率为77.78％，siRNA成功表达，混合稳转细胞系构建成功。成功构建小鼠胰岛素瘤INS-1-SEMS2单克隆细胞系。结论慢病毒介导RNAi沉默基因SGMS2的单克隆细胞系构建成功。
목적：탐구만병독개도RNAi침묵SGMS2기인적단극륭세포계구건중최가감염복수（ multiplicity of infection，MOI）급BSD기인사선항생소（ blasticidin）농도。방법형광표기소서SGMS2간우음성대조만병독병안조MOI치0、10、30、60、120（ TU number／cell）분별침염INS-1공백세포，배양72 h후사용형광현미경박조병계산세포적형광비솔（％）급사망솔（％），이학정최가MOI치。소서이도소류INS-1공백세포중가입0、1、2、3μg／mL blasticidin，제7천시채용MTT법검측세포적사망솔，이학정세포항생소민감농도。사용SGMS2간우음성대조만병독급SGMS2간우만병독（병독적도：1×108 TU／mL）안조최가MOI치침염세포，병용blasticidin민감농도진행양성세포사선，획득혼합계세포。당세포적형광솔체90％시，진행단극륭은전세포계적구건。결과최가MOI치위60，차시세포적형광솔체100％，단세포적사망솔＜0.5％，세포보지원유적형태。당blasticidin민감농도위2μg／mL，차시공백세포실거원유적첩벽성，전부사망。 INS-1-SEMS2세포제2차검측적Ct치28.21대우제1차검측적Ct치27.58，차siRNA적간우효솔위77.78％，siRNA성공표체，혼합은전세포계구건성공。성공구건소서이도소류INS-1-SEMS2단극륭세포계。결론만병독개도RNAi침묵기인SGMS2적단극륭세포계구건성공。
Objective To optimize multiplicity of infection ( MOI) and antibiotics ( blasticidin) concentration selecting BSD gene in construction of monoclonal stable cell line by lentivirus vector-mediated RNA interence silenced gene SGMS2.Methods The INS-1 cells were transfected by fluorescence labeled negative control SGMS2-siRNA lentivirus at MOI of 0, 10, 30, 60 and 120 TU number/cell.The cells were photographed under fluorescent microscopy after 72 h cultivation, then fluorescence ratio and apoptosis rate were calculated to determine optimal MOI.The INS-1 cells were treated by blasticidin with different concentrations of 0, 1, 2, and 3 μg/mL, and the apoptosis rate was observed to acquire optimal concentration of antibiotics.The INS-1 cells were transfected by negative control SGMS2-siRNA lentivirus and SGMS2-siRNA lentivirus (virus titer:1 ×108TU/mL) at optimal MOI and positive-transfected cells were selected by blasticidin at optimal concentration, then mixed cell lines were acquired.The monoclonal cell line was constructed at fluorescence ratio of 90%.Results The optimal MOI was 60 with 100% fluorescence ratio, less than 0.5% apoptosis rate and keep original cellular morphology.The optimal concentration of blasticidin was 2 μg/mL with cell adherence disappear and all cells apoptosis.The Ct value of INS-1-SEMS2 cells detected at the second time was 28.21, which was greater than 27.58 at the first time.The interfering efficiency of siRNA was 77.78% which indicated a successful expression of siRNA and construction of monoclonal stable cell line ( INS-1-SEMS2 ).Conclusion The monoclonal stable cell line was successfully constructed by lentivirus vector-mediated RNA interence silenced gene SGMS2.