CAJ | 학술논문

目的：利用3种不同组织金属蛋白酶抑制剂-1(tissue inhibitors of metalloproteinase-1，TIMP-1) siRNA转染大鼠系膜细胞，建立高效、稳定的TIMP-1低表达系膜细胞模型，为肾疾病研究奠定实验基础。方法分别设计合成3条TIMP-1特异性siRNA序列，并以标记了绿色荧光基团6-羧基荧光素(6-carboxy-fluorescein，FAM)的无义siRNA作为对照，利用脂质体转染方法转染原代大鼠系膜细胞，收集转染后48 h的细胞，利用荧光显微镜观察绿色荧光，明确其转染成功率；同时用Trizol法分离提取系膜细胞的总RNA，利用Taqman探针方法检测试验组及对照组TIMP-1的mRNA表达水平。结果荧光显微镜观察，利用脂质体转染方法，siRNA转染效率可以达到90%以上；Taqman探针荧光定量PCR结果显示，3种不同序列的siRNA均有抑制TIMP-1表达的作用(P＜0.01)，且siTIMP1-1的效果最佳。结论本研究利用siRNA转染技术，成功建立了TIMP-1低表达系膜细胞模型。
목적：이용3충불동조직금속단백매억제제-1(tissue inhibitors of metalloproteinase-1，TIMP-1) siRNA전염대서계막세포，건립고효、은정적TIMP-1저표체계막세포모형，위신질병연구전정실험기출。방법분별설계합성3조TIMP-1특이성siRNA서렬，병이표기료록색형광기단6-최기형광소(6-carboxy-fluorescein，FAM)적무의siRNA작위대조，이용지질체전염방법전염원대대서계막세포，수집전염후48 h적세포，이용형광현미경관찰록색형광，명학기전염성공솔；동시용Trizol법분리제취계막세포적총RNA，이용Taqman탐침방법검측시험조급대조조TIMP-1적mRNA표체수평。결과형광현미경관찰，이용지질체전염방법，siRNA전염효솔가이체도90%이상；Taqman탐침형광정량PCR결과현시，3충불동서렬적siRNA균유억제TIMP-1표체적작용(P＜0.01)，차siTIMP1-1적효과최가。결론본연구이용siRNA전염기술，성공건립료TIMP-1저표체계막세포모형。
Objective To compound three different small interference RNAs specific for TIMP-1 and establish low-expression model of TIMP-1 in mesangial cells of rats, in order to provide experimental basis for renal diseases.MethodsThree different small interference RNA sequences that were specific for TIMP-1 were designed and compounded. The nonsense siRNA marked with greenfluorescent perssad (6-carboxy-fluorescein, FAM) was used as control group and the three siRNA groups were transfected into cultured mesangial cells in rats by liposome transfection method, then the transfected cells were collected at 48 hours after transfection to identify the transfection efficiency withfluorescent microscope. At the same time, the total RNA in mesangial cells was extracted with Trizol, the TIMP-1 mRNA expression level of all four groups was detected by Taqman probe technique.Results Thefluorescent microscope revealed that the transfection efficiency was over than 90% in nonsense siRNA group, while the PCR results demonstrated that TIMP-1 mRNA expression level in all three groups was lower than control group (P<0.01), which suggested that our three siRNAs had inhibited the expression of TIMP-1, and siTIMP1-1 had the best effect for it showed the lowest expression level.Conclusion Our study has successfully established the low-expression cell model of TIMP-1 in primary MCs with siRNA transfection technique.