CAJ | 학술논문

目的:通过RNA干扰(RNAi)技术选择性下调高血压相关基因SLC7a8的表达,探讨对大鼠肾小管上皮细胞(NRK-52E)摄取左旋多巴的影响.方法:设计并合成3个针对大鼠SLC7a8基因的特异性siRNA片段(siRNA-1、siRNA-2、siRNA-3),以含非特异性编码序列的siRNA-con为对照.将上述3个片段及阴性对照通过阳离子脂质体法转染至NRK-52E内,通过流式细胞仪估算转染效率后经RT-PCR初步筛选高效率干扰片段,用Western blotting检测蛋白水平的干扰效率,采用紫外分光光度法检测空白对照组(blank)、siRNA-con转染组和SLC7a8基因特异性沉默组不同时点(6、12、24、36、48、60、72、120 min) NRK-52E内左旋多巴的浓度.结果:流式细胞仪测定siRNA转染NRK-52E效率为94%,RT-PCR初步筛选结果显示siRNA-1、3干扰效率较高,Western blotting检测显示siRNA-3组在蛋白水平的干扰效率较高,而对照组siRNA-con与空白对照组(blank)的SLC7a8 表达差异不明显.通过紫外分光光度法检测NRK-52E对左旋多巴的吸收结果显示干扰组不同时点(6、12、24、36、48、60、72、120 min)细胞内左旋多巴浓度均低于siRNA-con转染组和空白对照组;对照组siRNA-con与空白对照组相比则无明显差异.结论:RNA干扰技术能选择性下调大鼠肾小管上皮细胞SLC7a8的表达水平;SLC7a8基因特异沉默后NRK-52E对左旋多巴的摄取在各个不同时点(6、12、24、36、48、60、72、120 min)均降低.
목적:통과RNA간우(RNAi)기술선택성하조고혈압상관기인SLC7a8적표체,탐토대대서신소관상피세포(NRK-52E)섭취좌선다파적영향.방법:설계병합성3개침대대서SLC7a8기인적특이성siRNA편단(siRNA-1、siRNA-2、siRNA-3),이함비특이성편마서렬적siRNA-con위대조.장상술3개편단급음성대조통과양리자지질체법전염지NRK-52E내,통과류식세포의고산전염효솔후경RT-PCR초보사선고효솔간우편단,용Western blotting검측단백수평적간우효솔,채용자외분광광도법검측공백대조조(blank)、siRNA-con전염조화SLC7a8기인특이성침묵조불동시점(6、12、24、36、48、60、72、120 min) NRK-52E내좌선다파적농도.결과:류식세포의측정siRNA전염NRK-52E효솔위94%,RT-PCR초보사선결과현시siRNA-1、3간우효솔교고,Western blotting검측현시siRNA-3조재단백수평적간우효솔교고,이대조조siRNA-con여공백대조조(blank)적SLC7a8 표체차이불명현.통과자외분광광도법검측NRK-52E대좌선다파적흡수결과현시간우조불동시점(6、12、24、36、48、60、72、120 min)세포내좌선다파농도균저우siRNA-con전염조화공백대조조;대조조siRNA-con여공백대조조상비칙무명현차이.결론:RNA간우기술능선택성하조대서신소관상피세포SLC7a8적표체수평;SLC7a8기인특이침묵후NRK-52E대좌선다파적섭취재각개불동시점(6、12、24、36、48、60、72、120 min)균강저.
AIM: To investigate the effect of selective silencing of SLC7a8 on uptaking L-dopa in renal tubular epithelial cells of rat (NRK-52E). METHODS: The three siRNAs targeting SLC7a8 (siRNA-1, siRNA-2, siRNA-3) were designed and synthesized. A siRNA with nonspecific coding sequence (siRNA-con) was used for control. All siRNAs were transfected into NRK-52E cells. The siRNA-con transfected group, blank control group and gene-specific silencing SLC7a8 group were set up. The efficiency of transfection was estimated by flow cytometry. The efficiency of RNA interference was detected and screened by RT-PCR preliminarily, and was followed by Western blotting at protein level. The concentrations of L-dopa uptake into the NRK-52E cells were detected by ultraviolet spectrophotometry at different time points (6, 12, 24, 36, 48, 60, 72 and 120 min). RESULTS: The transfection efficiency was 94% detected by flow cytometry. The initial screening of RT-PCR showed that the efficiencies of RNA interference of siRNA-1 and siRNA-3 were higher, and siRNA-3 was the highest at protein level determined by Western blotting. No distinctive change was found between siRNA-con treated NRK-52E and blank control cells. The L-dopa uptake at different time points (6, 12, 24, 36, 48, 60, 72 and 120min) in siRNA-interference group was lower than that in siRNA-con transfected group and blank control group. No significant difference of L-dopa uptake between siRNA-con group and blank control group was observed. CONCLUSION: RNA interference technology selectively down-regulates SLC7a8 expression in rat renal tubular epithelial cells. The L-dopa uptake is also decreased after specifically silencing the slc7a8 expression.