CAJ | 학술논문

目的：探讨RNAi沉默NRP-1基因对人T细胞白血病细胞株增殖与凋亡的影响。方法：将我们前期构建好的pLB-NRP-1／shRNA重组慢病毒质粒，感染至Jurkat细胞中，用CCK-8法检测各组细胞的增殖情况及化疗药物表柔比星（ EPI）处理后细胞增殖情况；用AV／PI法结合流式细胞仪检测细胞凋亡、细胞周期。结果：细胞增殖结果：在48、72、96 h时间点NRP-1／shRNA干扰组的OD值均低于相应对照组，差异有统计学意义（P＜0.05），而阴性对照组与空白对照组差异无统计学意义（P＞0.05）；细胞凋亡率结果：与对照组比较，NRP-1／shRNA干扰组细胞的凋亡率明显升高，差异有统计学意义（P＜0.05）；对化疗药物表柔比星（ EPI）敏感性检测结果：EPI浓度为0.025、0.05、0.1、0.2、0.4μg／ml时， NRP-1／shRNA干扰组的细胞生长抑制率较相应对照组高，差异皆有统计学意义（ P＜0.05），并且选择IC50进行EPI诱导后，NRP-1／shRNA干扰组的细胞凋亡率明显升高，与对照组比较差异有统计学意义（ P＜0.05）；WB结果显示：与对照组相比，NRP-1／shRNA干扰组Bcl-2蛋白的表达水平明显下降， Bax的表达水平明显升高，差异均具有统计学意义（P＜0.05）；细胞周期结果：与对照组比较，NRP-1／shRNA干扰组G0／G1期细胞的比例增高，S期细胞的比例明显降低，差异均有统计学意义（ P＜0.05）。结论：RNAi沉默NRP-1基因可抑制Jurkat细胞增殖，促进其凋亡，提高对化疗药物的敏感性，其机制可能涉及Bcl-2／Bax途径的调节；并可将细胞周期阻滞在G0／G1期，降低细胞的增殖水平，诱导细胞进入凋亡期。
목적：탐토RNAi침묵NRP-1기인대인T세포백혈병세포주증식여조망적영향。방법：장아문전기구건호적pLB-NRP-1／shRNA중조만병독질립，감염지Jurkat세포중，용CCK-8법검측각조세포적증식정황급화료약물표유비성（ EPI）처리후세포증식정황；용AV／PI법결합류식세포의검측세포조망、세포주기。결과：세포증식결과：재48、72、96 h시간점NRP-1／shRNA간우조적OD치균저우상응대조조，차이유통계학의의（P＜0.05），이음성대조조여공백대조조차이무통계학의의（P＞0.05）；세포조망솔결과：여대조조비교，NRP-1／shRNA간우조세포적조망솔명현승고，차이유통계학의의（P＜0.05）；대화료약물표유비성（ EPI）민감성검측결과：EPI농도위0.025、0.05、0.1、0.2、0.4μg／ml시， NRP-1／shRNA간우조적세포생장억제솔교상응대조조고，차이개유통계학의의（ P＜0.05），병차선택IC50진행EPI유도후，NRP-1／shRNA간우조적세포조망솔명현승고，여대조조비교차이유통계학의의（ P＜0.05）；WB결과현시：여대조조상비，NRP-1／shRNA간우조Bcl-2단백적표체수평명현하강， Bax적표체수평명현승고，차이균구유통계학의의（P＜0.05）；세포주기결과：여대조조비교，NRP-1／shRNA간우조G0／G1기세포적비례증고，S기세포적비례명현강저，차이균유통계학의의（ P＜0.05）。결론：RNAi침묵NRP-1기인가억제Jurkat세포증식，촉진기조망，제고대화료약물적민감성，기궤제가능섭급Bcl-2／Bax도경적조절；병가장세포주기조체재G0／G1기，강저세포적증식수평，유도세포진입조망기。
Objective:To investigate the effect on proliferation and apoptosis of T-cell leukemia cells by silencing NRP-1 ( Jurkat cells).Methods:The lentivirus plasmid which expresses NRP1 gene specific shRNA was constructed in our preliminary ex-perimental.We transfected the lentivirus plasmid to human T-cell Lymphoma cells.The proliferation of Jurkat cells different groups and effect on cell proliferation after chemotherapy drug EPI-treated were found by CCK-8 kit.The proliferation level and apoptosis rate of the cells were detected by flow cytometry and Annexin-V-FITC/PI method.Results:The proliferation level of NRP-1 /shRNA interference group was decreased significantly in 48 h,72 h,96 h,which was compared with the control groups.The apoptosis rate of the NRP-1/shRNA interference group was increased compared with control groups.The chemotherapy drug sensitivity of epirubicin ( EPI ) test results showed that EPI concentration was 0.025,0.05,0.1,0.2,0.4 μg/ml,the NRP-1/shRNA interference group of cell growth inhibition rate was increased,the corresponding control group difference had statistical significance(P<0.05).We choose the drug con-centration of the EPI IC50 for next experiments.NRP-1/shRNA interference group cell apoptosis rate increased significantly after induction,compared with the control groups difference was statistically significant ( P<0.05 ).Compared with control group, the expression level of Bcl-2 protein was decreased and the expression level of bax protein was increased significantly after EPI induction.The percentage of cells at G0/G1 phase increased significantly,while those at S phase decreased significantly.Conclusion:Plasmid shRNA-NRP1 inhibited the expression of NRP1 in Jurkat cells and decreased the proliferation level of Jurkat cells and promote their apoptosis and enhance their drug sensitivity;the molecular mechanism may relate to down-regulation of Bcl-2 and up-regulation of Bax.and arrested the cell cycle at G0/G1 phase.