沉默NOTCHI基因对多发性骨髓瘤RPMI 8226细胞生物学的影响  

Effects of silencing NOTCH1 gene by eukaryotic vector in multiple myeloma RPMI 8226 cell line

作　　者：黄晓璐 马旭东 鹿全意[2] HUANG Xiaolu;MA Xudong;LU Quanyi(Department of Hematology, Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou 363000, Fujian, China;Department of Hematology, Zhongshan Affiliated Hospital of Xiamen University, Xiamen 361000, Fujian , China)

机构地区：[1]福建医科大学附属漳州市医院血液科 [2]厦门大学附属中山院血液科

出　　处：《中国临床药理学与治疗学》2017年第9期978-983,共6页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：国家自然科学基金项目(81172246)

摘　　要：目的:探讨沉默NOTCH1基因对多发性骨髓瘤细胞株RPMI 8226增殖和凋亡的影响。方法:针对人NOTCH1基因序列构建NOTCH1 shRNA真核表达载体,用LipofectamineTM2000脂质体转染RPMI 8226细胞,筛选出最佳干扰表达载体。观察NOTCH1 shRNA表达载体对RPMI 8226细胞增殖的影响;使用流式细胞仪分析细胞的周期分布;Western blot法检测NOTCH1 shRNA转染RPMI 8226细胞后NOTCH1蛋白及细胞周期蛋白依赖性激酶抑制蛋白P27、P21的表达含量变化,ELISA法检测NOTCH1 shRNA转染RPMI 8226细胞后对IL-6分泌量的影响。结果:沉默NOTCH1基因抑制细胞增殖,阻滞RPMI 8226细胞于G0/G1期,转染组、阴性对照组及空白组增殖率分别为(49.09±2.31)%、(91.73±2.67)%、(98.10±0.41)%,差异具有统计学意义(P<0.05);三组的G0/G1期细胞分别为(66.23±3.71)%、(42.27±3.65)%、(40.70±0.92)%(P<0.05);三组S期细胞分别为(31.03±1.49)%、(56.53±1.76)%、(51.83±1.45)%(P<0.05)。NOTCH1 shRNA诱导细胞凋亡,三组凋亡率分别为(46.67±1.31)%、(1.37±0.63)%、(0.85±0.43)%(P<0.05);转染组NOTCH1蛋白表达下调,P21、P27表达上调。NOTCH1 shRNA可下调与疾病相关的IL-6分泌能力。三组IL-6分泌的吸光度值分别为28.18±3.50、167.08±7.97及185.86±5.61(P<0.05)。结论:NOTCH1 shRNA能抑制RPMI 8226细胞增殖并诱导凋亡,下调与多发性骨髓瘤发病有关的IL-6,有望成为多发性骨髓瘤治疗的一个新靶点。AIM： To discuss the impact of NOTCH1 shRNA on cell proliferation and apoptosis in multiple myeloma RPMI 8226 cell line. METHODS： Eukaryotic expression vector for human NOTCH1 gene （NOTCH1 shRNA） was synthesized, and eukaryotic expression vector was transfected into RPMI 8226 cells by Lipofectamine^TM2000. The influence of cell growth by NOTCH1 shRNA was determined by MTT. Cell apoptosis and cell-cycle dis- tribution were measured by Flow cytometry. Western blot was used for the detection of NOTCH1, P21 and P27. ELISA was used for detecting IL-6 secretion affected by NOTCH1 shRNA. RESULTS ： NOTCH1 shRNA inhibited cell growth through blocking cell cycle in Go/Gl phase. The NOTCH1 shRNA group＇s cell growth rate was （49.09 ±2.31 ）%, Neg-shR- NA group was （91.73 ±2.67）%, and blank group was （98.10 ±0.41）%, （P 〈0.05）. The cell phase in G0/G1 with NOTCH1 shRNA group, Neg- shRNA group and blank group was （66.23 ± 3.71）%, （42.27 ±3.65）%, （40.70 ±0.92）% respectively （P 〈 0.05 ）. The S phase was （31.03 ± 1.49）%, （56.53 ±1.76）%, （51.83 ± 1,45 ） % , respectively （P 〈 0.05 ）. NOTCH1 shRNA induces cell apoptosis in RPMI 8226 cells. The cell apoptotic rate was （46.67 ± 1.31）% in NOTCH1 shRNA group, （ 1.37 ±0.63） % in NegshRNA group and （0.85 ± 0.43 ） % in Blank group （ P 〈 0.05 ） , respectively. The NOTCH1 protein was reduced and the expression of P27 and P21 protein were increased by transfected NOTCH1 shRNA. NOTCH1 shRNA downregulated IL-6 secretion. OD value of IL-6 secretion was 28.18 ± 3, 50, 167.08 ± 7.97 and 185.86 ± 5.61, respectively through three groups （ P 〈 0.05 ）. CONCLUSION： NOTCH1 shRNA inhibits cell growth and induces cell apoptosis RPMI 8226 cell line, which may be a new treatment target in multiple myeloma.

关 键 词：NOTCH1 RNA干扰 多发性骨髓瘤 RP-MI 8226细胞 

分 类 号：R965.2[医药卫生—药理学]