国际肿瘤学杂志

国际肿瘤学杂志 ›› 2015, Vol. 42 ›› Issue (8): 661-.doi: 10.3760/cma.j.issn.1673422X.2015.09.006

• 论著 • 上一篇    下一篇

Notch1表达下调对RPMI-8226骨髓瘤细胞荷瘤小鼠血管新生的抑制作用

李纯璞, 王静, 刘琰, 王玲, 李斑斑, 张开刚, 郭冬梅   

  1. 山东省泰安市中心医院骨外科
  • 出版日期:2015-09-08 发布日期:2015-08-12

Inhibition of RPMI8226 myeloma cell xenografted tumor angiogenesis by downregulation of Notch1

LI  Chun-Pu, WANG  Jing, LIU  Yan, WANG  Ling, LI  Ban-Ban, ZHANG  Kai-Gang, GUO  Dong-Mei   

  1. Department of Orthopaedics, Taian Central Hospital, Shandong Province
  • Online:2015-09-08 Published:2015-08-12

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1581

被引次数

5

摘要： 目的 探讨Notch1 siRNA对体内外RPMI8226人骨髓瘤细胞及荷瘤小鼠血管新生及肿瘤生长的影响。方法 体外采用Notch1 siRNA转染RPMI8226细胞，ELISA方法检测细胞上清VEGF分泌变化，Western blotting检测各组细胞Notch1、VEGF蛋白表达变化；将RPMI8226细胞皮下注射于糖尿病抵抗/重症联合免疫缺陷小鼠，建立人多发性骨髓瘤小鼠移植瘤模型，将成瘤小鼠随机分为3组：NS组（Notch1 siRNA转染）、CS组（Control siRNA转染）、UN组（未转染），观察各组肿瘤体积变化，免疫组织化学染色法观察Notch1、VEGF及CD34变化。结果 Notch1 siRNA有效下调RPMI8226细胞Notch1、VEGF蛋白表达；与CS组比较，在转染后48、72 h，NS组细胞培养上清液VEGF分泌量均明显下降［（120±25）ng/L∶（175±15）ng/L，t＝3.27，P＜0.05；（145±24）ng/L∶（295±17）ng/L，t＝8.83，P＜0.01）］；NS组肿瘤体积明显减小，13、17、21 d与CS组比较差异均有统计学意义 ［（1 548±218）mm3 ∶（1 820±64）mm3，t＝2.68，P＜0.05；（1 200±75）mm3∶（2 180±84）mm3，t＝19.46，P＜0.01；（1 150±88）mm3∶（2 250±145）mm3，t＝14.50，P＜0.01］；Notch1 siRNA降低移植瘤Notch1及VEGF蛋白表达，Notchl、VEGF在NS组和CS组表达的差异具有统计学意义［（16.33±2.52）% ∶（75.33±2.52）%，t＝28.71，P＜0.01；（5.00±1.00）%∶（29.67±2.08）%，t＝18.50，P＜0.01］；Notch1 siRNA降低移植瘤新生血管数量，NS组微血管密度显著少于CS组［（14.67±2.52）∶（30.00±5.00），t＝4.74，P＜0.01］。结论 体外实验证实Notch1 siRNA可以降低人骨髓瘤细胞RPMI8226细胞上清VEGF分泌，体内实验证实Notch1 siRNA可以减小荷瘤多发性骨髓瘤小鼠肿瘤体积、减少移植瘤新生血管数量，提示Notch1是抗骨髓瘤血管新生的有效分子靶点。

关键词: 多发性骨髓瘤/ 血管内皮生长因子类/ RNA, 小分子干扰/ 受体, Notch1

Abstract: ObjectiveTo investigate the effects of Notch1 siRNA on VEGF and angiogenesis of myeloma cell line RPMI8226 in vitro and in vivo. MethodsIn vitro, Notch siRNA was transfected into RPMI8226 cells, and then cell supernatant VEGF secretion was detected using ELISA method. Expression levels of Notch1 and VEGF proteins were assayed by Western blotting. RPMI8226 cells were subcutaneously transplanted in NOD/SCID mice, and then the tumor mice were divided into three groups randomly: NS group (Notch1 siRNAtransfected group), CS group (Control siRNAtransfected group) and UN group (Untransfected group), and the changes of tumor volume were observed. Immunohistochemical staining was used to detect the changes in expression levels of Notch1, VEGF and CD34. ResultsNotch1 and VEGF proteins expressions of RPMI8226 cells were significantly decreased by Notch1 siRNA. At 48 h and 72 h, VEGF secretion level in NS group was significantly different with CS group ［（120±25）ng/L∶（175±15）ng/L, t＝3.27, P＜0.05; （145±24）ng/L∶（295±17）ng/L, t＝8.83, P＜0.01］. At 13 d, 17 d and 21 d, tumor volume in NS group was significantly reduced, that was significantly different with CS group ［（1 548±218）mm3∶（1 820±64）mm3, t＝2.68, P＜0.05; （1 200±75）mm3∶（2 180±84）mm3, t＝19.46, P＜0.01; （1 150±88）mm3∶（2 250±145）mm3, t＝14.50, P＜0.01］. The expression levels of Notch1 and VEGF protein were decreased by Notch1 siRNA. The expression levels of Notchl and VEGF in NS group were different with CS group ［（16.33±2.52）%∶（75.33±2.52）%, t＝28.71, P＜0.01; （5.00±1.00）%∶29.67±2.08 %, t＝18.50, P＜0.01］. Notch1 siRNA reduced the number of transplanted tumor neovascularization in NS group. Microvascular density in NS group was significantly less than that in CS group ［(14.67±2.52)∶(30.00±5.00), t＝4.74, P＜0.01］. ConclusionIn vitro, Notch siRNA reduces human myeloma cell RPMI8226 cell supernatant VEGF secretion. In vivo, Notch siRNA can reduce tumor volume and the number of new blood vessels in transplantedmultiple myeloma mice. Thus, Notch1 is an effective molecular target for antiangiogenesis in myeloma.