下调脐静脉内皮细胞小窝蛋白1表达对RPMI8226细胞硼替佐米敏感性的影响

Sensitivity to bortezomib of RPIM8226 cells after co-cultured with down-regulated Cav-1 expression

目的探讨下调脐静脉内皮细胞（HUVEC）小窝蛋白1（Cav．1）表达对多发性骨髓瘤细胞硼替佐米敏感性的影响。方法构建靶向Cav-1基因的shRNA表达载体及阴性对照shRNA表达载体，转染HUVEC细胞株；以多发性骨髓瘤细胞系RPMl8226为对象，用MTT法检测不同浓度硼替佐米单独或联合50nmol／L地塞米松对单独培养及与HUVEC（转染干扰质粒或对照质粒）间接共培养的RPMl8226细胞增殖的抑制作用；用Western blot法检测Cav．1表达水平；用流式细胞术检测细胞周期、细胞凋亡率及活性氧（P．OS）水平变化。结果Cav．1shRNA．1转染的HuVEc（HuVEc。low）Cav-1蛋白表达水平与转染阴性对照载体组相比显著降低，其Cav-1蛋白相对表达水平为0．2199±0．0288对1．3195±0．2393（P〈0．01）；RPMl8226细胞单独培养及与HUVEC、HUVEC共培养，硼替佐米作用的IC50值分别为20、50、65nmol／L。HUVEC、HUVEC可使RPMl8226细胞周期阻滞，RPMl8226细胞单独培养及与HUVEC、HUVEC共培养后RPMl8226细胞G0／G1期比例分别为28．5％、30．4％和36．2％；HUVEC保护RPMl8226细胞免于凋亡，20nmol／L硼替佐米作用于单独培养及与HUVEC、HUVEC共培养的RPMl8226细胞24h后，其细胞凋亡和（或）死亡率分别为66．8％、10．7％和8．6％；RPMl8226细胞可诱导HUVEC的氧化应激，共培养前后RPMl8226细胞ROS水平由15．0％上升至35．2％，HUVEC的ROS水平由80．4％上升至91．0％，HUVEC ROS水平由84．6％上升至96．8％。结论下调HUVECCav一1表达可促进共培养的RPMl8226细胞增殖，抑制细胞凋亡，使其阻滞在静息期，并降低RPMl8226对硼替佐米的敏感性。

Objective To investigate the sensitivity to bortezomib of RPMI8226 cells after co-cultured with down-regulated Caveolin （Cav） -1 expression of HUVECs by transfeetion with Cav-1 shRNA （HUVEC ）. Methods Exposure to bortezomib with or without 50 nmol/L dexamethasone at different concentration, the proliferation of RPMI8226 was analyzed by MTT assay when it was cultured alone or co-cultured with HUVEC. Cav-1 expression was detected by using of Western blot and cell cycle, apoptosis and the level of reactive oxygen species （ROS） were analyzed by flow cytometry. Results Cav-1 expression was notably down-regulated in HUVECscav （0.2199±0.0288 vs 1.3195±0.2393 ） （P〈0.01）. The IC50 of bortezomib for RPMI8226 cultured alone, co-cultured with HUVECs or HUVECC were 20 nmol/L, 50 nmol/L and 65 nmol/L, respectively. The percentages of G0-G1 phase in RPMI8226 cultured alone, co-cultured with HUVECs and HUVECscavow were 28.49%, 30.41%, and 36.15% respectively. The protection of RPMI 8226 against apoptosis by HUVECs was demonstrated that the apoptosis/death rates were 66.8%, 10.7% and 8.6% in RPMI8226 cultured alone, co-cultured with HUVECs and HUVECsc after exposure to 20 nmol/L bortezomib for 24 h. RPMI8226 could induce the oxidative stress of HUVECs before and after co-culture. The ROS level was raised from 15.0% to 35.2% in RPMI8226, from 80.4% to 91.0% in HUVECs, and from 84.6% to 96.8% in HUVECs. Conclusion The down-regulated Cav- I expression of HUVECs could promote proliferation and induce apoptosis of RMPI8226 cells, lead to G,JG, phase arrest, and reduce the sensitivity to bortezomib.