【作者】 江莲； 张涛； 李梅； 赵孝先； 金江； 曲凡； 刁玉巧； 陈健；

【Author】 Jiang Lian The Fourth Hospital of Hebei medical university & Hebei provincial tumor hospital, Shijiazhuang, 050011, China

【机构】 河北医科大学第四医院暨河北省肿瘤医院儿科；

【摘要】 目的:通过体外培养人Burkitt’s淋巴瘤细胞系Raji 细胞,观察Raji细胞环氧化酶-2(COX-2)蛋白表达及其非选择性COX-2抑制剂吲哚美辛和选择性COX-2抑制剂塞来昔布对Rajii细胞COX-2蛋白表达、细胞增殖率、凋亡率、凋亡相关基因bcl-2蛋白表达及6-酮-前列腺素F1 α(6- Keto—PGF1 α)水平的影响,探讨COX-2及其抑制剂对恶性淋巴瘤的作用及可能机制,以期为恶性淋巴瘤的防治开辟新的途径。方法:体外培养人Burkitt’s淋巴瘤细胞系Raji细胞,分为对照组:细胞培养基中加入与实验组等体积的二甲基亚砜(DMSO);吲哚美辛组:细胞培养基中加入DMSO溶解的吲哚美辛,终浓度分别为50,100 μ mol／L;塞来昔布组:细胞培养基中加入DMSO溶解的塞来昔布,终浓度同上。各组分别于24小时、48小时、72小时收集细胞送检。应用流式细胞仪(FCM)检测Raji细胞COX-2蛋白表达、细胞凋亡率及Bcl-2蛋白表达,MTT还原法检测细胞增殖率;放射免疫法(RIA)测定6-Keto-PGF 1α水平。结果:1、 Raji 细胞COX-2蛋白呈阳性表达,吲哚美辛组COX-2表达, 在50,100,μmol／L浓度时分别为492．07±17．36、417．49 ±44．86;塞来昔布组分别为388．05±23．85、379．03± 37．90;与对照组相比均有显著性差异(P<0．05)。且随着药物浓度的提高作用更为明显。2、吲哚美辛和塞来昔布孵育 Raji细胞72小时均可抑制Raji细胞增殖。吲哚美辛各浓度组细胞的增殖率分别为57．75±3．00％,48．10±4．13％;塞来昔布各浓度组的细胞增殖率分别为57．38±4．33%,45．64 ±3．04％,与对照组相比均具有显著性差异(P<0．05),且随两组药物浓度的增加抑制作用明显增强。两实验组对Raji 细胞增殖率的影响,差异无显著性(P>0．05)。3、吲哚美辛和塞来昔布孵育Raji细胞24小时,各浓度组诱导的凋亡百分率吲哚美辛组分别为8.43±1．10％,9．37±0．72％;塞来昔布组分别为8．53±0．83％,9．50±1．51％,均明显高于对照组(P<0．05),且作用呈剂量依赖性。3、吲哚美辛和塞来昔布孵育Raji细胞24小时Bcl-2蛋白表达量下降。吲噪美辛各浓度组,FI值分别为0．80±0．04,0．72±0．06; 塞来昔布组FI值分别为0．79±0．02,0．70±0．02,与对照组相比均有显著性差异(P<0．05)。两实验组对Raji细胞 Bcl-2蛋白表达的影响,差异无显著性(P>0．05)。4、吲哚美辛和塞来昔布孵育Raji细胞24小时均可使6-Keto-PGF1 α生成减少,在100 μ mol／L浓度的吲哚美辛作用下,6- Keto-PGF1 α的生成量为133．4±9．6 pg／ml,和对照组相比具有显著性差异(P<0．05),50,100μ mol／L浓度的塞来昔布组,6-Keto-PGF1 α生成量分别为130．2± 15．7pg／ml,102．40±11．0 Pg／ml pg／ml,和对照组相比具有显著性差异(P<0．05)。50,100μmol／L浓度的塞来昔布抑制6-Keto-PGF1 α生成的作用比吲哚美辛明显增强 (P<0．05)。结论:1、吲哚美辛和塞来昔布均可剂量依赖性地抑制人Burkitt’s淋巴瘤细胞系Raji细胞COX-2表达, 抑制Raji细胞中6-Keto-PGF1 α生成,从而抑制淋巴瘤细胞的生长。认为此为COX-2抑制剂抗肿瘤作用的重要途径和机制之一。2、吲哚美辛和塞来昔布均可剂量依赖性地抑制人Burkitt’s淋巴瘤细胞系Raji细胞的增殖,但吲哚美辛组和塞来昔布组相比,对Raji细胞增殖率的影响无明显差异。 3、吲哚美辛和塞来昔布均剂量依赖性的提高Raji细胞的凋亡率,并下调抗凋亡基因Bcl-2蛋白的表达,认为COX-2抑制剂可通过下调Bcl-2蛋白表达而诱导Raji细胞凋亡,但两实验组之间并无显著性差异。更多还原

【Abstract】 Objective: To elucidate the possible anti-tumor mechanism of nonsteroidal anti-inflammatory drugs (NSAIDs), we used human Burkitt’s lymphoma Raji cells to investigate the effects of two NSAIDs, a nonseiective COX-2 inhibitior indomethacin and a selective COX-2 inhibitior celecoxib on proliferation, apoptosis, expression of COX-2 and prostaglandins (PGs) synthesis of cells . Methods: Burkitt’s lymphoma Raji cells cultured in vitro were divided into control group, indomethacin group: treated with indomethacin at concentrations of 50,100 μmol/l, celecoxib group: treated with celecoxib at concentrations of 50,100 μmol/l. Cells of each groups were collected at 24,48, and 72 hours. Proliferation of Raji cells were evaluated by MTT assay, apoptosis pencentage (AP) and the level of Bcl-2 protein were assed by cytometry (FCM) and 6-keto-PGF1 α was detected by Radioimmu-noassay (RIA). The expression of COX -2 protein in Raji cells was positive. At the concentratons of 50,100 μmol/L ,the FI of Raji cells theated with indomechacin was492.07 ± 17.3604, 417.49 ± 44.86, and the of FI in celecoxib group was388.05 ± 23.85, 379.03 ± 37.90 respectively, each was significantly lower than that of the control(P< 0.05), No remarkable difference was showed between- the two test groups by independent-sample t test(P>0.05). MTT assay demonstrated that incubation with indomechacin or celecoxib for 72 hours could resulted in growth inhibition in Raji cells. At concentra- tions of 50, 100 μmol/L , the proliferation percentage reduced to 57.75 ± 3.00%, 48.10 ± 4.13% in indomethacin group and the proliferation percentage was 57.38 ± 4.33% , 45.64 ± 3.04% in celecoxib group, respectively, and each is significantly lower than the control(P<0.05). There was no remarkable difference between the two test groups by independent-sample t test(P>0.05). FCM showed that treating Raji cells with indomechacin or celecoxib for 24 hours, the apoptosis percentage(AP) increased significantly. At concentrations of 50,100 μmol/L, the AP induced by indomechacin was 8.43 ± 1.10%, 9.37 ± 0.72%, and the AP was 8.53 ± 0.83%, 9.50 ± 1.51% induced by celecoxib, respectively, all of which were higher than that in control group (P<0.05). Compared between indometha-cin and celecoxib group, there was no remarkable difference existed by independent- sample t test(P>0.05). Treatment with indomechacin or celecoxib for 24 hours resulted in decreased expression of Bcl-2 protein in Raji cells. At the concentratons of 50,100 μ mol/L ,the FI of Raji cells theated with indomechacin was0.80 ± 0.04, 0.72 ± 0.06, and the of FI in celecoxib group was 0.79 ± 0.02, 0.70 ± 0.02 respectively, each was significantly lower than that of the control(P<0.05), No remarkable difference was showed between- the two test groups by independent-sample t test(P>0.05). Raji cells treated with indometha-cin or celecoxib for 24h resulted in a decrease of 6-keto-PGF1 α, which was detected by radioimmunoass更多还原