神经节苷脂GM3诱导人多发性骨髓瘤U266细胞凋亡及其作用机制研究

发布时间：2018-07-26 17:42

【摘要】：目的:多发性骨髓瘤(Multiple Myeloma,MM)是一种以浆细胞异常克隆为特征的B细胞肿瘤,其是第二大常见的血液系统恶性肿瘤。近年来,由于对MM生物学特征、细胞遗传学异常以及肿瘤发病机制的进行了深入研究,目前有一些新的靶向治疗药物如蛋白酶体抑制剂、免疫调节剂、CD38单克隆抗体等应用于MM患者,提高MM患者的缓解率,但目前仍是一种无法治愈的恶性疾病。神经节苷脂GM3是鞘糖脂的一种,其特征是含有唾液酸,并且有研究发现其与肿瘤形成有关。探讨外源性神经节苷脂GM3对人多发性骨髓瘤U266细胞生长的影响及初步探讨诱导U266细胞凋亡的可能机制。方法:1.观察神经节苷脂GM3对U266细胞的生长抑制作用:收集U266细胞(对数生长期)加入不同浓度(0μmol/L、20μmol/L、40μmol/L、80μmol/L、160μmol/L)神经节苷脂GM3作用48 h后应用MTT法检测其增殖抑制率。2.检测GM3对U266细胞诱导凋亡的作用:利用流式细胞术检测不同浓度(0μmol/L、20μmol/L、40μmol/L、80μmol/L、160μmol/L)神经节苷脂GM3对U266细胞的诱导凋亡作用及对其细胞周期的影响。3.检测GM3对U266细胞Bcl-2 mRNA和Bax mRNA表达水平的影响:采用实时荧光定量PCR检测不同浓度(0μmol/L、20μmol/L、40μmol/L、80μmol/L、160μmol/L)神经节苷脂GM3引起的U266细胞Bcl-2 mRNA和Bax mRNA表达水平的变化。结果:1.神经节苷脂GM3具有抑制U266细胞增殖作用。在20~160μmol/L范围内GM3对U266细胞增殖具有抑制作用。不同浓度GM3实验组与空白对照组相比,差异具有统计学意义(P0.05);且神经节苷脂GM3对U266细胞的生长抑制作用具有剂量依赖性。2.GM3作用48 h后U266细胞的凋亡情况。U266细胞经神经节苷脂GM3作用后,对照组(0μmol/L)、20、40、80和160μmol/L组细胞早期凋亡率分别为(1.34±0.18)%,(29.13±1.15)%,(32.88±0.78)%,(42.2±7.19)%,(4.46±1.22)%,在GM3浓度范围为20~160μmol/L内,U266细胞随其浓度增加早期凋亡率增高。不同浓度GM3实验组与空白对照组相比,差异具有统计学意义(P0.05)。3.GM3对U266细胞Bcl-2 mRNA和Bax mRNA表达水平的影响。U266细胞经神经节苷脂GM3作用后,随着药物浓度的增加,促凋亡基因Bax mRNA表达水平呈上升趋势,而抗凋亡基因Bcl-2 m RNA呈下降趋势。不同浓度GM3实验组与空白对照组相比,差异具有统计学意义(P0.01)。结论:1.神经节苷脂GM3能够抑制U266细胞增殖。在0~160μmol/L药物浓度范围内,其抑制率随着神经节苷脂GM3浓度增加而增高。2.GM3能够诱导U266细胞凋亡。利用流式细胞术(Annexin-Ⅴ/PI双标)检测不同浓度GM3对U266细胞的凋亡影响,本实验结果证实GM3可诱导U266细胞凋亡及使其阻滞在S期,且在一定范围内(20~160μmol/L),呈剂量效应关系。3.GM3诱导U266细胞凋亡的作用机制可能与Bcl-2 mRNA和Bax mRNA表达水平有关。GM3可能通过上调Bax m RNA表达水平和下调Bcl-2 mRNA表达水平诱导U266细胞凋亡。
[Abstract]:Objective: multiple myeloma (Multiple Myeloma MM) is a B-cell tumor characterized by abnormal plasmacyte cloning. It is the second most common malignant tumor of the blood system. In recent years, due to the in-depth study of MM biological characteristics, cytogenetic abnormalities and tumor pathogenesis, there are some new targeted therapeutic drugs such as proteasome inhibitors. The immunomodulator CD38 monoclonal antibody is used in MM patients to improve the remission rate of MM patients, but it is still an incurable malignant disease. Ganglioside GM3 is a sphingolipids characterized by sialic acid and has been found to be associated with tumor formation. To investigate the effect of exogenous ganglioside GM3 on the growth of human multiple myeloma cell line U266 and the possible mechanism of inducing U266 cell apoptosis. Method 1: 1. To observe the inhibitory effect of ganglioside GM3 on the growth of U266 cells: the proliferation inhibition rate of U266 cells was measured by MTT assay after 48 hours of treatment with ganglioside GM3 at different concentrations (0 渭 mol / L ~ (20) 渭 mol / L ~ (-1) or 40 渭 mol / L ~ (80) 渭 mol / L ~ (160) 渭 mol/L. To detect the effect of GM3 on U266 cell apoptosis: flow cytometry was used to detect the effect of ganglioside GM3 on apoptosis and cell cycle of U266 cells at different concentrations (0 渭 mol / L ~ (20) 渭 mol / L ~ (40) 渭 mol 路L ~ (-1) and 80 渭 mol / L ~ (60) 渭 mol / L ~ (160 渭 mol/L). To detect the effect of GM3 on the expression of Bcl-2 mRNA and Bax mRNA in U266 cells: the changes of Bcl-2 mRNA and Bax mRNA expression in U266 cells induced by ganglioside GM3 at different concentrations (0 渭 mol / L ~ (20) 渭 mol / L ~ (40) 渭 mol / L ~ (40) and 80 渭 mol / L ~ (160) 渭 mol/L were detected by real-time quantitative PCR. The result is 1: 1. Ganglioside GM3 can inhibit the proliferation of U266 cells. The proliferation of U266 cells was inhibited by GM3 in the range of 20 ~ 160 渭 mol/L. Compared with the blank control group, the experimental group of different concentrations of GM3, The inhibitory effect of ganglioside GM3 on the growth of U266 cells was dose-dependent. 2. The apoptosis of U266 cells was induced by GM3 for 48 h. U266 cells were treated with ganglioside GM3. In the control group (0 渭 mol/L), the early apoptotic rate was (1.34 卤0.18), (29.13 卤1.15), (32.88 卤0.78), (42.2 卤7.19), (4.46 卤1.22) in the control group (0 渭 mol/L) and 160 渭 mol/L, respectively. The early apoptosis rate of U266 cells increased with the increase of GM3 concentration within 20160 渭 mol/L. The effect of GM3 on the expression of Bcl-2 mRNA and Bax mRNA in U266 cells was statistically significant compared with the control group. After treated with ganglioside GM3, the effect of GM3 on the expression of Bcl-2 mRNA and Bax mRNA in U266 cells increased with the increase of the concentration of GM3. The expression level of pro-apoptotic gene Bax mRNA increased, while that of anti-apoptotic gene Bcl-2 m RNA decreased. The difference of GM3 concentration between the experimental group and the blank control group was statistically significant (P0.01). Conclusion 1. Ganglioside GM3 could inhibit the proliferation of U266 cells. In the range of 0 ~ 160 渭 mol/L, the inhibitory rate increased with the increase of ganglioside GM3 concentration. 2. GM3 could induce apoptosis of U266 cells. Flow cytometry (Annexin- 鈪，

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