【摘要】：目的:多發(fā)性骨髓瘤(Multiple Myeloma,MM)是一種以漿細胞異常克隆為特征的B細胞腫瘤,其是第二大常見的血液系統(tǒng)惡性腫瘤。近年來,由于對MM生物學特征、細胞遺傳學異常以及腫瘤發(fā)病機制的進行了深入研究,目前有一些新的靶向治療藥物如蛋白酶體抑制劑、免疫調節(jié)劑、CD38單克隆抗體等應用于MM患者,提高MM患者的緩解率,但目前仍是一種無法治愈的惡性疾病。神經(jīng)節(jié)苷脂GM3是鞘糖脂的一種,其特征是含有唾液酸,并且有研究發(fā)現(xiàn)其與腫瘤形成有關。探討外源性神經(jīng)節(jié)苷脂GM3對人多發(fā)性骨髓瘤U266細胞生長的影響及初步探討誘導U266細胞凋亡的可能機制。方法:1.觀察神經(jīng)節(jié)苷脂GM3對U266細胞的生長抑制作用:收集U266細胞(對數(shù)生長期)加入不同濃度(0μmol/L、20μmol/L、40μmol/L、80μmol/L、160μmol/L)神經(jīng)節(jié)苷脂GM3作用48 h后應用MTT法檢測其增殖抑制率。2.檢測GM3對U266細胞誘導凋亡的作用:利用流式細胞術檢測不同濃度(0μmol/L、20μmol/L、40μmol/L、80μmol/L、160μmol/L)神經(jīng)節(jié)苷脂GM3對U266細胞的誘導凋亡作用及對其細胞周期的影響。3.檢測GM3對U266細胞Bcl-2 mRNA和Bax mRNA表達水平的影響:采用實時熒光定量PCR檢測不同濃度(0μmol/L、20μmol/L、40μmol/L、80μmol/L、160μmol/L)神經(jīng)節(jié)苷脂GM3引起的U266細胞Bcl-2 mRNA和Bax mRNA表達水平的變化。結果:1.神經(jīng)節(jié)苷脂GM3具有抑制U266細胞增殖作用。在20~160μmol/L范圍內GM3對U266細胞增殖具有抑制作用。不同濃度GM3實驗組與空白對照組相比,差異具有統(tǒng)計學意義(P0.05);且神經(jīng)節(jié)苷脂GM3對U266細胞的生長抑制作用具有劑量依賴性。2.GM3作用48 h后U266細胞的凋亡情況。U266細胞經(jīng)神經(jīng)節(jié)苷脂GM3作用后,對照組(0μmol/L)、20、40、80和160μmol/L組細胞早期凋亡率分別為(1.34±0.18)%,(29.13±1.15)%,(32.88±0.78)%,(42.2±7.19)%,(4.46±1.22)%,在GM3濃度范圍為20~160μmol/L內,U266細胞隨其濃度增加早期凋亡率增高。不同濃度GM3實驗組與空白對照組相比,差異具有統(tǒng)計學意義(P0.05)。3.GM3對U266細胞Bcl-2 mRNA和Bax mRNA表達水平的影響。U266細胞經(jīng)神經(jīng)節(jié)苷脂GM3作用后,隨著藥物濃度的增加,促凋亡基因Bax mRNA表達水平呈上升趨勢,而抗凋亡基因Bcl-2 m RNA呈下降趨勢。不同濃度GM3實驗組與空白對照組相比,差異具有統(tǒng)計學意義(P0.01)。結論:1.神經(jīng)節(jié)苷脂GM3能夠抑制U266細胞增殖。在0~160μmol/L藥物濃度范圍內,其抑制率隨著神經(jīng)節(jié)苷脂GM3濃度增加而增高。2.GM3能夠誘導U266細胞凋亡。利用流式細胞術(Annexin-Ⅴ/PI雙標)檢測不同濃度GM3對U266細胞的凋亡影響,本實驗結果證實GM3可誘導U266細胞凋亡及使其阻滯在S期,且在一定范圍內(20~160μmol/L),呈劑量效應關系。3.GM3誘導U266細胞凋亡的作用機制可能與Bcl-2 mRNA和Bax mRNA表達水平有關。GM3可能通過上調Bax m RNA表達水平和下調Bcl-2 mRNA表達水平誘導U266細胞凋亡。
[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 鹵0.18), (29.13 鹵1.15), (32.88 鹵0.78), (42.2 鹵7.19), (4.46 鹵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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