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目的:观察外源性类Smac小分子SmacN7对人胰腺癌细胞株SW1990凋亡的影响,以期找到治疗胰腺癌的新方法。方法:使用固相多肽合成技术(SPPS)和反相高效液相层析法(RP-HPLC)制备SmacN7;Heochst 33342染色法观察SW1990分别与PBS液和500ng/mL TRAIL、500μg/mL SmacN7作用24h的细胞凋亡形态;流式细胞术(FCM)检测SW1990分别与500μg/mL SmacN7和500ng/mL TRAIL作用24h的细胞凋亡率(CAR)并观察细胞周期分布;四甲基偶氮唑盐法(MTT法)检测SW1990分别与50、100、200和500μg/mL SmacN7作用24、48和72h的细胞生长抑制率(CGIR);MTT法检测500μg/mL SmacN7分别与200、500、1 000和2 500ng/mL TRAIL或10、20、40和60μmol/L吉西他滨(GEM)联用,与SW1990作用24h的CGIR。结果:SmacN7纯度≥95%,相对分子质量3 278.08,质谱鉴定结果与预期结果完全一致。SW1990与500μg/mL SmacN7作用24h和与500ng/mL TRAIL作用24h细胞形态变化类似,细胞体积和细胞核增大,轻度肿胀,细胞变为短梭形,细胞核呈亮蓝色,分叶或碎片状,边缘集中,且数目更多;细胞周期均显示G0/G1期阻滞,S期比例下降,细胞生长变缓;CAR分别为5.64%和15.30%。SW1990分别与50、100、200和500μg/mL SmacN7作用24、48和72h,CGIR不同,且随SmacN7浓度增加和作用时间延长,CGIR升高,P<0.05;500μg/mL SmacN7分别联合200、500、1 000和2 500ng/mL TRAIL,与SW1990作用24h,SW1990的CGIR为18.11%、37.67%、42.63%和67.60%;分别联合10、20、40和60μmol/L GEM与SW1990作用24h,SW1990的CGIR分别为17.65%、31.85%、40.11%和74.99%。两组均随TRAIL和GEM浓度的增加,SW1990的CGIR升高。结论:SmacN7能促使SW1990凋亡,且有浓度和作用时间依赖性。其作用机制与XIAP表达量降低,细胞色素C和Caspase-3活性裂解片段p17表达量升高有关。SmacN7有可能成为治疗胰腺癌的新方法。
Objective: To observe the effect of exogenous Smac SmacN7 on the apoptosis of human pancreatic cancer cell line SW1990 in order to find a new method for the treatment of pancreatic cancer. Methods: SmacN7 was prepared by solid phase peptide synthesis (SPPS) and reverse-phase high performance liquid chromatography (RP-HPLC). The effect of SW1990 on PBS and 500ng / mL TRAIL and 500μg / mL SmacN7 was observed by Heochst 33342 staining (FCM) was used to detect the apoptosis rate (CAR) of SW1990 treated with 500μg / mL SmacN7 and 500ng / mL TRAIL for 24 h respectively. The cell cycle distribution was also observed. (MTT assay) was used to detect the cell growth inhibition rate (CGIR) of SW1990 treated with 50, 100, 200 and 500μg / mL SmacN7 for 24,48 and 72h, respectively. 500 ng / mL TRAIL or 10, 20, 40 and 60 μmol / L gemcitabine (GEM) for 24 h with SW1990. Results: The purity of SmacN7 was ≥95% and the relative molecular mass was 3 278.08. The results of mass spectrometry were in good agreement with the expected results. SW1990 and 500μg / mL SmacN7 24h and 500ng / mL TRAIL effect 24h cell morphology changes, cell volume and nucleus increased, mild swelling, the cells become short spindle, the nucleus was bright blue, lobulated or fragmented , With more concentrated margins. The cell cycle showed G0 / G1 phase arrest, S phase proportion decreased, cell growth slowed; CAR were 5.64% and 15.30% respectively. SW1990 with 50, 100, 200 and 500μg / mL SmacN7 respectively for 24,48 and 72h, with different CGIR. CGIR increased with the increase of SmacN7 concentration and duration of action, P <0.05; 500μg / mL SmacN7 combined with 200,500 , 1 000 and 2 500ng / mL TRAIL, respectively. The results showed that the CGIR of SW1990 was 18.11%, 37.67%, 42.63% and 67.60% respectively when treated with SW1990 for 24h, and SW1990 treated with 10, 20, 40 and 60μmol / CGIR were 17.65%, 31.85%, 40.11% and 74.99% respectively. With increasing concentration of TRAIL and GEM, CGIR of SW1990 increased in both groups. Conclusion: SmacN7 can induce apoptosis of SW1990 cells in a time-and concentration-dependent manner. The mechanism of action is related to the decrease of XIAP expression and the increase of p17 expression of cytochrome C and Caspase-3 active fragment. SmacN7 may be a new way to treat pancreatic cancer.