【摘要】 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。更多还原

【Abstract】 OBJECTIVE:To observe the effect of SmacN7 of exogenous Smac small molecule group on the apoptosis of human pancreatic cancer cell line SW1990 and wish to look for a new option for pancreatic cancer treatment.METHODS: 1)SmacN7 was prepared through solid-phase peptide synthesis technique and RP-HPLC;2)Apoptosis cell morphology of SW1990 treated with 500 μg/mL SmacN7 and TRAIL for 24 hours was observed with Heochst 33342 staining.3)Cell circle distribution SW1990 treated with SmacN7 and TRAIL was assayed with flow cytometry;4)SW1990 growth inhibition rate from different concentration and acting time of SmacN7 were assayed with MTT.RESULTS: 1)The purity of SmacN7 was more than 95%,with molecular weight of 3 278.08;2)Treatment of the SW1990 cells for 24 h with 500 μg/mL of SmacN7 and 500 ng/mL of TRAIL yielded similar results,including cell volume and nuclei enlargement with mild swelling,cells shorten and spindled,blue bright of nuclei and leaf-like or fragmented with edge concentration.Cell circle analysis showed that cells were blocked at G0/G1 stage,the proportion of S phage was decreased and cell growth was slowed down.3)The CAR of SW1990 was 15.3% after 24 h SmacN7 treatment,significantly higher than that of TRAIL treatment(5.64%).4)Along with different concentration of SmacN7(50,100,200 and 500 μg/mL) at different duration of treatment(24,48 and 72 h),CGIR were elevated with concentration increment and elongation of time(P<0.05).5)Treatment with 500 μg/mL of SmacN7 combined with different concentration of TRAIL(200,500,1 000 and 2 500 ng/mL) and GEM(10,20,40 and 60 μmol/L),the CGIR percentage were increased with the concentration elevation(18.11%,37.67%,42.63% and 67.60% in TRAIL,17.65%,31.85%,40.11% and 74.99% in GEM respectively).CONCLUSIONS: SmacN7 can induce pancreatic cancer cell apoptosis in a concentration-and time-dependent manner,decreased expression of XIAP and increased expression of Cyto C and p17 of Caspase-3 bio-active fragment may be the underlying mechanism.SmacN7 may become a new option for pancreatic cancer treatment.更多还原