尼古丁抑制穿孔素/颗粒酶B诱导的A549肺癌细胞凋亡

摘要

目的:首次探讨尼古丁对穿孔素/颗粒酶B(perforin/granzyme B)诱导的A549肺癌细胞凋亡的抑制作用及其机制.方法:以人肺癌细胞株A549为研究对象,采用四甲基偶氮噻唑蓝(MTT)比色法检测细胞活性,吖啶橙/溴化乙啶(AO/EB)双染荧光显微镜下观察细胞凋亡形态学变化,异硫氰酸荧光素标记的膜联蛋白V/碘化丙啶(Annexin V-FITC/PI)染色后流式细胞术定量检测细胞凋亡率,RT-PCR法检测X连锁凋亡抑制蛋白(XIAP)及存活素(survivin)mRNA的表达量.结果:(1)不同浓度尼古丁单独作用A549细胞时未见明显诱导凋亡现象,可显著抑制细胞生长(P< 0.01),其抑制率与作用时间和剂量呈正相关.(2)0.125 mg/L perforin作用于A549细胞(1×106个)时,PI染核率可达到90%以上.而当加入1 μL granzyme B(1 mg/L)孵育6 h时,perforin/granzyme B组细胞可呈现90%以上的凋亡现象.(3)6 μmol/L尼古丁作用于perforin/granzyme B预处理的细胞6 h时可见明显凋亡抑制现象,当检测其凋亡抑制蛋白XIAP和survivin mRNA的含量时,发现尼古丁组表达量较高,perforin/granzyme B组表达量明显降低,而当尼古丁联合perforin/granzyme B时表达量最高.结论:尼古丁可明显抑制perforin/granzyme B诱导的A549肺癌细胞凋亡现象,这种作用可能与凋亡抑制蛋白XIAP和survivin的表达上调有关.%AIM: To investigate the mechanism that nicotine inhibits perforin/granzyme B - induced apoptosis in A549 lung cancer cells.METHODS: MTT assay, acridine orange( AO Vethidium bromide( EB )staining, Annexin V -FITC/PI staining and RT - PCR were applied to identify the viability, the morphological characteristics of apoptosis, the apoptotic rate and the mRNA expression of X - linked inhibitor of apoptosis protein( XIAP ) and survivin, respectively.RESULTS: No significant apoptosis was observed when nicotine was used alone, and the proliferation of A549 lung cancer cells was inhibited by nicotine in a concentration - and time - dependent manner ( P ＜0.01 ).The positive rate of PI staining in A549 cells treated with 0.125 mg/L perform was up to 90％.When 1 μL of granzyme B ( 1 mg/L ) was added to the cells for 6 h, more than 90％ apoptotic cells were found.Combination of nicotine ( 6 μmol/L ) with perforin/granzyme B for 6 h significantly inhibited apoptosis.Furthermore, higher XIAP and survivin mRNA was detected when used nicotine alone, and the lowest XIAP and survivin mRNA was measured when used perforin/granzyme B alone.However, the highest XIAP and survivin mRNA was observed when used perforin/granzyme B combined with nicotine.CONCLUSION: Nicotine inhibits perforin/granzyme B - induced apoptosis in A549 lung cancer cells.This effect may be related to up - regulation of the inhibitor of apoptosis proteins, XIAP and survivin.