Fas/FasL信号传导途径在启动尘肺中的作用机制

Mechanism of Fas/FasL signal transduction pathway on initiating pneumoconiosis

目的探讨Fas/FasL信号传导途径在尘肺发生发展中的作用机制。方法以中国煤炭工人北戴河疗养院无其他肺部疾病的0+接尘工人及Ⅰ期、Ⅱ期尘肺患者为研究对象,将支气管肺泡灌洗液(BALF)中的肺泡巨噬细胞(AMs)分5组培养(分别在信号传导途径的不同环节进行抑制),即对照组(未抑制任何途径介导的细胞凋亡过程)、SOD组(在AMs凋亡过程上游保护AMs,抑制AMs细胞膜脂质过氧化过程从而抑制凋亡过程,观察SOD对凋亡细胞的保护作用)、Fas/FasL组(抑制三条传导途径中的另两条即TNFR/TNF-α、TRAILR/TRAIL途径,观察Fas/FasL信号传导途径介导的细胞凋亡情况)、FasL抑制组(同时抑制TNFR/TNF-α、TRAILR/TRAIL和Fas/FasL三条途径,观察Fas/FasL信号传导途径上游抑制后的细胞凋亡情况),caspase-8抑制组(同时抑制TNFR/TNF-α、TRAILR/TRAIL途径和Fas/FasL途径的下游蛋白caspase-8的活性,观察Fas/FasL信号传导途径下游抑制后的细胞凋亡情况)。分别采用Western blot(WB)、琼脂糖凝胶电泳(AGE)和TDT介导的dUTP缺口末端标记技术(TUNEL)方法检测肺泡巨噬细胞(AMs)的凋亡情况、DNA片段的改变、信号蛋白Fas、FasL、caspase-8、caspase-3的表达水平。结果SOD组、FasL抑制组、caspase-8抑制剂组的凋亡指数均低于对照组,差异均有统计学意义。对照组的AMs出现凋亡细胞特征性"梯状带",SOD组未出现,FasL抑制组、caspase-8抑制剂组的DNA条带明显弱于对照组。SOD组的Fas、FasL、caspase-8、caspase-3蛋白的表达均低于对照组,差异均有统计学意义(P<0·05);FasL抑制组的caspase-8、caspase-3的表达低于Fas/FasL组与对照组,差异均有统计学意义(P<0·05);caspase-8抑制组的caspase-3表达低于对照组,差异有统计学意义(P<0·05)。结论尘肺的发生发展可能与Fas/FasL信号传导途径介导的AMs凋亡有关。

Objective The purpose of this was to study the mechanism of Fas/FasL signal transduction on triggering and promoting pneumoconiosis. Methods Phase 0^＋, I and Ⅱ pneumoconiosis cases without other lung diseases admitted in Beidaihe Coal-Workers＇ Sanatorium were selected as subjects, collecting their BALF, the alveolar macrophages （AMs） in BALF were separately cultivated in 5 groups： A, control group; B, Fast/FasL group; C, SOD group; D, FasL inhibitor group and E, caspase-8 inhibitor group. The apoptosis was measured by TUNEL; agarose gel electrophoresis was used to evaluate the alternation of DNA fragment; the expressions of Fas, FasL, caspase-8, caspase-3 were detected by Western blot （WB）. Results The results showed that the apoptosis indices in SOD group, FasL inhibitor group and caspase-8 inhibitor group were all lower than that of control group; the expressions of Fas, FasL, caspase-8, caspase-3 in SOD group were decreased compared with control group （P 〈 0. 05）; the levels of caspase-8, caspase-3 in FasL inhibitor group were decreased compared with control group and Fas/FasL group （P 〈0. 05）; and the caspase-3 level in caspase-8 inhibitor group was also decreased compared with control group （P 〈0. 05）. The AMs＇ DNA electrophoresis strips of control group characterized by distinctive ladder pattern were well observed, but wasn＇t observed in SOD group, the other groups were also illegible. Conclusion It is suggested that Fas/FasL signal transduction pathway might be essential in triggering and promoting pneumoconiosis triggered by AMs apoptosis.