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目的 观察木材烟雾凝集物(WSC)对人气道平滑肌细胞(HASMC)增殖与坏死的影响.方法 原代培养的HASMC取2至8代细胞,分为对照组、WSC组和烟草烟雾凝集物(CSC)组,每组4个复孔.用细胞毒性检测试剂盒检测细胞活性,用核酸类似物标记检测细胞增殖试剂盒和流式细胞术检测增殖期(S期)细胞比例并分析细胞周期,以荧光定量PCR和Western blot法定量检测细胞周期蛋白D1(cyclin D1)的表达.用膜联蛋白-V/碘化丙啶双荧光染色法鉴别细胞坏死和凋亡.多组间比较采用单因素方差分析,两两比较采用LSD-t检验.结果 细胞活性峰浓度时,WSC组和CSC组的细胞活性[(126±12)％和(142±11)％]均显著高于对照组[(100±0)％],细胞活性抑制浓度时(WSC 10 mg/L、CSC 60 mg/L),WSC组和CSC组的细胞活性(86％和76％)均显著低于对照组[(100±0)％],差异均有统计学意义(q值为3.63～9.33,均P＜0.05).WSC 1 mg/L组细胞增殖比例和cyclin D1蛋白表达量[(124±20)％和1.31±0.64]显著高于对照组[(100±0)％和1.0±0.0],差异均有统计学意义(q值分别为5.85和5.91,均P＜0.05),S+G2/M期细胞比例和cyclin D1的mRNA表达量[(103±4)％和1.18±0.21]与对照组[(100±0)％和1.0±0.0]比较,差异均无统计学意义(q值分别为1.16和2.05,均P＞0.05)；CSC 10 mg/L组细胞增殖比例、cyclin D1蛋白表达量、S+G2/M期细胞比例和cyclin D1的mRNA表达量分别为(204±45)％、1.80±0.25、(140±6)％和1.48 ±0.2,均显著高于WSC 1 mg/L组和对照组[(100±0)％、1.0±0.0、(100±0)％和1.0±0.0],WSC 10 mg/L组坏死细胞比例[(13.39±0.15)％]均显著高于对照组[(1.57±0.41)％]和CSC60 mg/L组[(6.61±1.91)％],CSC 40 mg/L组凋亡细胞比例[(61.8±10.6)％]均显著高于对照组[(0.0±0.0)％]和WSC 10 mg/L组[(1.7±0.4)％],差异均有统计学意义(q值为3.33～18.03,均P ＜0.05).结论 WSC对HASMC的效应与CSC不同,表现为一定剂量下的弱促增殖效应,高剂量暴露时则表现出强有力的细胞坏死效应,但凋亡效应不明显.

Objective To investigate the effect of wood smoke condensate (WSC) on proliferation and necrosis of human airway smooth muscle cells (HASMCs).Methods Primary cultured HASMCs between passage 2 and 8 were divided into 3 groups:a control group,a WSC group and a cigarette smoke condensate (CSC) group.The viability of cells was examined by the CCK8 assays.The ratio of cellular proliferative stage (S phase) and cell cycle index were examined by fluorescent-labeled thymidine analogue uptake assays and flow cytometry.The expression of cyclin D1 was detected by quantitative reverse transcriptase polymerase chain reaction (Q-PCR) and Western blot.Cell apoptosis and necrosis were observed by the annexin-V and PI staining.Statistical analysis was performed by using the One-way ANOVA and LSD-t test.Results Cell viability reached peak at WSC 1 mg/L[(126 ± 12) ％] and at CSC 10 mg/L exposure level [(142 ± 11) ％] respectively.While at WSC 10 mg/L and CSC 60 mg/L exposure levels,cell viability decreased significantly to 86％ and 76％,respectively,as compared with that of the blank control group [(100 ± 0) ％] (q =3.63-9.33,P ＜ 0.05).In the WSC 1 mg/L group,the cell proliferation ratio and the expression of cyclin D1 protein were (124 ± 20)％ and 1.31 ± 0.64,respectively,the differences being significant as compared with the blank control group [(100 ± 0) ％,1.0 ± 0.0] (q =5.85,5.91,P＜0.05),while the expression of cyclin D1 mRNA and the percentage of S + G2M phase were 1.18 ±0.21 and (103 ±4)％,respectively,not significantly different as compared to the control group [(100±0)％,1.0±0.0],(q =1.16,2.05,P＞0.05).In the CSC 10 mg/L group,the abovementioned values were (204 ± 45) ％,1.80 ± 0.25,(140 ± 6) ％,1.48 ± 0.2,respectively,which were significantly higher than those in the blank control group (q =5.38-16.51,P ＜ 0.05) and in the WSC group (q =3.33-15.35,P ＜0.05).However,when HASMCs were exposed to WSC 10 mg/L,the cell death ratio was (13.39 ± 0.15) ％,higher than that of the blank control group [(1.57 ± 0.41) ％] and the CSC group [(6.61 ± 1.91)％] (q =18.03,10.34,P ＜0.05).Apoptosis ratio in the CSC 40 mg/L group was [(61.8 ± 10.6) ％],higher than that of the blank control group [(0.0 ±0.0) ％] and the WSC group [(1.7±0.4)％] (q =17.44,16.95,P ＜0.05).Conclusions Exposure to WSC caused a weak proliferation of HASMCs,but resulted in cell necrosis instead of apoptosis at high doses.There was a slight difference in cell effects between the WSC group and the CSC group.