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目的探讨煤焦沥青对中国仓鼠肺(CHL)细胞c-jun、fos和表皮生长因子受体(EGFR)m RNA表达和细胞周期和凋亡的影响。方法常规培养CHL细胞,常规消化细胞后,将细胞接种于6孔板中,待其融合度达到40%左右时,加入含有煤焦沥青的培养液,调整浓度至0、20、40 mg/L,分别于作用24、72 h后提取细胞总RNA,采用半定量RT-PCR技术检测c-jun、fos和EGFR m RNA的表达水平;将等量的细胞接种在50 ml的培养瓶中,待细胞融合度达到40%左右时,加入含煤焦沥青的培养液,调整浓度为0、20、40 mg/L,每组设置6个重复样品,分别加入谷胱甘肽(GSH)和N-乙酰半胱氨酸(NAC),调整浓度至0、2.5、10 mmol/L,作用120 h后收集细胞,采用流式细胞术检测细胞凋亡率和S期细胞的构成比。结果在20 mg/L煤焦沥青作用24 h后,CHL细胞的fos、c-jun和EGFR m RNA的表达水平均高于0 mg/L剂量组,差异均有统计学意义(P<0.05);作用72 h后c-jun和EGFR的表达水平显著高于0 mg/L剂量组(P<0.05)。在40 mg/L剂量组作用24和72 h后,fos、c-jun和EGFR m RNA的表达水平均高于0 mg/L剂量组,差异均有统计学意义(P<0.05)。煤焦沥青作用后,细胞凋亡率降低,在GSH作用下,凋亡率有所增加,NAC作用下的凋亡率增加的幅度更加明显,但以上差异均无统计学意义(P>0.05);20 mg/L的煤焦沥青作用120 h后,S期的细胞构成显著增加(P<0.05);GSH和NAC可以显著抑制S期的细胞构成改变,差异有统计学意义(P<0.05)。结论煤焦沥青可以诱导fos、c-jun和EGFR m RNA的表达水平和S细胞周期的构成增加、抑制细胞凋亡率,而GSH和NAC可以抑制这种诱导效应。
Objective To investigate the effects of coal tar pitch on the expression of c-jun, fos and epidermal growth factor receptor (EGFR) m RNA and cell cycle and apoptosis in Chinese hamster lung (CHL) cells. Methods CHL cells were cultured routinely. After routinely digesting the cells, the cells were inoculated into a 6-well plate. When the degree of fusion reached about 40%, a medium containing coal tar pitch was added to adjust the concentration to 0, 20 and 40 mg / L , Total cellular RNA was extracted 24 and 72 hours after treatment respectively. The expression of c-jun, fos and EGFR m RNA was detected by semi-quantitative RT-PCR. The same amount of cells were inoculated into a 50 ml culture flask until When the degree of cell fusion reached about 40%, the medium containing coal tar pitch was added to adjust the concentration to 0, 20, and 40 mg / L. Six replicate samples were set in each group and glutathione (GSH) and N- Acetyl cysteine (NAC) was added to the cells and the cells were harvested at concentrations of 0, 2.5 and 10 mmol / L for 120 h. Flow cytometry was used to detect the ratio of apoptotic cells to S phase cells. Results The expression levels of fos, c-jun and EGFR m RNA in CHL cells were significantly higher than those in 0 mg / L group after 20 mg / L coal tar pitch for 24 h (P 0. 05) The expression of c-jun and EGFR was significantly higher than that of 0 mg / L group after 72 h (P <0.05). The levels of fos, c-jun and EGFR m RNA in the 40 mg / L group were significantly higher than those in the 0 mg / L group (P <0.05) at 24 and 72 h. After treated with coal tar pitch, the apoptotic rate was decreased. Under the action of GSH, the apoptosis rate was increased and the apoptosis rate increased more significantly under the action of NAC, but the above differences were not statistically significant (P> 0.05) (P <0.05); GSH and NAC could significantly inhibit the changes of cell constitution in S phase after treated with 20 mg / L coal tar pitch for 120 h (P <0.05) . CONCLUSION Coal tar pitch can induce the expression of fos, c-jun and EGFR m RNA and the increase of the S cell cycle, and inhibit the apoptosis rate. However, GSH and NAC can inhibit this induction effect.