【摘要】 目的 观测乳酸通过胆固醇对非小细胞肺癌细胞中Hedgehog(Hh)信号通路活性的影响。方法 本研究用乳酸（0.0 mM、7.5 mM、15.0 mM、20.0 mM）和胆固醇（0μg/mL、25μg/mL、50μg/mL）处理A549、NCI-H1703细胞6、24 h，即刻检测Hh信号通路靶基因、胆固醇代谢合成酶相关基因或蛋白表达水平，并测定细胞内总胆固醇水平；为排除pH值对研究结果的影响，用相同浓度乳酸钠（7.5 mM）处理A549、NCI-H1703细胞24 h，用qPCR法检测胆固醇代谢合成酶相关基因表达水平；通过胆固醇合成酶抑制剂辛伐他汀（0.2μM）和乳酸（0.0 mM、7.5 mM、15.0 mM、20.0 mM）同时处理A549细胞24 h，检测Hh信号通路靶基因表达水平；同时用RTCA DPlus法检测增殖及迁移情况。结果 经乳酸、胆固醇分别处理后的A549、NCl-H1703组细胞与对照组细胞比，前者GLI1、PTCH1 mRNA水平上调（P<0.05）；经乳酸处理后的A549细胞总胆固醇水平和HMGCR、FDFT1、SQLE mRNA水平均升高（P<0.001），同时HMGCR、FDFT1蛋白水平增高；经乳酸及乳酸钠分别处理后的A549、NCl-H1703细胞HMGCR、FDFT1 mRNA水平升高（P<0.01）。用0.2μM辛伐他汀下调胆固醇水平（P<0.05）后，GLI1、PTCH1 mRNA水平下调（P<0.01）；乳酸能够逆转辛伐他汀对GLI1和PTCH1 mRNA水平的抑制作用，对A549细胞增殖（F=3.941,P=0.020）、迁移（F=5.851,P=0.003）能力的抑制作用。结论 乳酸可能通过上调非小细胞肺癌细胞的胆固醇水平，促进Hh信号通路活性，并提高细胞增殖、迁移能力，提示抑制乳酸生成可能会改善辛伐他汀针对非小细胞肺癌的疗效。更多还原

【Abstract】 Objectives To explore the effects of lactic acid via cholesterol on Hedgehog(Hh)signaling pathway activity in non-small cell lung cancer cells.Methods Lactic acid(0.0 mM,7.5 mM,15.0 mM,20.0 mM)and cholesterol(0,25 μg/mL,50 μg/mL)were adopted to treat A549 and NCI-H1703 cells for 6 and 24hours respectively to detect the expression levels of Hh signaling pathway target genes,genes or proteins related to cholesterol biosynthesis enzymes,and to measure the total cholesterol level in the cells.In order to exclude the effects of pH value on the study results,A549 and NCI-H1703 cells were treated with the same concentration of sodium lactate(7.5 mM)for 24 h.The expression levels of genes related to cholesterol biosynthesis enzymes were detected by qPCR method.A549 cells were treated with a cholesterol synthetase inhibitor simvastatin(0.2 μM)and lactic acid(0.0 mM,7.5 mM,15.0 mM,20.0 mM)for 24 h to detect the expression level of Hh signaling pathway target genes.Besides,RTCA DPlus method was used to detect proliferation and migration.Results The mRNA levels of GLI1 and PTCH1 in A549 and NCl-H1703 cells treated with lactic acid and cholesterol were up-regulated compared with those in control group(P<0.05).After lactic acid treatment,the total cholesterol level and mRNA levels of HMGCR,FDFT1 and SQLE in A549 cells were increased(P<0.001).The protein levels of HMGCR and FDFT1 were increased as well.The mRNA levels of HMGCR and FDFT1 were increased in A549 and NCl-H1703 cells treated with lactic acid and sodium lactate respectively(P<0.01).The mRNA levels of GLI1 and PTCH1 were down-regulated by 0.2 μM simvastatin(P<0.05)after cholesterol level was down-regulated(P<0.01).Lactic acid could reverse the inhibitory effects of simvastatin on GLI1 and PTCH1mRNA levels,while inhibit the proliferation(F=3.941,P=0.020)and migration(F=5.851,P=0.003)of A549cells.Conclusions Lactic acid may up-regulate the cholesterol level of NSCLC cells to promote the activity of Hh signaling pathway,and enhance cell proliferation and migration ability,suggesting that inhibiting lactic acid may improve the efficacy of simvastatin in NSCLC.更多还原