p38MAPK基因对PM2.5染毒HK-2细胞部分癌基因和凋亡相关基因表达的影响

目的：探讨p38MAPK基因对PM_2.5染毒人肾上皮细胞（HK-2）部分癌基因和凋亡相关基因表达的影响。方法：根据GenBank提供的p38MAPK mRNA序列，设计合成干扰序列，将重组慢病毒载体转染HK-2细胞，构建p38MAPK基因沉默细胞株。分别采用实时荧光定量PCR （qPCR）和Western blot法检测p38MAPK mRNA和蛋白的表达水平鉴定沉默效果。用50 μg/mL的PM_2.5混悬液分别染毒正常HK-2细胞和p38MAPK基因沉默细胞24 h，利用荧光定量PCR和Western blot检测癌基因c-myc，c-fos、p53和凋亡相关基因Caspase-8、Caspase-9、Bcl-2的mRNA和蛋白的相对表达水平。结果：qPCR和Western blot检测结果显示，与正常HK-2细胞比较，p38MAPK基因沉默细胞中p38MAPK mRNA的表达下降了58.50%，p38MAPK蛋白表达水平下降了51.33%（P<0.01）。PM_2.5混悬液对HK-2细胞和p38MAPK基因沉默HK-2细胞染毒后，qPCR检测结果显示，与正常HK-2细胞未染毒组比较，正常HK-2细胞PM_2.5染毒组中c-myc、c-fos、Caspase-8和Casepase-9基因表达分别升高39.89%、15.12%、19.47%和15.45%，p53和Bcl-2基因表达分别下降21.54%和31.77%，差异均具有统计学意义（P<0.05）；与正常HK-2细胞PM_2.5染毒组比较，p38MAPK基因沉默HK-2细胞PM_2.5染毒组中c-myc、c-fos、p53、Caspase-8和Caspase-9 mRNA表达分别下降了84.55%、63.55%、34.49%、37.19%和54.97%，差异均具有统计学意义（P<0.05）。Western blot检测结果显示，与正常HK-2细胞未染毒组比较，正常HK-2细胞PM_2.5染毒组中的c-myc和Caspase-8蛋白表达增加，Bcl-2蛋白表达减少；与正常HK-2细胞PM_2.5染毒组比较，p38MAPK基因沉默HK-2细胞PM_2.5染毒组中的c-myc、Caspase-8和Bcl-2蛋白表达减少（均为P<0.05）。结论：成功构建了p38MAPK基因沉默细胞株，PM_2.5可引起HK-2细胞癌基因和凋亡相关基因表达水平升高，p38MAPK基因可能参与PM_2.5对HK-2细胞的毒性作用过程。

Effects of p38MAPK gene on PM2.5-induced expression of oncogenes and apoptosis genes in HK-2 cells

OBJECTIVE: To investigate effects of p38MAPK gene on expression of oncogenes and apoptosis related genes in human kidney epithelial cells (HK-2 cells) which had been exposed to PM_2.5. METHODS: According to the mRNA sequence of p38MAPK gene provided by GenBank,interference sequences were designed and synthesized,and a recombinant lentiviral vector was constructed and was transfected into HK-2 cells to construct p38MAPK gene-silenced cells. Real-time fluorescent quantitative PCR (qPCR) and western blot were used to identifyeffects from p38MAPK gene silencing. HK-2 cells and p38MAPK gene-silenced cells were treated with 50 μg/mL PM_2.5 suspension for 24 h,and qPCR and western blot were used to detect their expression of oncogenes (c-myc,c-fos and p53) and apoptosis-related genes (Caspase-8,Caspase-9 and Bcl-2). RESULTS: p38MAPK mRNA expression levels in p38MAPK-silenced cells were inhibited by 58.50% and p38MAPK protein expression levels by 51.33% when compared with the normal HK-2 cells (P<0.01). These results indicate that the p38MAPK silenced cells were successfully constructed. The qPCR results showed that when compared with the HK-2 cells in the control group,the PM_2.5-exposed cells indicated the mRNA of c-myc,c-fos,Caspase-8 and Casepase-9 in expressions were significantly increased by 39.89%,15.12%,19.47% and 15.45%,respectively,and p53 and Bcl-2 mRNA expressions were significantly decreased by 21.54% and 31.77% respectively (P<0.05). When compared with the PM_2.5 exposed HK-2 cells,the PM_2.5-exposed p38MAPK-silenced cells showed that the mRNA of c-myc,c-fos,p53,Caspase-8 and Caspase-9 expressions were significantly decreased by 84.55%、63.55%、34.49%、37.19% and 54.97% respectively,(P<0.05). At the protein level,when compared with the HK-2 cells in the control group,the PM_2.5 exposed cells showed that the protein levels of c-myc,and Caspase-8 were increased and that of Bcl-2 were decreased. When compared with the PM_2.5-exposed HK-2 cells,the PM_2.5-exposed p38MAPK-silenced cells showed that the mRNA levels of c-myc,Caspase-8 and Bcl-2 were decreased. CONCLUSION: p38MAPK-silenced cells were successfully constructed in this study. Our data show that PM_2.5 promoted the expression of oncogenes and apoptotic-related genes in HK-2 cells,and the p38MAPK gene was involved in the cytotoxicity of PM_2.5.