摘要目的 观察全氟辛酸(PFOA)对体外培养的人恶性胚胎横纹肌瘤(RD)细胞的增殖、迁移及侵袭的影响,并初步探讨其作用机制.方法 应用不同浓度PFOA作用体外培养的RD细胞一定时间后,使用细胞增殖与毒性检测试剂盒(CCK8)检测细胞活力并确定PFOA作用浓度;使用该浓度PFOA处理RD细胞,通过划痕实验和细胞体外侵袭(transwell)实验评估细胞的侵袭和迁移能力,流式细胞术检测细胞周期的分布,实时荧光定量PCR(qPCR)检测相关基因mRNA表达差异,蛋白质免疫印迹法(WB)检测相关蛋白水平表达差异.结果 CCK8结果 显示不同浓度PFOA作用RD细胞72 h后,呈现出低浓度PFOA(1、10、50、100 μmol/L)促进、高浓度PFOA(250、500 μmol/L)抑制细胞增殖的趋势,与对照组相比,50 μmol/L PFOA组对RD细胞的增殖呈现促进作用(P＜0.001).流式细胞术结果显示PFOA (50 μmol/L)作用RD细胞72 h后,G0/G1期细胞差异无统计学意义,S期细胞减少,G2/M期细胞增加.S期及G2/M期相对比例在PFOA组及对照组之间差异均有统计学意义(均P＜0.01).qPCR结果显示在mRNA水平,对照组与PFOA(50 μmol/L)处理组的细胞周期蛋白依赖性蛋白激酶2(CDK2)相对表达量分别为0.97±0.07、2.64±0.11,差异有统计学意义(t=12.60,P＜0.001);细胞周期蛋白E2 (cyclin E2)相对表达量分别为1.33±0.17、3.35±0.22,差异有统计学意义(t=7.42,P＜0.001);WB结果显示,对照组与PFOA(50 μmol/L)处理组的CDK2相对表达量分别为0.35±0.01、0.84±0.03,差异有统计学意义(t=14.60,P＜0.001);cyclin E2相对表达量分别为0.67±0.04、0.86±0.01,差异有统计学意义(t=4.88,P＜0.01).p21、p53在mRNA及蛋白表达差异均无统计学意义(均P＞0.05).划痕实验结果显示,PFOA (50 μmol/L)组的划痕愈合速度明显加快,其相对迁移面积大于对照组(P＜0.001);transwell侵袭试验显示与对照组相比,PFOA (50 μmol/L)组细胞穿过小孔数增多(t=54.40,P＜0.001),侵袭能力明显增强.qPCR结果显示在mRNA水平,对照组与PFOA(50 μmol/L)处理组的波形蛋白(vimentin)相对表达量分别为0.71 ±0.03、2.53±0.16,差异有统计学意义(t=1 1.00,P＜0.001);基质金属蛋白酶2(MMP2)相对表达量分别为1.09±0.04、10.73±1.20,差异有统计学意义(t=8.04,P＜0.001).WB结果显示,对照组与PFOA(50 μmol/L)处理组的vimentin相对表达量分别0.55±0.06、0.81±0.01,差异有统计学意义(t=4.50,P＜0.05);MMP2相对表达量分别为0.64±0.04、1.03±0.13,差异有统计学意义(t=2.94,P＜0.05).结论 低剂量PFOA(50 μmol/L)暴露可通过上调细胞周期相关蛋白及MMP2、vimentin促进横纹肌肉瘤细胞的增殖、侵袭转移.

abstractsObjective This study aimed to explore the effect of perfluorooctanoate acid (PFOA)on the proliferation,migration and invasion of the human muscle rhabdomyosarcoma RD cell line and its related mechanisms.Methods RD cells were cultured and exposed to PFOA of different concentrations with 6-72 hours.The cell viability was assessed by cell counting kit-8 (CCK-8) assay.Wound healing and transwell filter assay were used to evaluated the migration and invasion ability of the RD cells respectively.The cell cycles were detected by Flow cytometry.Quantitative real-time PCR and Western blot were used to quantify the mRNA and protein expression difference of related genes,respectively.Results CCK-8 assay showed that,after treated the RD cell with different dose of PFOA for 72 h,low dose PFOA (1,10,50,100 μ mol/L) promotes the proliferation of RD cells while high dose PFOA (250,500 mol/L) inhibits the proliferation (P＜0.001).Flow cytometry showed that compared with the control group,there was no significant difference in G0/G1 phase,while cells in S phase deceased and G2/M phase cells increased after treated with PFOA (50 μmol/L) for 72 h.The relative proportions of S and G2/M were significantly different between the two groups (P＜0.01).The results of qPCR showed that the mRNA relative expression of CDK2 of the control group and the PFOA (50 μ mol/L) group were 0.97±0.07 and 2.64±0.11 respectively,and there was a significant difference (t=12.60,P＜0.001);The mRNA relative expression of cyclin E2 of the control group and the PFOA (50 μ mol/L) group were 1.33 ± 0.17 and 3.35 ± 0.22 respectively,and there was a significant difference (t=7.42,P＜0.001);The results of Western blot showed that the protein relative expression of CDK2 of the control group and the PFOA (50 μmol/L) group were 0.35±0.01 and 0.84±0.03 respectively,and there was a significant difference (t=14.60,P＜0.001);The protein relative expression of cyclin E2 of the control group and the PFOA (50 μmol/L) group were 0.67±0.04 and 0.86±0.01 respectively,and there was a significant difference (t=4.88,P＜0.01);There was no significant difference in the mRNA and protein expression of p21 and p53 between the PFOA and control group (P＞0.05).The wound healing rate of the PFOA (50 μmol/L) group was faster than that of the control group,and the relative migration area of the PFOA group was larger accordingly (P＜0.001).After PFOA (50 μmol/L) treated,the number of the cell through the membranes was much more than the control group (t=54.40,P＜0.001),which means PFOA significantly stimulated the invasion ability of the RD cells.The results of qPCR showed that the mRNA relative expression of vimentin of the control group and the PFOA (50 μmol/L) group were 0.71 ±0.03 and 2.53±0.16 respectively,and there was a significant difference (t=ll.00,P＜0.001);The mRNA relative expression of MMP2 of the control group and the PFOA (50 μ mol/L) group were 1.09±0.04 and 10.73± 1.20 respectively,and there was a significant difference (t=8.04,P＜0.001).The results of Western blot showed that the protein relative expression of vimentin of the control group and the PFOA (50 μmol/L) group were 0.55±0.06 and 0.81±0.01 respectively,and there was a significant difference (t=4.50,P＜0.05).The protein relative expression of cyclin E2 of the control group and the PFOA (50 μmol/L) group were 0.64±0.04 and 1.03±0.13 respectively,and there was a significant difference (t=2.94,P＜0.05).Conclusions Low dose PFOA (50 μmol/L) exposure promotes cell proliferation,migration and invasion in the human muscle rhabdomyosarcoma cell line through inducing the expressions of MMP2,vimentin and cell cycle related genes.