3-溴丙酮酸调控JAK/STAT信号通路抗人卵巢癌细胞株A2780增殖的实验研究

3-brominated Pyruvate Regulates JAK/STAT Signaling Pathway Against Proliferation of Human Ovarian Cancer Cell Line A2780

目的探讨3-溴丙酮酸对人卵巢癌细胞株A2780增殖的抑制作用,并观察其对Janus激酶/信号转导子及转录激活子(JAK/STAT)信号通路的调控作用。方法取人卵巢癌细胞株A2780,培养至对数期,常规分装(1.5×106个/孔)至6孔板中,设置低、中、高剂量组和对照组,每组各包含5个复孔。继续培养待细胞贴壁生长后,3剂量组分别加入25、50、100μmol/L浓度的3-溴丙酮酸,对照组加入等量磷酸盐缓冲液(PBS),培养72h。对比各组培养期间细胞形态学变化,培养24h、48h和72h增殖抑制率变化,培养72h后各组细胞周期分布结果,培养72h后各组JAK2mRNA、STAT3mRNA表达,培养72h后各组JAK2、p-JAK2、STAT3、p-STAT3蛋白表达。结果在透射电镜下检查发现对照组各时刻均表现出细胞形态结构规则、染色质均匀分布、细胞膜完整的形态,低中高剂量组细胞形态学均出现明显的异常,其中高剂量组形态学异常最为显著,中剂量组形态学明显异常,低剂量组稍有异常,且均呈时间依赖性;各组培养24h、48h、72h增殖抑制率对比差异均有统计学意义(P<0.05),且均呈时间依赖性和剂量依赖性,本组内每2个时刻、各时刻每2组间增殖抑制率对比差异均有统计学意义(P<0.05);各组培养72h后G0/G1期、G2/M期结果对比差异均有统计学意义(P<0.05),其中对照组G0/G1期最高,低剂量组次之,中剂量组更低,高剂量组最低,高剂量组G2/M期最高,中剂量组次之,低剂量组更低,对照组更低;各组培养72h后JAK2mRNA、STAT3mRNA表达对比差异均无统计学意义(P>0.05),且每2组间对比差异均无统计学意义(P>0.05);各组培养72h后p-JAK2、p-STAT3蛋白表达、p-JAK2/JAK2、p-STAT3/STAT3对比差异均有统计学意义(P<0.05),其中对照组均最高,低剂量组均次之,中剂量组均更低,高剂量组均最低。结论3-溴丙酮酸能够抑制人卵巢癌细胞株A2780增殖,阻滞于G2/M期,推测与调控JAK/STAT信号通路,下调p-JAK2、p-STAT3蛋白表达有关。

Objective To investigate the inhibitory effect of 3-bromopyruvate on the proliferation of human ovarian cancer cell line A2780,and to observe its regulation on Janus kinase/signal transducer and activator of transcription(JAK/STAT)signaling pathway.Methods Human ovarian cancer cell line A2780 was cultured in logarithmic phase,routinely packed(1.5×106 cells/hole),each group with 5 holes.After adherent cell growth,3-Bromopyruvic acid at25,50 and 100 mmol/L was added to the three dose groups,and phosphate buffer(PBS)was added to the control group for 72 hours.Morphological changes in each group during culture,changes of proliferation inhibition rate after 24 h,48 h and 72 hof culture,the results of cell cycle distribution after 72 hof culture,the expressions of JAK2 mRNA and STAT3 mRNA in each group after 72 hof culture and the expressions of JAK2,p-JAK2,STAT3 and p-STAT3 proteins in each group after 72 hof culture were compared.Results Transmission electron microscopy showed that the control group showed regular cell morphology,uniform distribution of chromatin and intact cell membrane morphology at all times.Morphological abnormalities were observed in the low,middle and high dose groups.Morphological abnormalities were most significant in the high dose group,significantly abnormal in the middle dose group,slightly abnormal in the low dose group,and all in a time-dependent manner.The proliferation inhibition rates after 24 h,48 hand 72 hof culture in each group were statistically different(P<0.05).All of them were time dependent and dose-dependent.There were significant differences in the proliferation inhibition rates between the two groups at every two time points(P<0.05).There were significant differences in G0/G1 phase,G2/M phase results among the groups after 72 hof culture(P<0.05).The G0/G1 in the control group was the highest,low dose group was lower,middle dose group was lower,and high dose group was the lowest.The G2/M in the high dose group was the highest,followed by the middle dose group,the low dose group,the control group.There was no significant difference in JAK2 mRNA and STAT3 mRNA expressions among the groups 72 hours after culture(P>0.05),and there was no significant difference between each two groups(P>0.05).There were significant differences in p-JAK2,p-STAT3 protein expressions,p-JAK2/JAK2,p-STAT3 after 72 hof culture(P<0.05).The p-JAK2,p-STAT3 protein expressions,p-JAK2/JAK2,pSTAT3/STAT3 protein expressions in the control group were the highest,followed by the low dose group,the middle dose group,the high dose group.Conclusion 3-Bromopyruvic acid can inhibit the proliferation of human ovarian cancer cell line A2780,block the G2/M phase,presumably related to the regulation of JAK/STAT signaling pathway,downregulation of p-JAK2,p-STAT3 protein expressions.