海参多糖调控JAK2/STAT3/survivin通路对肝癌细胞增殖和凋亡的影响

Effects of sea cucumber polysaccharide regulating JAK2/STAT3/survivin pathway on proliferation and apoptosis of hepatocellular carcinoma cells

目的研究海参多糖对肝癌细胞增殖和凋亡的影响并探究其对JAK2/STAT3/survivin通路的作用。方法将人肝癌细胞株Hep G2培养至对数生长期后将细胞分别置入24孔板中,然后分别加入不同浓度(0、50、100、200μg/mL)的海参多糖进行处理。采用MTT法检测不同浓度海参多糖处理后12 h、24 h、36 h时对细胞增殖的影响;采用流式细胞仪分析海参多糖处理后36 h时对细胞凋亡的影响;采用实时定量PCR方法检测海参多糖处理后36 h时细胞中JAK2、STAT3和survivin mRNA表达水平,同时采用免疫印迹方法检测JAK2、STAT3、survivin及其磷酸化蛋白表达水平。然后将经不同浓度(0、50、100、200μg/mL)的海参多糖处理后的人肝癌HepG2细胞异种移植于裸鼠,观察海参多糖对裸鼠肿瘤组织生长的影响,同时采用免疫组织化学染色方法检测肿瘤组织中磷酸化JAK2、STAT3及survivin蛋白表达情况。结果经不同浓度海参多糖处理后,HepG2细胞增殖抑制率均随着时间延长和海参多糖浓度的增加而升高(P<0.05)。培养后36 h时的细胞经不同浓度的海参多糖处理后,细胞凋亡率总体比较差异有统计学意义(F=117.110,P<0.001)且观察范围内在200μg/mL海参多糖时最高;JAK2、STAT3、survivin及其磷酸化蛋白的蛋白表达水平均随着海参多糖浓度的增加而逐渐降低(P<0.05),然而没有发现其mRNA表达水平比较差异有统计学意义(P>0.05)。随着海参多糖浓度的增加,裸鼠肿瘤体积逐渐变小(P<0.05),同时免疫组织化学染色结果显示,肿瘤组织中磷酸化JAK2、STAT3及survivin蛋白表达阳性率均随着海参多糖浓度的增加而降低(P<0.05)。结论从本研究初步研究结果看,海参多糖可抑制肝癌细胞的增殖并促进其凋亡,其可能是通过调控JAK2/STAT3/survivin通路中JAK2、STAT3、survivin蛋白磷酸化表达而实现的。

Objective To study the effects of sea cucumber polysaccharide(SCPS)on the proliferation and apoptosis of hepatocellular carcinoma(HCC)cells,and to explore its effect on JAK2/STAT3/survivin pathway.Methods The human HCC cell lines HepG2 were placed into 24-well plates after culturing to the logarithmic phase,then dealed with different concentrations(0,50,100,200μg/mL)of SCPS.The MTT assay was used to detect the effects of different concentrations of SCPS on cell proliferation at 12 h,24 h,and 36 h.The effect of SCPS on cell apoptosis was analyzed by flow cytometry.The mRNA and protein expression levels of JAK2,STAT3,and survivin were detected by real-time quantitative PCR(qRT-PCR)and Western blot at 36 h after treatment with SCPS,respectively.Then,the human HepG2 cells treated with different concentrations(0,50,100,200μg/mL)of SCPS were subcutaneously xenografted into nude mice to observe the effect of SCPS on the growth of tumor tissues in nude mice.At the same time,the expressions of phosphorylated JAK2,STAT3,and survivin proteins in tumor tissues were detected by immunohistochemical method.Results After treatment with different concentrations of SCPS,the proliferation inhibition rate of HepG2 cells increased over time and increased SCPS concentrations(P<0.05).After 36 h cultivation time,the apoptosis rate of cells treated with different concentrations of SCPS was statistically significant(F=117.110,P<0.001)and increased with the increase of SCPS concentrations(P<0.05).The protein expression levels of JAK2,STAT3,survival and their phosphorylated proteins decreased gradually with the increase of SCPS concentrations(P<0.05),but there was no significant difference in the mRNA expression level(P>0.05).With the increase of SCPS concentration,the tumor volume of nude mice gradually reduced(P<0.05).At the same time,the results of immunohistochemical detection showed that the positive expression rates of phosphorylated JAK2,STAT3,and survivin proteins in tumor tissues decreased with the increase of SCPS concentrations(P<0.05).Conclusion From preliminary results of this study,SCPS could inhibit the proliferation of HCC cells and promote their apoptosis,which might be achieved by regulating the phosphorylated expressions of JAK2,STAT3,and survivin in the JAK2/STAT3/survivin pathway.