钩吻总碱通过JAK2/STAT3/Survivin通路调控人舌癌细胞株Tca8113增殖、凋亡的作用探讨

目的观察钩吻总碱对人舌癌细胞株Tca8113增殖、凋亡的作用,并探讨其对酪氨酸蛋白激酶2(JAK2)/信号传导及转录激活因子3(STAT3)/Survivin通路的调控作用。方法取人舌癌细胞株Tca8113,培养至对数期,分装至6孔板中,分为对照组、A组、B组和C组,每组设置5个复孔。A组、B组和C组分别加入25μmol/L、50μmol/L、100μmol/L浓度的钩吻总碱处理(20μl),对照组加入等量PBS缓冲液。以倒置显微镜观察72 h后各组细胞形态;以MTT法检测24 h、48 h、72 h后细胞增殖抑制率;以流式细胞仪检测72 h后细胞凋亡率;以RT-PCR检测72 h后JAK2、STAT3、Survivin mRNA相对表达量;以WB检测72 h后IL-6蛋白、JAK2、STAT3、Survivin蛋白表达并计算p-JAK2/JAK2、p-STAT3/STAT3、p-Survivin/Survivin。结果72 h后,倒置显微镜下观察结果显示对照组细胞紧密,轮廓清晰,贴壁生长状态良好;A组细胞有浮起、变圆,B组和C组可见不同程度细胞壁皱缩和细胞碎片,其中C组变化最为明显,B组次之,A组变化最轻;各组24 h、48 h、72 h后增殖抑制率比较差异均有统计学意义(P<0.05),各组增殖抑制率均随时间延长显著增长,且呈时间依赖性和剂量依赖性;各组72 h后细胞凋亡率比较差异有统计学意义(P<0.05),各组凋亡率均随剂量升高显著增高;各组72 h后JAK2、STAT3、Survivin mRNA相对表达量比较差异均无统计学意义(P>0.05);各组p-JAK2/JAK2、p-STAT3/STAT3、p-Survivin/Survivin比较差异均有统计学意义(P<0.05),每两组间比较也可见差异具有统计学意义(P<0.05),其中对照组均最高,A组均次之,B组均稍低,C组均最低;各组IL-6蛋白表达比较差异无统计学意义(P>0.05)。结论钩吻总碱能够呈剂量依赖性抑制人舌癌细胞株Tca8113增殖、促进凋亡,推测与直接调控JAK2/STAT3/Survivin通路,抑制JAK2、STAT3、Survivin蛋白磷酸化有关。

Effects of Gelsemium elegans Benth on proliferation and apoptosis of human tongue cancer cell line Tca8113 via by the regulation JAK2/STAT3/Survivin pathway

Objective To observe the effects of Gelsemium elegans Benth on proliferation and apoptosis of human tongue cancer cell line Tca8113,and to explore the regulation of JAK2/STAT3/Survivin pathway.Methods A human tongue cancer cell line Tca8113 was cultured to the logarithmic phase,which were divided into 6 hole plates,divided into control group,A group,B group and C group,with 5 holes in each group.The group A,group B and group C were treated with 25μmol/L,50μmol/L,100μmol/L concentration of Gelsemium elegans Benth(20μl).The control group added the same amount of PBS buffer.The morphology of cells in each group after 72 h was observed by inverted microscope.The inhibition rates of cell proliferation after 24 h,48 h and 72 h were detected by MTT.The apoptosis rates of 72 h after flow cytometry were detected by flow cytometry.The relative expressions of JAK2,STAT3 and Survivin mRNA after 72 h were detected by RT-PCR.The expressions of IL-6,JAK2,STAT3 and Survivin were detected by WB at 72 h and p-JAK2/JAK2,p-STAT3/STAT3,p-Survivin/Survivin were calculated.Results After 72 h,the inverted microscope showed that the cells in the control group were compact,clear outline and well adhered to the wall.The cells in group A were floating and rounded,the cells in group B and group C showed varying degrees of cell wall shrinkage and cell fragments.Among them,the C group had the most obvious change,the B group took the second place,and the A group changed the lightest.There were significant differences in proliferation inhibition rate after 24 h,48 h and 72 h(P<0.05),and it was time dependent and dose dependent.There was significant difference in apoptosis rates after 72 h among each group(P<0.05),and in a dose-dependent manner.There was no significant difference in the relative expression of JAK2,STAT3 and Survivin mRNA between the two groups 72 hours later(P>0.05).There were significant differences in p-JAK2/JAK2,p-STAT3/STAT3,p-Survivin/Survivin among the three groups(P<0.05).The differences between each 2 groups were also statistically significant(P<0.05).The p-JAK2/JAK2,p-STAT3/STAT3,p-Survivin/Survivin in the control group were the highest,followed by the A group,the B group and the C group.There was no significant difference in IL-6 protein expressions among all groups(P>0.05).Conclusion The Gelsemium elegans Benth can inhibit the proliferation and promote apoptosis of human tongue cancer cell line Tca8113,which may be related to the direct regulation of JAK2/STAT3/Survivin pathway and the inhibition of JAK2,STAT3,Survivin protein phosphorylation.