芍药汤含药血清对结肠癌HT29细胞凋亡克隆迁移和侵袭的影响

Effect of Shaoyao Decoction on apoptosis,cloning,migration and invasion of HT29 cells of colon cancer

目的探讨芍药汤(SYD)含药血清是否可以诱导结肠癌HT29细胞的凋亡,抑制其克隆、迁移和侵袭。方法体外培养HT29细胞,取对数生长期的细胞进行相关实验。制备SYD含药血清,CCK8法检测,5%、10%、15%、20%浓度的SYD含药血清对HT29细胞增殖的影响,筛选出最佳浓度用于后续实验。分为对照组、SYD含药血清组。首先,显微镜下观察HT29细胞的形态变化。然后,进行克隆实验,划痕实验,Transwell实验,探究SYD含药血清是否抑制HT29细胞的克隆和迁移、侵袭能力。接着,采用流式分析的方法,Annexin V/PI标记HT29细胞检测对凋亡的影响,以及Western Blot技术,检测了HT29细胞相关凋亡蛋白Cleaved Caspase-3、Survivin和EMT相关蛋白Vimentin、E-cadherin和N-cadherin的表达。结果SYD含药血清对HT29细胞有明显抑制作用,且受抑制情况与作用时间呈正相关(P<0.01)。选用10%、15%浓度的含药血清进行后续实验。克隆实验显示,与对照组相比,SYD含药血清组明显抑制HT29细胞克隆形成的数目(P<0.01),划痕实验和Transwell实验结果显示,SYD含药血清组抑制HT29细胞迁移和侵袭(P<0.05)。流式凋亡实验证实,SYD含药血清能够诱导HT29细胞早期凋亡(P<0.01)。此外,Westernblot也发现,SYD含药血清组细胞凋亡相关蛋白Survivin表达降低(P<0.01),Cleaved Caspase-3表达升高(P<0.01)。EMT相关蛋白N-cadherin、Vimentin表达降低(P<0.01),E-cadherin表达升高(P<0.01)。结论芍药汤含药血清能够诱导结肠癌HT29细胞凋亡并抑制其克隆,通过调控EMT抑制HT29细胞的迁移和侵袭。

Objective To investigate whether the drug-containing serum of Shaoyao Decoction(SYD)can induce apoptosis of HT29 cells of colon cancer and inhibit their cloning,migration and invasion.Methods HT29 cells were cultured in vitro,and the cells of logarithmic growth stage were selected for related experiments.SYD drug-containing serum was prepared,the effect of 5%,10%,15%and 20%SYD drug-containing serum on the proliferation of HT29 cells was detected by CCK8 method,and the optimal concentration was selected for subsequent experiments.They were divided into control group and SYD drug-containing serum group.First,the morphological changes of HT29 cells were observed under microscope.Then,cloning experiment,scratch experiment and Transwell experiment were conducted to explore whether SYD drug-containing serum inhibited the cloning,migration and invasion ability of HT29 cells.Then,flow analysis was used to detect the effect of Annexin V/PI tagged HT29 cells on apoptosis,and Western Blot technique was used.Expression levels of Cleaved Caspase-3,Survivin,and EMT-related proteins Vimentin,E-cadherin,and N-cadherin in HT29 cells were measured.Results The drug-containing serum of SYD had obvious inhibitory effect on HT29 cells,and the inhibitory state was positively correlated with the time of action(P<0.01).Use 10%,15%concentration of drug-containing serum for follow-up experiments.The clone experiment showed that compared with the control group,the number of HT29 cell clone formation was significantly inhibited in the SYD drug-containing serum group(P<0.01).Scratch test and Transwell test showed that the migration and invasion of HT29 cells were inhibited in the SYD drug-containing serum group(P<0.05).Flow cytometry showed that SYD drug-containing serum could induce early apoptosis of HT29 cells(P<0.01).In addition,Western blot analysis showed that apoptosis-related protein Survivin expression decreased(P<0.01),Cleaved Caspase-3 expression increased(P<0.01)in the SYD drug-containing serum group.The expression of EMT-related proteins N-cadherin and Vimentin was decreased(P<0.01),and the expression of E-cadherin was increased(P<0.01).Conclusion Shaoyao Decoction serum can induce apoptosis and inhibit the cloning of HT29 cells of colon cancer,and inhibit the migration and invasion of HT29 cells by regulating EMT.