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目的:探讨健脾化瘀法含药血清对肝癌细胞Smad3蛋白磷酸化及上皮间质转移标志蛋白E-cadherin/Ncadherin表达水平及侵袭转移能力的影响。方法:分别以不同浓度(10%和20%)健脾化瘀法中药含药血清及10%小牛血清对照组培养肝癌细胞MHCC97-H,实时定量PCR检测细胞Smad3和Snail mRNA水平,Western blot检测Smad3、p-Smad3、Snail、E-cadherin和N-cadherin蛋白表达水平。划痕法和Transwell小室检测肝癌细胞侵袭/迁移能力。结果:实时定量PCR结果显示高浓度组(20%含药血清组)、低浓度组(10%含药血清组)和对照组(10%小牛血清对照)Smad3的mRNA水平分别为0.87±0.24、0.97±0.28和1.18±0.34(P>0.05);高浓度组Snail的mRNA水平低于对照组(0.49±0.05 vs.1.10±0.21,P=0.034)。与对照组相比,高浓度含药血清组48h的p-Smad3、Snail和N-cadherin明显降低,E-cadherin明显升高(P<0.05)。与对照组相比,低浓度含药血清组48h的E-cadherin升高,N-cadherin降低(P<0.05);而Smad3、p-Smad3及snail的结果与对照组无明显差异(P>0.05)。划痕法结果显示,与对照组比较,20%含药血清组及10%含药血清组肝癌细胞的划痕愈合较慢,差异均有统计学意义(P<0.05)。Transwell显示与对照组相比,20%含药血清组及10%含药血清组MHCC97-H细胞的迁移受抑制,差异有统计学意义(P<0.05)。结论:健脾化瘀法中药能抑制肝癌细胞侵袭转移,可能与抑制Smad3蛋白磷酸化及上皮间质转化有关。
Objective: To investigate the effect of Jianpi Huayu Decoction-containing serum on the phosphorylation of Smad3 protein and the expression of E-cadherin / Ncadherin and the invasion and metastasis of hepatocellular carcinoma cells. Methods: Liver cancer cells MHCC97-H were cultured in Chinese medicated serum with different concentration (10% and 20%) and control group with 10% fetal calf serum respectively. Smad3 and Snail mRNA were detected by real-time quantitative PCR. Western blot The expression of Smad3, p-Smad3, Snail, E-cadherin and N-cadherin were detected. Scratch assay and Transwell chambers to detect hepatocellular carcinoma cell invasion / migration ability. Results: The results of real-time PCR showed that the mRNA levels of Smad3 in high concentration group (20% serum group), low concentration group (10% serum group) and control group (10% fetal bovine serum control group) were 0.87 ± 0.24 , 0.97 ± 0.28 and 1.18 ± 0.34 respectively (P> 0.05). The mRNA level of Snail in high concentration group was lower than that in control group (0.49 ± 0.05 vs.1.10 ± 0.21, P = 0.034). Compared with the control group, p-Smad3, Snail and N-cadherin were significantly decreased and E-cadherin was significantly increased at 48h (P <0.05). Compared with the control group, E-cadherin and N-cadherin in low-concentration group were significantly increased at 48h (P <0.05), but not in Smad3, p-Smad3 and snail group (P> 0.05 ). Scratch test results showed that compared with the control group, the wound healing of 20% drug-containing serum group and 10% drug-containing serum group was slow, the differences were statistically significant (P <0.05). Transwell showed that compared with the control group, the migration of MHCC97-H cells in 20% drug-containing serum group and 10% drug-containing serum group was inhibited, with statistical significance (P <0.05). Conclusion: Chinese herbal medicine for invigorating the spleen and removing blood stasis can inhibit the invasion and metastasis of hepatocellular carcinoma cells, which may be related to the inhibition of Smad3 phosphorylation and epithelial-mesenchymal transition.