地龙提取物通过抑制wnt/β-catenin通路促进人卵巢癌细胞凋亡的实验研究

Experimental Study of Dilong(Pheretima)Extract Promoting Apoptosis of Human Ovarian Cancer Cells by Inhibiting Wnt/β-Catenin Signaling Pathway

目的探讨地龙提取物对人卵巢癌细胞凋亡的影响及生物学机制。方法获取正常人卵巢上皮细胞和卵巢癌细胞,应用免疫组化检测细胞内wnt3a、β-catenin、c-Myc、Cyclin D1蛋白表达,应用PCR检测细胞wnt3a mRNA、β-catenin mRNA、c-Myc mRNA、Cyclin D1mRNA相对表达量,应用western-blot检测细胞wnt3a、β-catenin、c-Myc、Cyclin D1蛋白表达;分别应用终浓度400 mg/L和800 mg/L地龙提取物干预卵巢癌细胞生长72 h后、应用免疫组化检测细胞内wnt3a、β-catenin、c-Myc、Cyclin D1蛋白表达,应用PCR检测细胞wnt3a mRNA、β-catenin mRNA、c-Myc mRNA、Cyclin D1 mRNA相对表达量,应用western-blot检测细胞wnt3a、β-catenin、c-Myc、Cyclin D1蛋白表达,应用Tunel法分别检测卵巢癌细胞24、48、72 h凋亡率。结果与正常人卵巢上皮细胞比较,免疫组化结果显示卵巢癌细胞表达蛋白wnt3a[(5.61±1.13)分vs(0.79±0.21)分,t=9.60,P=0.00]、β-catenin[(5.27±1.40)分vs(0.74±0.25)分,t=7.12,P=0.00]、c-Myc[(4.15±1.70)分vs(1.10±0.30)分,t=3.88,P=0.01]、Cyclin D1[(5.42±1.90)分vs(1.00±0.38)分,t=5.07,P=0.00]均增多,PCR结果显示卵巢癌细胞wnt3amRNA[(7.20±2.12)倍vs(1.00±0.10)倍,t=6.92,P=0.00]、β-catenin mRNA[(6.44±1.27)倍vs(0.99±0.09)倍,t=10.03,P=0.00]、c-Myc mRNA[(3.65±1.10)倍vs(1.00±0.10)倍,t=5.26,P=0.00]、Cyclin D1mRNA[(6.50±1.72)倍vs(1.00±0.10)倍,t=20.44,P=0.00]相对表达量均增高,western-blot结果显示卵巢癌细胞wnt3a[(0.61±0.20)vs(0.18±0.10),t=4.00,P=0.00]、β-catenin[(1.24±0.43)vs(0.32±0.09),t=4.88,P=0.00]、c-Myc[(0.80±0.27)vs(0.11±0.08),t=4.95,P=0.00]、Cyclin D1[(0.51±0.18)vs(0.10±0.07),t=11.19,P=0.00]均增多;经地龙提取物干预后,免疫组化结果显示卵巢癌细胞表达蛋白wnt3a[0 mg组(5.80±1.40)分,400 mg组(3.10±1.00)分,800 mg组(1.60±0.30)分,F=22.28,P=0.00]、β-catenin[0 mg组(5.00±1.60)分,400 mg组(3.70±1.10)分,800 mg组(2.40±1.00)分,F=5.31,P=0.02]、c-Myc[0 mg组(4.60±2.11)分,400 mg组(3.58±1.75)分,800 mg组(1.20±0.40)分,F=6.49,P=0.01]、Cyclin D1[0 mg组(5.70±1.80)分,400 mg组(2.61±1.20)分,800 mg组(1.82±0.54)分,F=12.91,P=0.00]均降低,PCR结果显示卵巢癌细胞wnt3a mRNA[0 mg组(1.00±0.12),400 mg组(0.63±0.18),800 mg组(0.20±0.09),F=40.00,P=0.00]、β-catenin mRNA[0 mg组(1.00±0.09),400 mg组(0.41±0.09),800 mg组(0.10±0.08),F=112.10,P=0.00]、c-Myc mRNA[0 mg组(1.00±0.07),400 mg组(0.71±0.22),800 mg组(0.30±0.09),F=31.84,P=0.00]、Cyclin D1mRNA[0 mg组(1.00±0.10),400 mg组(0.48±0.20),800 mg组(0.23±0.09),F=40.83,P=0.00]相对表达量均减少,western-blot结果显示卵巢癌细胞表达蛋白wnt3a[0 mg组(1.33±0.42),400 mg组(0.81±0.30),800 mg组(0.10±0.08),F=20.96,P=0.00]、β-catenin[0 mg组(1.81±0.52),400 mg组(0.90±0.27),800 mg组(0.38±0.19),F=19.87,P=0.00]、c-Myc[0 mg组(1.41±0.40),400 mg组(0.28±0.10),800 mg组(0.10±0.06),F=40.83,P=0.00]、Cyclin D1[0 mg组(1.10±0.38),400 mg组(0.78±0.26),800 mg组(0.11±0.06),F=20.79,P=0.00]均减少,Tunel结果显示72h卵巢癌细胞凋亡率增高[0 mg组(2.17±1.10)%,400 mg组(17.20±3.65)%,800mg组(28.64±7.11)%,F=40.62,P=0.00]。结论地龙提取物可以通过抑制wnt/β-catenin通路来促进人卵巢癌细胞的凋亡,并且以终浓度800 mg/L为佳。

Objective To explore the effect of Dilong(Pheretima)extract on apoptosis of human ovarian cancer cells and its biological mechanism.Methods The levels of wnt3a,β-catenin,c-Myc and Cyclin D1 protein in normal ovarian epithelial cells and ovarian cancer cells were detected by immunohistochemistry(IHC)and Western blot,and the relative expression levels of wnt3a mRNA,β-catenin mRNA,c-Myc mRNA and Cyclin D1mRNA in cells were detected by PCR.The levels of wnt3a,β-catenin,c-Myc and Cyclin D1 protein in ovarian cancer cells were detected by IHC and Western blot after using the Dilong(Pheretima)extract with final concentration of 400 mg/L and 800 mg/L to interfere with the growth of ovarian cancer cells for 72 hours.PCR was used to detect the relative expression levels of wnt3a mRNA,β-catenin mRNA,c-Myc mRNA and Cyclin D1 mRNA,and Tunel method was used to detect the apoptosis rate of ovarian cancer cells at 24 h,48 h and 72 h respectively.Results Compared with those of the normal group,the IHC results showed that the levels of wnt3a[(5.61±1.13)score vs(0.79±0.21)score,t=9.60,P=0.00],β-catenin[(5.27±1.40)score vs(0.74±0.25)score,t=7.12,P=0.00],c-Myc[(4.15±1.70)score vs(1.10±0.30)score,t=3.88,P=0.01],Cyclin D1[(5.42±1.90)score vs(1.00±0.38)score,t=5.07,P=0.00]were higher in A2780 group.PCR results showed that the relative folds of wnt3a mRNA[(7.20±2.12)fold vs(1.00±0.10)fold,t=6.92,P=0.00],β-catenin mRNA[(6.44±1.27)fold vs(0.99±0.09)fold,t=10.03,P=0.00],c-Myc mRNA[(3.65±1.10)fold vs(1.00±0.10)fold,t=5.26,P=0.00],Cyclin D1mRNA[(6.50±1.72)fold vs(1.00±0.10)fold,t=20.44,P=0.00]were higher in A2780 group.The results of Western blot showed that the levels of wnt3a[(0.61±0.20)vs(0.18±0.10),t=4.00,P=0.00],β-catenin[(1.24±0.43)vs(0.32±0.09),t=4.88,P=0.00],c-Myc[(0.80±0.27 vs 0.11±0.08,t=4.95,P=0.00],Cyclin D1[(0.51±0.18)vs(0.10±0.07),t=11.19,P=0.00]were higher in A2780 group.After intervention by Dilong(Pheretima)extract,the IHC results showed that the levels of wnt3a[0 mg(5.80±1.40)score,400 mg(3.10±1.00)score,800 mg(1.60±0.30)score,F=22.28,P=0.00],β-catenin[0 mg(5.00±1.60)score,400 mg(3.70±1.10)score,800 mg(2.40±1.00)score,F=5.31,P=0.02],c-Myc[0 mg(4.60±2.11)score,400 mg(3.58±1.75)score,800 mg(1.20±0.40)score,F=6.49,P=0.01],Cyclin D1[0 mg(5.70±1.80)score,400 mg(2.61±1.20)score,800 mg(1.82±0.54)score,F=12.91,P=0.00]in A2780 group decreased.PCR results showed that the relative folds of wnt3amRNA[0 mg(1.00±0.12),400 mg(0.63±0.18),800 mg(0.20±0.09),F=40.00,P=0.00],β-catenin mRNA[0 mg(1.00±0.09),400 mg(0.41±0.09),800 mg(0.10±0.08),F=112.10,P=0.00],c-Myc mRNA[0 mg(1.00±0.07),400 mg(0.71±0.22),800 mg(0.30±0.09),F=31.84,P=0.00],Cyclin D1mRNA[0 mg(1.00±0.10),400 mg(0.48±0.20),800 mg(0.23±0.09),F=40.83,P=0.00]in A2780 group decreased.The results of Western blot showed that the levels of wnt3a[0 mg(1.33±0.42),400 mg(0.81±0.30),800 mg(0.10±0.0),F=20.96,P=0.00],β-catenin[0 mg(1.81±0.52),400 mg(0.90±0.27),800 mg(0.38±0.1),F=19.87,P=0.00],c-Myc[0 mg(1.41±0.40),400 mg(0.28±0.10),800 mg(0.10±0.06),F=40.83,P=0.00],Cyclin D1[0 mg(1.10±0.38),400 mg(0.78±0.26),800 mg(0.11±0.06),F=20.79,P=0.00]in A2780 group decreased.Tunel results showed that the apoptosis rate of ovarian cancer cells increased after 72 hours[0 mg(2.17±1.10)%,400 mg(17.20±3.65)%,800 mg(28.64±7.11)%,F=40.62,P=0.00].Conclusion The Dilong(Pheretima)extract could promote the apoptosis of human ovarian cancer cells by inhibiting Wnt/β-catenin pathway,and the final concentration of 800 mg/L was better.