蟾酥诱发人成骨肉瘤MG-63细胞凋亡的作用机制研究

Toad venom induces apoptosis of human osteosarcoma MG-63 cells via down-regulating Integrin α4

目的探究蟾酥对人骨肉瘤MG-63细胞增殖、凋亡、侵袭、转移的影响及可能作用机制。方法取雄性C57BL/6J小鼠30只,制备空白血清和不同浓度(2.5%和5%)的蟾酥含药血清。取传代培养MG-63细胞,实验分为3组:2.5%蟾酥含药血清组和5%蟾酥含药血清组分别加入相应浓度的蟾酥含药血清培养,对照组加入等体积PBS培养。CCK-8法检测MG-63细胞增殖活性,PNPP法检测细胞中碱性磷酸酶(ALP)活性,茜素红法检测细胞成骨能力,Transwell法检测MG-63细胞侵袭、转移情况,流式细胞仪检测细胞凋亡率及MG-63细胞中整合素α4(Integrinα4)表达情况。结果不同浓度蟾酥含药血清组各时间点细胞增殖率均明显低于对照组(P均<0.05),且5%蟾酥含药血清组均明显低于同期2.5%蟾酥含药血清组(P均<0.05);不同浓度蟾酥含药血清组细胞凋亡率、ALP活性均明显高于对照组(P均<0.05),且5%蟾酥含药血清组均明显高于2.5%蟾酥含药血清组(P<0.05);茜素红染色显示,不同浓度含药血清组有橘红色结节、边界清楚细胞增多;不同浓度蟾酥含药血清组侵袭性细胞和转移性细胞数量均明显少于对照组(P均<0.05),Integrinα4表达占比均明显低于对照组(P均<0.05),且5%蟾酥含药血清组细胞数量均明显少于2.5%蟾酥含药血清组(P均<0.05),Integrinα4表达占比明显低于2.5%蟾酥含药血清组(P<0.05)。结论蟾酥可以促进MG-63细胞凋亡,抑制细胞增殖、侵袭和转移,改善细胞分化和成骨功能,机制可能与下调Integrinα4表达有关。

Objective It is to explore the effects and possible mechanisms of action of toad venom on proliferation,apoptosis,invasion and metastasis of human osteosarcoma MG-63 cells.Methods Thirty male C57BL/6J mice were taken to prepare blank serum and different concentrations(2.5% and 5%)of medicated serum containing toad venom.The passaged cultured MG-63 cells were divided into 3 groups:the 2.5% toad venom medicated serum group and 5%toad venom medicated serum group were cultured with the corresponding concentrations of toad venom serum,and the control group was cultured with an equal volume of PBS.The proliferative activity of MG-63 cells was detected by CCK-8 assay,the activity of alkaline phosphatase(ALP)in the cells was detected by PNPP assay,the osteogenic ability of cells was detected by alizarin red assay,the invasion and metastasis of MG-63 cells were detected by Transwell assay,and the apoptosis rate and the expression of integrin α4 in MG-63 cells were detected by flow cytometry.Results The cell proliferation rates of toad venom medicated serum groups of different concentrations were significantly lower than those of the control group at all time points(all P<0.05),and the 5% toad venom medicated serum group was significantly lower than that of the 2.5% toad venom medicated serum group at the same period(all P<0.05);the apoptosis rates and ALP activities of the toad venom medicated serum groups of different concentrations were significantly higher than that of the control group(all P<0.05),and the 5%toad venom medicated serum group was significantly higher than that of the 2.5%toad venom medicated serum group(P<0.05);Transwell staining results showed that there were orange-red nodules and increased cells with clear boundaries in toad venom medicated serum groups of different concentration;the numbers of invasive cells and metastatic cells in the toad venom medicated serum groups of different concentrations were significantly less than those in the control group(all P<0.05),and the proportion of Integrinα4 expression was significantly lower than that in the control group(P<0.05),and the numbers of invasive cells and metastatic cells in the 5% toad venom medicated serum group were significantly less than those in the 2.5% toad venom medicated serum group(all P<0.05),and the proportion of Integrinα4 expression in the 5% toad venom medicated serum group was significantly lower than that in the 2.5% toad venom medicated serum group(P<0.05).Conclusion Toad venom can promote apoptosis,inhibit proliferation,invasion and metastasis,and improve differentiation and osteogenesis of MG-63 cells,and the mechanism may be related to down-regulating expression of Integrinα4.