桑黄多糖抑制胶质瘤细胞增殖与迁移作用机制研究

目的桑黄多糖(phellinus igniarius polysaccharide,PP)属于真菌类多糖,具有抑制肿瘤细胞生长和转移的作用,并能够促进肿瘤细胞凋亡。目前关于桑黄多糖对胶质瘤细胞增殖与凋亡的影响尚罕见相关报道。因此,本研究探讨桑黄多糖对胶质瘤细胞增殖、转移与凋亡的影响及其可能的作用机制。方法使用不同浓度PP处理胶质瘤细胞,应用MTT比色法测胶质瘤细胞的增殖抑制率,采用划痕实验和Transwell细胞侵袭实验检测胶质瘤细胞的迁移和侵袭能力,使用流式细胞计数测定胶质瘤细胞的凋亡及细胞周期,并使用蛋白印迹法及实时荧光定量PCR(quantitative realtime PCR,qRT-PCR)测定PI3K、Akt、p-PI3K、p-Akt、Cyclin A、CDK4、Cyt-C和Caspase3的变化。结果 MTT检测结果表明,与对照组比较,经PP处理24h后各组细胞增殖抑制率差异有统计学意义,F=34.230,P<0.05;经PP处理48h后各组细胞增殖抑制率差异有统计学意义,F=1 260.663,P<0.05。划痕及Transwell细胞侵袭实验显示,经PP处理48h后的细胞比对照组细胞的迁移率明显降低,穿膜细胞数明显减少,F值分别为91.381和111.711,均P<0.05。经PP处理48h后胶质瘤U251细胞凋亡结果显示,对照组、A组、B组、C组细胞凋亡率差异有统计学意义,F=175.669,P<0.05;G0/G1期比例显著降低,F=175.440,P<0.05;而S期比例升高,F=72.839,P<0.05。蛋白质印迹及qRT-PCR结果显示,与对照组比较,A、B、C组U251细胞中Cyclin A、CDK4蛋白(F值分别为138.592、208.075,均P<0.05)及mRNA(F值分别为238.489、29.200,均P<0.05)明显下降,Cyt-C、Caspase3蛋白(F值分别为107.956、19.818,P<0.05)及mRNA(F值分别为76.297、46.933,均P<0.05)的表达明显增加;经PP处理48h后胶质瘤U251细胞中PI3K、Akt、p-PI3K、p-Akt蛋白(F值分别为155.631、167.037、167.691和153.383,均P<0.05)及PI3K、Akt mRNA(F值分别为288.840、46.960,均P<0.05)的表达明显降低。结论桑黄多糖可能是通过PI3K/Akt信号通路抑制胶质瘤细胞的增殖与转移。

Mechanism of inhibitory effect of Phelinus linteus polysaccharide on glioma cell proliferation and migration through PI3K/Akt signaling pathway

OBJECTIVE Phellinus igniarius polysaccharide(PP)is a kind of fungal polysaccharide,which can inhibit the growth and metastasis of tumor cells and promote the apoptosis of tumor cells.However,the effect of PP on glioma cells has not been reported yet.The effect of PP on the proliferation and metastasis of glioma cells was studied,and explored its possible mechanism in the study.METHODS Glioma cells were treated with different concentrations of PP for 48 h.Application of MTT colorimetry in the detection of proliferation inhibition rate of glioma cells.Using scratch test and Transwell cell invasion test to detect the migration and invasion ability of glioma cells.The apoptosis and cell cycle of glioma cells were measured by flow cytometry.Western blotting and quantitative real-time PCR(qRT-PCR)were used to detect the changes of PI3K,Akt,p-PI3K,p-Akt,Cyclin A,CDK4,Cyt-C and Caspase 3.RESULTS MTT results showed that there was a significant difference in cell viability between groups treated with PP for 24 h(F=34.230,P<0.05).Also there was a significant difference in proliferation inhibition rate between groups treated with PP for 48 h(F=1 260.663,P<0.05).The results of scratch test and Transwell cell invasion test showed that the migration rate and the number of transmembrane cells after 48 hours of treatment with PP was significantly lower than those of control group(F=91.381,P<0.05;F=111.711,P<0.05).After 48 hours of PP treatment,the apoptosis rates of U251 cells in the control group,A group,B group and C group were significantly different,and the proportion of G0/G1 phase was significantly reduced while that of S phase was increased(Fapoptosis=175.669,P<0.05;FG0/G1=175.440,P<0.05;FS phase=72.839,P<0.05).Western blotting and qRT-PCR results showed that the expressions of Cyclin A,CDK4 protein(F=138.592,208.075,P<0.05)and mRNA(F=238.489,29.200,P<0.05)in U251 glioma cells were decreased significantly after 48 hours of treatment with PP compared with the control group,while the expressions of Cyt-C,Caspase 3 protein(F=107.956,19.818,P<0.05)and mRNA(F=76.297,46.933,P<0.05)were increased significantly.Western blotting and fluorescence quantitative PCR showed that the expressions of PI3K,Akt,p-PI3K,p-Akt protein(F values were 155.631,167.037,167.691 and 153.383,all P<0.05)and PI3K,Akt mRNA(Fvalues were 288.840 and 46.960,both P<0.05)in U251 glioma cells treated with PP were significantly lower than that of control group.CONCLUSION The PP may inhibit the proliferation and metastasis of glioma cells through PI3K/Akt signaling pathway.