肝癌细胞自分泌外泌体通过TGF-β1调控自身细胞学行为

Autocrine exosomes in liver cancer cells are regulated by TGF-β1 autologous cell behavior

目的:研究肝癌Huh7细胞外泌体促进自身转移的机制。方法:培养肝癌Huh7细胞,分离提取外泌体并进行鉴定。将分离的外泌体与Huh7细胞共培养,Transwell比较细胞迁移和侵袭能力;检测外泌体中TGF-β1水平;实时荧光定量PCR和Western blot分别检测TGF-β1、SMAD2/3、EMT相关分子表达。结果:提取的外泌体经Western blot检出CD63、TSG101条带;透射电镜检测出双侧膜结构小体;纳米粒径分析显示颗粒大小集中在100 nm左右。外泌体中检出TGF-β1。外泌体与Huh7细胞共培养,免疫荧光显示外泌体成功进入Huh7细胞。与对照组相比,共培养组细胞迁移和侵袭大幅增加,TGF-β/Smad通路相关分子活化,上皮标志物E-钙黏蛋白、p-Smad7表达下降,间充质标志物-波形蛋白表达升高。结论:肝癌Huh7细胞自分泌的外泌体通过运输TGF-β1上调TGF/Smad通路进而影响肝癌转移,探明了肝癌发展机制,为肝细胞癌治疗提供了新思路。

Objective:To study mechanism of liver cancer Huh7 cell exosomes to promote self-metastasis.Methods:Liver can-cer Huh7 cells were cultured,exosomes were isolated,extracted and identified.Isolated exosomes were co-cultured with Huh7 cells,and Transwell was used to compare cell migration and invasion capabilities;TGF-β1 level in exosomes was detected;real-time fluores-cent quantitative PCR and Western blot were used to detect expressions of TGF-β1,SMAD2/3,EMT related molecules.Results:Extracted exosomes was detected CD63 and TSG101 bands by Western blot;transmission electron microscopy detected double-sided membrane structure small bodies;nanometer particle size analysis showed that particle size was concentrated around 100 nm.TGF-β1 was detected in exosomes.Exosomes were co-cultured with Huh7 cells,and immunofluorescence showed that exosomes successfully entered Huh7 cells.Compared with control group,co-culture group has significantly increasing cell migration and invasion,activatal TGF-β/Smad pathway related molecules,decreasing expressions of epithelial marker E-cadherin and p-Smad7,while increasing expression of mesenchymal marker-vimentin.Conclusion:Autocrine exosomes of liver cancer Huh7 cells up-regulate TGF/Smad pathway by transporting TGF-β1 and then affect liver cancer metastasis,further explores mechanism of liver cancer development and provides new ideas for treatment of hepatocellular carcinoma.