摘要
目的评估肝癌HepG2细胞分泌的Exosome对间充质干细胞(MSC)分化为肿瘤相关成纤维细胞(CAF)的影响及其相互作用机制。方法在人脂肪MSC培养体系中加入HepG2细胞分泌的Exosome进行培养,采用Western blot法检测CAF特征性蛋白的表达情况。将已鉴定为CAF样条件培养基(CAF-CM)和牛血清白蛋白对照组分别加至HepG2培养基中,采用Western blot法和定量PCR检测上皮间质转化相关基因的表达,MTS法检测细胞增殖,Transwell法检测HepG2细胞的迁移和侵袭。结果 HepG2细胞分泌的Exosome表达CD63、热休克蛋白(HSP)70和HSP90。将Exosome加入MSC培养体系后14 d,可检测到α平滑肌肌动蛋白、成纤维细胞激活蛋白α、白细胞介素(IL)-6、IL-8和IL-1β等CAF特征性蛋白的表达。MTS法检测CAF-CM组的细胞增殖OD值为1.075±0.104,明显高于对照组的0.874±0.066(P=0.023)和MSC条件培养基组的0.649±0.034(P=0.0005)。CAF-CM组迁移的HepG2细胞数为(42.5±9.1)个,明显高于对照组的(18.5±3.1)个(P=0.001);CAF-CM组侵袭细胞数为(29.0±3.5)个,明显高于对照组的(13.1±3.7)个(P=0.009)。CAF-CM组Smad交互蛋白1(P=0.040)、β-catenin(P=0.038)、纤连蛋白(P=0.029)和Vimentin(P=0.013)的表达较对照组显著上调;而CAF-CM组的紧密连接蛋白表达显著低于对照组(P=0.010)。结论肝癌HepG2细胞系分泌的Exosome可诱导脂肪组织来源的MSC分化为CAF,后者又可以促进HepG2细胞的增殖、迁移和侵袭。
Objective To investigate the effect of human hepatocellular carcinoma HepG2 cell-derived Exosome on the differentiation of mesenchymal stem cells（MSC） into cancer-associated myofibroblasts（CAF）and the impacts of CAF on liver cancer cell proliferation,migration,and invasion. Methods The protein expression of HepG2 cell-derived Exosome was detected by Western blotting. MSCs were separated from human adi-pose tissue and cultured with HepG2 cell-derived Exosome（100 ng/nl） to initiate differentiation. The expressions of mesenchymal markers and several interleukins were also detected by Western blotting. HepG2 cells were co-cultured with the conditioned media（CM）,in which HepG2 Exosome induced the differentiation of MSC into CAF. The expressions of epithelial and mesenchymal markers were detected by real-time polymerase chain reaction（PCR） and Western blotting. Cell proliferation was assessed using MTS assay. Transwell chambers were used in the in vitro migration and invasion assay. Results HepG2 cell-derived particles expressed CD63,70 kilodalton heat shock proteins, and 90 kilodalton heat shock proteins. With the treatment of HepG2 cell-derived Exosome,the expressions of mesenchymal marker α-smooth muscle actin, fibroblast activation proteinα,interleukin（IL）-6,IL-8,and IL-1β were up-regulated,while vascular endothelial growth factor had no significant change. The conditioned media which HepG2 Exosome induced MSC differentiation CAF（CAFCM） could significantly promote Hep G2 cells proliferation（1. 075 ± 0. 104）, compared to BSA control（0. 874 ± 0. 066,P = 0. 023） and MSC-CM（0. 649 ± 0. 034,P = 0. 0005）. CAF-CM could significantly enhance cell migration [（42. 5 ± 9. 1） cells vs.（18. 5 ± 3. 1） cells,P = 0. 001]and invasion [（29. 0 ± 3. 5） cells vs.（13. 1 ± 3. 7） cells,P = 0. 009] compared to its control group. Moreover the conditioned medium which HepG2 Exosome induced MSC to differentiate into CAF could also promote the expressions of mesenchyme-related genes Smad interacting protein 1（P = 0. 040）,β-catenin（P = 0. 038）,fibronectin（P = 0. 029）,and Vimentin（P = 0. 013） and inhibit the expression of epithelial related genes zonula ocdudens-1（P =0. 010）. Conclusions Exosome extracted from HepG2 cells can induce human adipose-derived MSC to differentiate into cancer-associated myofibroblasts. CAF-like cells can promote the migration of the liver cancer cell line HepG2.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2017年第3期312-317,共6页
Acta Academiae Medicinae Sinicae
基金
国家自然科学基金(81472785
61435001)~~
作者简介
通信作者：白春梅电话：010-69158315,电子邮件：baichunmei1964@126.com
