摘要
目的肝癌细胞的上皮间质转化（EMT）与其恶性行为密切相关。而肿瘤微环境对肿瘤细胞的EMT的调控尤为重要。探明肿瘤相关巨噬细胞（TAM）调控Hep3B肝癌细胞发生EMT的可能机制，为肝细胞癌（HCC）的l临床研究和治疗提供新的方案。方法将人白血病单核细胞系（THP-1）成功诱导为TAM。以TAM为靶细胞，分别用Hep3B肝癌细胞的上清液以及与Hep3B细胞共培养的形式干预，用蛋白质印迹法（Western blot）及RT—PCR方法检测TAM的Toll样受体（TLR）4表达变化。用质粒瞬时转染shRNA方法下调TAM的TLR4表达。以Hep3B肝癌细胞为靶细胞，分别用TAM上清液以及转染了shRNATLR4质粒的TAM的上清液干预，用Western blot方法检测Hep3B肝癌细胞的E—cadherin、N—cadherin及Vimentin蛋白表达。两独立样本均数的比较采用双侧f检验。结果THP-1细胞被成功诱导为TAM。Western blot结果显示，与对照组比较，TAM干预组（TAM—CM组）的Hep3B细胞E-cadherin蛋白表达降低、N-cadherin及Vimentin蛋白表达升高。当下调TAM的TLR4表达后，用其培养液干预Hep3B细胞，此时，Hep3B细胞的E-Cadherin表达量较TAM—CM组明显增加，而N—cadherin及Vimentin蛋白表达明显减少。此外，Westernblot及RT-PCR结果显示，TAM的TLR4受体表达水平受Hep3B肝癌细胞的影响。TAM组TLR4基因的表达高于TAM＋HCC—CM组，t=6．321，P=0．0032，差异具有统计学意义；TAM＋Co—HCC组的表达高于TAM＋HCC—CM组，t=3．189，P=0．0332，差异具有统计学意义。结论TAM能促进肝癌细胞Hep3B的上皮间质转化。下调TAM的TLR4表达可削弱TAM对Hep3B肝癌细胞上皮间质转化的影响。表明TAM表面的TLR4受体是TAM与Hep3B肝癌细胞相互作用的关键分子。
Objective To investigate the possible mechanisms of tumor-associated macrophages （TAMs） in regulating epithelia-mesenchymal transition （EMT） of Hep3B hepatoma cells, since EMT is closely associated with the malignancy of hepatoma cells and tumor microenvironment plays an important role in regulating EMT of hepatoma cells, and to provide new regimens for the clinical studies and treatment of liver cancer. Methods Human monocytic leukemia THP-1 cells were successfully induced to TAMs. With TAMs as target cells, they were co-cultured with the supernatant of Hep3B hepatoma cells or Hep3B hepatoma cells, and Western blot and RT-PCR were used to measure the change in the expression of Toll- like receptor 4 （TLR4） in TAMs. The expression of TLR4 in TAMs was downregulated by transient plasmid transfection with shRNA. With Hep3B hepatoma cells as target ceils, the supematants of TAMs and TAMs transfected with shRNA TLR4 plasmid were used for intervention, and Western blot was used to measure the protein expression of E-cadherin, N-cadherin, and vimentin. The two-sided t-test was used for comparison of the means of two independent samples. Results THP-1 cells were successfully induced to TAMs. According to the results of Western blot, compared with the control-CM group, the TAM-CM group had a significant reduction in the protein expression of E-cadherin and significant increases in the protein expression of N-cadherin and vimentin in Hep3B cells. After the expression of TLR4 in TAMs was downregulated, the culture solution of TAMs was used for the intervention of Hep3B cells （shRNA group）, and compared with the TAM-CM group, the shRNA group had a significant increase in the expression of E-cadherin and significant reductions in the protein expression of N-cadherin and vimentin in Hep3B cells. Western blot and RT-PCR showed that the expression of TLR4 in TAMs was influenced by Hep3B cells. Conclusion TAMs can promote EMT of Hep3B hepatoma cells, and downregulation of the expression of TLR4 in TAMs may reduce
作者
姚蓉蓉
王咪咪
王艳红
Yao Rongrong;Wang Mimi;Wang Yanhong(Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, Chin)
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2018年第4期305-309,共5页
Chinese Journal of Hepatology
关键词
癌
肝细胞
肿瘤相关巨噬细胞
TOLL样受体4
上皮间质转化
Carcinoma, hepatocellular
Tumor-associated macrophages
Toll-receptor 4
Epithelial mesenchymal transition
