肝癌细胞促进骨髓间充质干细胞向肿瘤相关成纤维细胞转化

Hepatoma cells conditioned medium promoting transition of bone marrow mesenchymai stem cells to tumor associated fibroblasts

目的探讨在体外实验条件中肝癌细胞能否通过旁分泌途径促进骨髓间充质千细胞（BMSCs）向肿瘤相关成纤维细胞（TAF）转化。方法提取肝癌肿瘤细胞HepG2的培养液上清（CM），实验组为10％胎牛血清＋HepG2-CM，对照组为10％胎牛血清＋DMEM，应用细胞计数试剂盒（CCK-8）分别检测12、24、48、72h细胞增殖，细胞免疫荧光染色、Westernblot法检测旺．平滑肌肌动蛋白（α-SMA）、肌间线蛋白（Desmin）、成纤维细胞活化蛋白（FAP）、凝血酶敏感蛋白1（Tsp-1）、成纤维细胞特异蛋白（FSP）等TAF特异相关蛋白的表达。结果实验组的BMSC细胞增殖较对照组明显增加（48h：0．9772±0．1280比0．6928士0．0784；72h：1．4206±0．2642tLo．8456±0．1294），填差肄有统计学意义（P〈0．05）；细胞免疫荧光染色、Westernblot结果提示实验组α-SMA、Desmin、FAP、Tsp-1、FSP蛋白较对照组明显上调，且随着时间的增加表达增加（P〈0．05）。结论肝癌细胞可促进骨髓间充质干细胞的增殖，并通过旁分泌机制促进骨髓间充质干细胞中TAF特异相关蛋白α-SMA、Desmin、FAP、Tsp-1、FSP等的表达，进而促进骨髓间充质干细胞向肿瘤相关成纤维细胞转化。

Objective To investigate if the hepatoma cells conditioned media （CM） can promote transition of bone marrow mesenchymal stem cells （BMSCs） to the tumor associated fibroblasts （TAF） through paracrine pathway in vitro. Methods BMSCs were isolated and cultured, and CM were collected form culture supernatant of liver tumor cells HepG2. 10% fetal bovine serum and HepG2 - CM was added in experimental group, and 10% fetal bovine serum and DMEM were added in the control group. Cell pro- liferation was determined at 12, 24, 48, and 72 h by cell counting kit- 8 （CCK- 8） method. Immunoflu- orescence staining and Western blotting methods were used for the detection of α - smooth muscle actin （α -SMA）, Desmin, fibroblast activation protein （FAP）, thrombospondin - 1 （Tsp - 1 ）, fibroblast - specific protein （FSP） of specific TAF related protein. Results The proliferation of BMSCs in experimen- tal group was increased significantly compared to the control group （ at 48 h： 0. 977 2± 0. 128 0 vs. 0. 692 8± 0. 078 4 ; at 72 h ： 1. 420 6 ± 0. 264 2 vs. 0. 845 6 ±0. 129 4, P 〈 0. 05）. The results of immu- nofluorescence staining and Western blotting indicated that alpha - SMA, Desmin, FAP, Tsp - 1 and FSP proteins in experimental group was up - regulated significantly as compared with the control group, and with the time prolongation, the expression of those proteins increased （ P 〈 0.05 ）. Conclusion Hepatoma cells CM can promote proliferation of BMSCs, and the expression of TAF related proteins alpha -SMA, Desmin, FAP, Tsp - 1 and FSP in BMSCs through paracrine mechanism. Hepatoma cells CM can promote transition of BMSCs to the tumor associated fibroblasts.