S180骨肉瘤细胞TLRs表达及其与肿瘤耐受的相关性

Expression of TLRs in S180 Osteosarcoma and Relationship between TLRs and Tumor Tolerance

目的探讨TLRs在诱导S180骨内瘤细胞免疫耐受中的作用,为肿瘤临床的免疫治疗提供新的思路。方法建立S180腹水癌早晚期模型,采用RT-PCR方法检测体外S180骨肉瘤细胞和荷瘤鼠体内早晚期S180骨肉瘤细胞TLR1-9mRNA的表达水平,以及LPS体外刺激S180骨肉瘤细胞前后TGF-β和IL-10mRNA表达变化,ELISA方法检测LPS体外刺激S180骨肉瘤细胞前后肿瘤上清液中TGF-β蛋白分泌的含量。结果体外S180骨肉瘤细胞表达TLR1-7和TLR9,TLR8不表达。荷瘤鼠体内早晚期S180骨肉瘤细胞TLRs的表达不同,TLR4、9表达早期高于晚期,其中TLR4的高表达有统计学意义(P<0.05);TLR1、2、3、5、6和7表达晚期高于早期,但差异无统计学意义(P>0.05)。10μg/ml的LPS在不同时间点体外刺激S180骨肉瘤细胞后IL-10mRNA表达水平均上调,且有时间依赖关系,TGF-β在mRNA和蛋白水平也均有增加。结论体外S180骨肉瘤细胞表达多种TLRs,荷瘤鼠体内早晚期S180骨肉瘤细胞TLRs表达的不同在肿瘤发展的不同时期可能发挥不同的作用,S180骨肉瘤细胞TLR4信号通路活化后可能通过促进TGF-β和IL-10的分泌从而参与肿瘤的免疫耐受。

Objective To examine the expression of TLRs in 8180 osteosarcoma cells in vitro and at earlier and advanced stages of tumor-bearing mouse in vivo; To reveal the expression of TGF-β and IL-10 after stimulating the S180 osteosarcoma cells by LPS in vitro; And to explore the mechanisms of tumor tolerance induced by TLRs express in tumor cells. Methods S180 ascites carcinoma models were established. The expression of TLRs mRNA in S180 osteosarcoma cells in vitro, and at earlier and advanced stages of tumor-bearing mouse in vivo as well as the changes of TGF-β and IL-10 after stimulating the S180 osteosarcoma cells by LPS were analyzed by semi-quantitative RT-PCR. The changes of TGF-β protein were detected by ELISA. Results S180 osteosarcoma cells could express TLR1-7 and TLR 9,while couldn＇t express TLR8 in vitro. The expression of TLR4 and TLR9 was higher in earlier stage than that in ad vanced stage. The expression of TLR4 was in significant difference in different stages（P〈0. 05）. The express of TLRI, TLR2,TLR3,TLR5,TLR6, and TLR7 was lower in earlier stage than that in advanced stage, while showed no significant difference（P〉0. 05）. The expression of TGF-β and IL-10 increased and depended on time after stimulating S180 osteosarcoma cells using LPS at 10 μg/ml level. Conclusion S180 in osteosarcoma cells could express many kinds of TLRs, and the TLRs expression changed in different stage. Thus the express of TLRs in S180 osteosarcoma cells played different roles in different stages during the development of tumor. The activation of TLR4 could promote the expression of TGF-β and IL-10,and then enhanced the tolerance of tumor cells.