Li Xiang, Gong Zhi-yong, Li Hong, Wei Qiang, Shi Ming, Yang Yu-ru
Department of Urology, Huaxi Hospital of Sichuan University, Chengdu 610041, China.
Zhonghua Wai Ke Za Zhi. 2004 Feb 7;42(3):177-81.
To investigate the effect of bacillus Calmette-Guerin (BCG) on Toll-like receptors (TLRs) expression and cytokine production in human bladder cancer cell line T24.
Human transitional carcinoma cell line T24 was cultured in RPMI1640 medium with 10% FBS, BCG was added into the cell culture with various doses in bacteria-cell ratio. After T24 cells were stimulated by BCG for 1 hour, total RNA of cells was extracted. RT-PCR procedure was conducted with the primer of TLR-2 and TLR-4, and the products were analyzed with agarose gels electrophoresis. Then the expression of TLR-2 and TLR-4 in T24 cells was assessed by the analyzing system of gel imaging. After the stimulation culture for 12 hours, the supernatant of cells was collected. The levels of IL-4 and IL-12 in each sample were assayed by ELISA method.
The expression level of either TLR-2 or TLR-4 was increased by the stimulation of BCG in a dose-dependent manner, the effects reached the maximal level at the dose of BCG as 10 bacilli per cell. The production of IL-12 in T24 cells was also increased gradually by the stimulation of BCG in a dose-dependent manner, and the dose of BCG obtained maximal effect was 10 bacilli per cell, which is coincident with the results observed in the expression of TLRs. There was no difference showed on the production of IL-4 between T24 cells stimulated with BCG and control.
The stimulation of BCG not only up-regulated the expression of TLR-2 and TLR-4, but also increased the production of IL-12 in bladder cancer T24 cell line. The expression of TLRs and the production of cytokine in bladder cancer cells may be related to the BCG-induced immunol response to human bladder cancer.
探讨卡介苗（BCG）对人膀胱癌细胞系T24中Toll样受体（TLRs）表达及细胞因子产生的影响。
人移行癌细胞系T24在含10%胎牛血清的RPMI1640培养基中培养，按细菌与细胞比例加入不同剂量的BCG。BCG刺激T24细胞1小时后，提取细胞总RNA。用TLR-2和TLR-4引物进行逆转录聚合酶链反应（RT-PCR），产物经琼脂糖凝胶电泳分析。然后通过凝胶成像分析系统评估T24细胞中TLR-2和TLR-4的表达。刺激培养12小时后，收集细胞上清液。采用酶联免疫吸附测定（ELISA）法检测各样本中白细胞介素-4（IL-4）和白细胞介素-12（IL-12）的水平。
BCG刺激可使TLR-2或TLR-4的表达水平呈剂量依赖性增加，当BCG剂量为每细胞10个菌时，作用达到最大水平。BCG刺激也使T24细胞中IL-12的产生呈剂量依赖性逐渐增加，BCG获得最大效应的剂量为每细胞10个菌，这与TLRs表达的观察结果一致。BCG刺激的T24细胞与对照相比，IL-4产生无差异。
BCG刺激不仅上调了膀胱癌细胞系T24中TLR-2和TLR-4的表达，还增加了IL-12的产生。膀胱癌细胞中TLRs的表达及细胞因子的产生可能与BCG诱导的人膀胱癌免疫反应有关。
