CAJ | 학술논문

背景与目的已有的研究表明：Toll样受体5（toll-likereceptor5,TLR5）在肿瘤起始和发展中发挥重要作用。我们前期研究发现，TLR5在非小细胞肺癌（non-smallcelllungcancer,NSCLC）组织中高表达，但其在NSCLC高表达后的信号通路活化情况的研究并不多见。本研究旨在探讨TLR5在不同NSCLC细胞株上的表达，及其在NSCLC细胞中活化的机制。方法用免疫荧光、RT-PCR和Westernblot方法检测TLR5在三种不同NSCLC细胞株中的表达。分别用0μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL、5μg/mL、10μg/mL的鞭毛蛋白刺激，用NF-κB荧光素酶报告基因质粒瞬时转染后，检测细胞内NF-κB荧光素酶的活性。选择TLR5表达最高的SPC-A-1细胞株为实验对象，选择0.1μg/mL的鞭毛蛋白，分别用0μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL、10μg/mL的TLR5抗体抑制通路活化，检测细胞内NF-κB荧光素酶的活性，验证TLR5活化通路。构建TLR5-shRNA，转染SPC-A-1细胞48h后，以0.1μg/mL浓度鞭毛蛋白分别刺激SPC-A-1细胞及转染的SPC-A-1细胞，在刺激0min、10min、30min、60min，用Westernblot方法比较TLR5信号通路因子p-IKBα、p-ERK1/2、p-JNK、IKBα、ERK1/2的变化。结果TLR5在肺腺癌细胞株SPC-A-1中呈高表达，且主要表达在细胞膜上。三种细胞株中SPC-A-1细胞NF-κB荧光素酶的活性最高，呈浓度依赖性，0.1μg/mL鞭毛蛋白即可明显增强NF-κB荧光素酶的活性（P<0.05）；而SPC-A-1细胞内NF-κB荧光素酶的活性可被TLR5抗体抑制，与TLR5抗体浓度负相关（P<0.05）。与0min相比较，SPC-A-1细胞内p-IKBα、p-ERK1/2、p-JNK水平在鞭毛蛋白刺激10min即明显增高，30min达到高峰，60min开始下降（P<0.05），且与10min和60min组相比，p-IKBα、p-ERK1/2、p-JNK水平在30min增高（P<0.05）；而IKBα、ERK1/2的水平无明显变化（P>0.05）。以适合浓度鞭毛蛋白刺激转染的SPC-A-1细胞，p-IKBα、p-JNK蛋白均未检出，IKBα、ERK1/2蛋白的水平无明显变化（P>0.05）， p-ERK1/2蛋白水平随着时间延长明显增高（P<0.05）。结论外源性配体鞭毛蛋白可激活NSCLC细胞株TLR5蛋白，启动下游信号通路，可能与NSCLC的发生发展有关。揹景與目的已有的研究錶明：Toll樣受體5（toll-likereceptor5,TLR5）在腫瘤起始和髮展中髮揮重要作用。我們前期研究髮現，TLR5在非小細胞肺癌（non-smallcelllungcancer,NSCLC）組織中高錶達，但其在NSCLC高錶達後的信號通路活化情況的研究併不多見。本研究旨在探討TLR5在不同NSCLC細胞株上的錶達，及其在NSCLC細胞中活化的機製。方法用免疫熒光、RT-PCR和Westernblot方法檢測TLR5在三種不同NSCLC細胞株中的錶達。分彆用0μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL、5μg/mL、10μg/mL的鞭毛蛋白刺激，用NF-κB熒光素酶報告基因質粒瞬時轉染後，檢測細胞內NF-κB熒光素酶的活性。選擇TLR5錶達最高的SPC-A-1細胞株為實驗對象，選擇0.1μg/mL的鞭毛蛋白，分彆用0μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL、10μg/mL的TLR5抗體抑製通路活化，檢測細胞內NF-κB熒光素酶的活性，驗證TLR5活化通路。構建TLR5-shRNA，轉染SPC-A-1細胞48h後，以0.1μg/mL濃度鞭毛蛋白分彆刺激SPC-A-1細胞及轉染的SPC-A-1細胞，在刺激0min、10min、30min、60min，用Westernblot方法比較TLR5信號通路因子p-IKBα、p-ERK1/2、p-JNK、IKBα、ERK1/2的變化。結果TLR5在肺腺癌細胞株SPC-A-1中呈高錶達，且主要錶達在細胞膜上。三種細胞株中SPC-A-1細胞NF-κB熒光素酶的活性最高，呈濃度依賴性，0.1μg/mL鞭毛蛋白即可明顯增彊NF-κB熒光素酶的活性（P<0.05）；而SPC-A-1細胞內NF-κB熒光素酶的活性可被TLR5抗體抑製，與TLR5抗體濃度負相關（P<0.05）。與0min相比較，SPC-A-1細胞內p-IKBα、p-ERK1/2、p-JNK水平在鞭毛蛋白刺激10min即明顯增高，30min達到高峰，60min開始下降（P<0.05），且與10min和60min組相比，p-IKBα、p-ERK1/2、p-JNK水平在30min增高（P<0.05）；而IKBα、ERK1/2的水平無明顯變化（P>0.05）。以適閤濃度鞭毛蛋白刺激轉染的SPC-A-1細胞，p-IKBα、p-JNK蛋白均未檢齣，IKBα、ERK1/2蛋白的水平無明顯變化（P>0.05）， p-ERK1/2蛋白水平隨著時間延長明顯增高（P<0.05）。結論外源性配體鞭毛蛋白可激活NSCLC細胞株TLR5蛋白，啟動下遊信號通路，可能與NSCLC的髮生髮展有關。
배경여목적이유적연구표명：Toll양수체5（toll-likereceptor5,TLR5）재종류기시화발전중발휘중요작용。아문전기연구발현，TLR5재비소세포폐암（non-smallcelllungcancer,NSCLC）조직중고표체，단기재NSCLC고표체후적신호통로활화정황적연구병불다견。본연구지재탐토TLR5재불동NSCLC세포주상적표체，급기재NSCLC세포중활화적궤제。방법용면역형광、RT-PCR화Westernblot방법검측TLR5재삼충불동NSCLC세포주중적표체。분별용0μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL、5μg/mL、10μg/mL적편모단백자격，용NF-κB형광소매보고기인질립순시전염후，검측세포내NF-κB형광소매적활성。선택TLR5표체최고적SPC-A-1세포주위실험대상，선택0.1μg/mL적편모단백，분별용0μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL、10μg/mL적TLR5항체억제통로활화，검측세포내NF-κB형광소매적활성，험증TLR5활화통로。구건TLR5-shRNA，전염SPC-A-1세포48h후，이0.1μg/mL농도편모단백분별자격SPC-A-1세포급전염적SPC-A-1세포，재자격0min、10min、30min、60min，용Westernblot방법비교TLR5신호통로인자p-IKBα、p-ERK1/2、p-JNK、IKBα、ERK1/2적변화。결과TLR5재폐선암세포주SPC-A-1중정고표체，차주요표체재세포막상。삼충세포주중SPC-A-1세포NF-κB형광소매적활성최고，정농도의뢰성，0.1μg/mL편모단백즉가명현증강NF-κB형광소매적활성（P<0.05）；이SPC-A-1세포내NF-κB형광소매적활성가피TLR5항체억제，여TLR5항체농도부상관（P<0.05）。여0min상비교，SPC-A-1세포내p-IKBα、p-ERK1/2、p-JNK수평재편모단백자격10min즉명현증고，30min체도고봉，60min개시하강（P<0.05），차여10min화60min조상비，p-IKBα、p-ERK1/2、p-JNK수평재30min증고（P<0.05）；이IKBα、ERK1/2적수평무명현변화（P>0.05）。이괄합농도편모단백자격전염적SPC-A-1세포，p-IKBα、p-JNK단백균미검출，IKBα、ERK1/2단백적수평무명현변화（P>0.05）， p-ERK1/2단백수평수착시간연장명현증고（P<0.05）。결론외원성배체편모단백가격활NSCLC세포주TLR5단백，계동하유신호통로，가능여NSCLC적발생발전유관。
Background and objective It has been proven that toll-like receptor 5 (TLR5) plaied an important role in the development of tumor. In our previous study, we found that the expression of TLR5 was remarkably higher in non-small cell lung cancer (NSCLC) tissues than that in normal tissues, but the activation of TLR5 signaling pathway in NSCLC was still unknown. Te aim of this study is to investigate the expression of TLR5 in diferent types of NSCLC cell lines, and analyze the activity of the signaling pathway afer stimulated by its specific exogenous ligand fiagellin. Methods Te TLR5 protein was detected by immunofiuorescence and Western blot in three kinds of NSCLC cell lines, and the TLR5 mRNA was detected by RT-PCR. Select the cell line of TLR5 highest expression as the research object, and select the suitable concentration of fiagel-lin. NF-κB luciferase activity was detected to validate the TLR5 activation pathway through inhibitory signaling pathways by 0 μg/mL, 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL TLR5 antibody. Te chosen cell line was transfected by TLR5 shRNA plasmid, and p-IKBα, IKBα, p-ERK1/2, ERK1/2 and p-JNK of untrasfected and transfected cells were detected in the activ-ity of TLR5 signaling pathway by Western blot at 0 min, 10 min, 30 min and 60 min, respectively. Results Te expression of TLR5 was the highest in the lung adenocarcinoma cell line SPC-A-1 by immunofiuorescence, mainly expressed on the cell membrane. NF-κB luciferase activity of SPC-A-1 cells was the highest, and the activity was increased in a dose-dependent man-ner. 0.1 μg/mL fiagellin could significantly increase the NF-κB luciferase activity (P<0.05), while its activity could be inhibited by the TLR5 antibody in a negative correlation. Treated by 0.1 μg/mL fiagellin, compared with that of 0 min group, the levels of p-IKBα, p-ERK1/2, p-JNK of SPC-A-1 cells increased significantly afer 10 min, reached the peak at 30 min, and declined at 60 min (P<0.05). Compared with that of 10 min and 60 min group, the levels of p-IKBα, p-ERK1/2, p-JNK significantly increased at 30 min (P<0.05). While the levels of IKBα, ERK1/2 at 0 min, 10 min, 30 min and 60 min had no significant changes (P>0.05). SPC-A-1 cells transfected TLR5-shRNA were also stimulated by fiagellin (0.1 μg/mL). At 0 min, 10 min, 30 min and 60 min, p-IKBα and p-JNK proteins could not be detected, and the levels of IKBα and ERK1/2 had no significant changes (P>0.05), but the levels of p-ERK1/2 significantly increased as time went on (P<0.05). Conclusion Exogenous ligand fiagellin can ac-tivate TLR5 protein in NSCLC cell lines and initiate downstream signaling pathways. It may be relative to the development of NSCLC.