Compound C誘導(dǎo)人膽管癌細(xì)胞發(fā)生自噬的機(jī)制.pdf

1、目的:研究AMPK抑制劑Compound C誘導(dǎo)人膽管癌(cholangiocarcinoma，CCA)細(xì)胞發(fā)生自噬的分子機(jī)制;
　　方法:實驗通過培養(yǎng)人膽管癌細(xì)胞株QBC939和RBE細(xì)胞，采用Compound C進(jìn)行處理，采用自噬抑制劑3-MA、Bafilomycin A1(BAF)單獨或與Compound C聯(lián)合處理細(xì)胞、Akt/mTOR抑制劑LY294002(LY)/Rapamycin(Rap)單獨或與Compound C

2、聯(lián)合處理細(xì)胞，JNK抑制劑SP600125(SP)、p53抑制劑Pifithrin-α（PFT-α）或轉(zhuǎn)染siRNA干擾JNK、p53的表達(dá)、p38MAPK抑制劑SB203580(SB)單獨或與Compound C聯(lián)合處理細(xì)胞，通過細(xì)胞免疫熒光染色檢測細(xì)胞自噬，用乳酸脫氫酶細(xì)胞毒性檢測試劑盒檢測細(xì)胞毒性，免疫印跡(Western blot)分析自噬和凋亡相關(guān)分子LC3A/B、PARP、p53等蛋白表達(dá)水平及實時熒光定量PCR檢測p53、

3、MDM2的mRNA表達(dá)水平。
　　結(jié)果:(1)Compound C誘導(dǎo)人CCA細(xì)胞發(fā)生死亡:乳酸脫氫酶(LDH)釋放結(jié)果表明Compound C有效誘導(dǎo)QBC939和RBE細(xì)胞死亡。Westernblot結(jié)果表明Compound C誘導(dǎo)QBC939和RBE細(xì)胞中凋亡分子標(biāo)記物PARP的活化剪切，與細(xì)胞死亡結(jié)果有較好的一致性。這些結(jié)果提示Compound C誘導(dǎo)人CCA細(xì)胞發(fā)生死亡，且呈劑量-時間依賴性;(2)Compound C誘

4、導(dǎo)人CCA細(xì)胞發(fā)生自噬:細(xì)胞免疫熒光結(jié)果表明CompoundC處理后人CCA細(xì)胞出現(xiàn)LC3A/B熒光顆粒，且呈劑量-時間依賴性;此外，Western blot結(jié)果說明Compound C誘導(dǎo)LC3A/B表達(dá)。結(jié)果提示Compound C誘導(dǎo)人CCA細(xì)胞發(fā)生自噬。(3)抑制自噬促進(jìn)Compound C誘導(dǎo)人CCA細(xì)胞死亡:LDH釋放結(jié)果表明自噬抑制劑3-MA和BAF阻斷自噬發(fā)生后顯著增加Compound C誘導(dǎo)的QBC939和RBE細(xì)胞死

5、亡。此外，Western blot結(jié)果也表明用3-MA和BAF阻斷自噬發(fā)生后顯著增加Compound C誘導(dǎo)的QBC939和RBE細(xì)胞凋亡，與LDH結(jié)果一致。結(jié)果提示自噬的誘導(dǎo)減弱Compound C誘導(dǎo)的人CCA細(xì)胞死亡(4) CompoundC誘導(dǎo)人CCA細(xì)胞發(fā)生的自噬不依賴于Akt/mTOR通路:Western blot結(jié)果表明Compound C處理QBC939和RBE細(xì)胞后p-Akt上調(diào)，p-p70s6k未見明顯變化。此外，L

6、Y/Rap分別阻斷Akt/mTOR通路后，Compound C誘導(dǎo)的QBC939和RBE細(xì)胞自噬未受影響。結(jié)果提示Akt/mTOR通路不參與Compound C誘導(dǎo)的人CCA細(xì)胞自噬的調(diào)節(jié)。(5)阻斷JNK抑制CompoundC對人CCA細(xì)胞自噬的誘導(dǎo):Western blot結(jié)果表明SP陽斷細(xì)胞JNK的活性后，明顯減弱Compound C誘導(dǎo)的QBC939和RBE細(xì)胞自噬。轉(zhuǎn)染siRNA干擾JNK的表達(dá)也明顯減弱Compound C誘

7、導(dǎo)的QBC939和RBE細(xì)胞自噬。結(jié)果提示JNK參與Compound C誘導(dǎo)的人CCA細(xì)胞自噬的調(diào)節(jié)。(6)抑制p38MAPK促進(jìn)Compound C對人CCA細(xì)胞自噬的誘導(dǎo):Western blot結(jié)果表明盡管Compound C激活QBC939和RBE細(xì)胞p38MAPK活性，但是SB阻斷細(xì)胞p38MAPK的活性后，促進(jìn)了CompoundC對細(xì)胞自噬的誘導(dǎo)。結(jié)果提示p38MAPK參與Compound C誘導(dǎo)人CCA細(xì)胞發(fā)生自噬的調(diào)節(jié)。

8、(7)抑制p38MAPK通過增強(qiáng)JNK促進(jìn)Compound C對人CCA細(xì)胞自噬的誘導(dǎo):Western blot結(jié)果表明SB阻斷細(xì)胞p38MAPK的活性后，促進(jìn)了QBC939和RBE細(xì)胞中JNK活性上調(diào);此外，SP阻斷細(xì)胞JNK的活性后，減弱了抑制p38MAPK促進(jìn)Compound C誘導(dǎo)QBC939和RBE細(xì)胞自噬。這些結(jié)果提示JNK活化參與了抑制p38MAPK促進(jìn)Compound C誘導(dǎo)人CCA細(xì)胞發(fā)生自噬的調(diào)節(jié)。(8)阻斷p53抑

9、制Compound C對人CCA細(xì)胞自噬的誘導(dǎo):實時熒光定量PCR和Western blot檢測結(jié)果表明Compound C誘導(dǎo)QBC939和RBE細(xì)胞p53表達(dá)。此外，Western blot結(jié)果說明PFT-α阻斷細(xì)胞p53的活性后，明顯減弱Compound C誘導(dǎo)的QBC939和RBE細(xì)胞自噬。轉(zhuǎn)染siRNA干擾p53的表達(dá)也明顯減弱Compound C誘導(dǎo)的QBC939和RBE細(xì)胞自噬。結(jié)果提示p53參與Compound C誘導(dǎo)人

10、CCA細(xì)胞發(fā)生自噬的調(diào)節(jié)。(9) Compound C誘導(dǎo)人CCA細(xì)胞凋亡不依賴于Akt/mTOR通路:Western blot結(jié)果表明LY/Rap分別阻斷細(xì)胞Akt/mTOR通路后，Compound C誘導(dǎo)QBC939和RBE細(xì)胞凋亡未受影響。結(jié)果提示Akt/mTOR通路不涉及Compound C誘導(dǎo)的人CCA細(xì)胞的細(xì)胞凋亡調(diào)節(jié)。
　　結(jié)論:Compound C誘導(dǎo)人CCA細(xì)胞發(fā)生死亡;Compound C誘導(dǎo)人CCA細(xì)胞發(fā)生自