本文選題：鎘 + WRL68　； 參考：《南京師范大學(xué)》2014年碩士論文

【摘要】：本實(shí)驗(yàn)室已有研究證明了低濃度鎘(10μM)可以誘導(dǎo)肝細(xì)胞自噬,并沒(méi)有誘導(dǎo)細(xì)胞凋亡。但是鎘誘導(dǎo)肝細(xì)胞自噬與內(nèi)質(zhì)網(wǎng)應(yīng)激的關(guān)系當(dāng)前還不清楚。本研究旨在探究鎘是否經(jīng)內(nèi)質(zhì)網(wǎng)應(yīng)激引起WRL68細(xì)胞自噬,以及在WRL68細(xì)胞中自噬與凋亡的關(guān)系。本研究分為三部分：①探究鎘誘導(dǎo)WRL68細(xì)胞自噬與內(nèi)質(zhì)網(wǎng)應(yīng)激之間的關(guān)系；②分析胞內(nèi)鈣穩(wěn)態(tài)的變化對(duì)鎘經(jīng)內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)細(xì)胞自噬的影響；③分析WRL68細(xì)胞自噬與凋亡的關(guān)系。 本實(shí)驗(yàn)運(yùn)用細(xì)胞培養(yǎng)、透射電子顯微鏡、Western blot、流式細(xì)胞術(shù)等細(xì)胞和分子生物學(xué)技術(shù),研究分析了低濃度(0～10μM)鎘誘導(dǎo)人肝細(xì)胞的自噬標(biāo)志蛋白LC3B-Ⅱ/Ⅰ和內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志蛋白GRP78的表達(dá)；探究胞內(nèi)鈣離子與鎘誘導(dǎo)的細(xì)胞自噬的關(guān)系；用流式細(xì)胞術(shù)和Western blot檢測(cè)3-MA(自噬抑制劑)對(duì)WRL68細(xì)胞自噬與凋亡的影響,分析鎘致肝細(xì)胞自噬和凋亡之間的關(guān)系。具體實(shí)驗(yàn)結(jié)果如下： 1.鎘經(jīng)內(nèi)質(zhì)網(wǎng)應(yīng)激途徑誘導(dǎo)WRL68細(xì)胞自噬 在本實(shí)驗(yàn)室研究的基礎(chǔ)上,WRL68細(xì)胞暴露于低濃度鎘后,細(xì)胞形態(tài)基本完好,但發(fā)生細(xì)胞自噬。在透射電鏡下觀察到自噬泡和內(nèi)質(zhì)網(wǎng)腫脹,并有脫顆�，F(xiàn)象；加入內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑4-PBA后,發(fā)現(xiàn)GRP78和LC3B-Ⅱ/Ⅰ的表達(dá)量減少；GFP-LC3B在熒光顯微鏡下點(diǎn)狀聚集也減少。 2.鈣穩(wěn)態(tài)變化與內(nèi)質(zhì)網(wǎng)應(yīng)激引起的WRL68自噬的關(guān)系 WRL68細(xì)胞暴露于低濃度鎘(0～10gM)12h,引起細(xì)胞自噬,同時(shí)用酶標(biāo)儀檢測(cè)到胞內(nèi)鈣離子濃度升高；當(dāng)加入內(nèi)質(zhì)網(wǎng)鈣庫(kù)抑制劑2-APB后,Western blot檢測(cè)到GRP78和LC3B-Ⅱ/Ⅰ的表達(dá)量減少。 3.鎘誘導(dǎo)WRL68細(xì)胞的自噬與凋亡的關(guān)系 WRL68細(xì)胞加入自噬抑制劑3-MA后,流式細(xì)胞儀檢測(cè)結(jié)果顯示,加入3-MA后細(xì)胞凋亡率增高,Western blot檢測(cè)發(fā)現(xiàn)LC3B-Ⅱ/Ⅰ的表達(dá)量減少,而Cleaved Caspase-3的表達(dá)量增加。 本研究在WRL68細(xì)胞暴露于低濃度鎘(0～10μM)后細(xì)胞形態(tài)基本完好的基礎(chǔ)上進(jìn)行研究。低濃度鎘可以經(jīng)內(nèi)質(zhì)網(wǎng)應(yīng)激途徑誘導(dǎo)WRL68細(xì)胞自噬,且胞內(nèi)鈣離子參與了內(nèi)質(zhì)網(wǎng)應(yīng)激引起的自噬,此外,加入3-MA后細(xì)胞凋亡增加。
[Abstract]:It has been demonstrated in our laboratory that low concentration of cadmium (10 渭 M) can induce autophagy of hepatocytes, but do not induce apoptosis.However, the relationship between cadmium induced hepatocyte autophagy and endoplasmic reticulum stress is unclear.The aim of this study was to investigate whether cadmium induced autophagy of WRL68 cells through endoplasmic reticulum stress and the relationship between autophagy and apoptosis in WRL68 cells.This study was divided into three parts: 1 to explore the relationship between cadmium induced autophagy and endoplasmic reticulum stress in WRL68 cells. 2 to analyze the effect of intracellular calcium homeostasis on cadmium induced autophagy by endoplasmic reticulum stress. 3 to analyze the relationship between autophagy and apoptosis of WRL68 cells.Cell culture, transmission electron microscopy (TEM), Western blot, flow cytometry and other cell and molecular techniques were used in this study.The expression of autophagy marker protein LC3B- 鈪，

本文編號(hào)：1748631