發(fā)布時(shí)間：2018-05-19 03:35

  本文選題：咖啡醇 + 結(jié)腸癌��； 參考：《昆明醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[目的]本研究從體外細(xì)胞水平出發(fā),探尋咖啡醇對(duì)結(jié)腸癌細(xì)胞HCT116增殖的影響及其量效關(guān)系,通過檢測(cè)自噬標(biāo)志物確定咖啡醇誘導(dǎo)結(jié)腸癌細(xì)胞產(chǎn)生自噬性死亡,進(jìn)而通過檢測(cè)不同自噬通路上的關(guān)鍵靶基因深入探索咖啡醇誘導(dǎo)自噬的關(guān)鍵通路和作用機(jī)理。通過探索咖啡醇抑制結(jié)腸癌的生物學(xué)功效,有助于進(jìn)一步開發(fā)咖啡醇資源,使咖啡醇在結(jié)腸癌的防控中發(fā)揮作用,對(duì)于推動(dòng)藥物治療的發(fā)展,促進(jìn)基礎(chǔ)研究對(duì)慢性病預(yù)防控制的關(guān)鍵作用有重大意義。[方法]培養(yǎng)細(xì)胞HCT116,加入不同濃度咖啡醇(OμM、10μM、20μM、40μM、80μM、160μM)與細(xì)胞孵育48h后,通過MTT實(shí)驗(yàn)檢測(cè)咖啡醇對(duì)結(jié)腸癌細(xì)胞HCT116增殖的影響及其量效關(guān)系,通過透射電鏡檢測(cè)自噬標(biāo)志物,進(jìn)而運(yùn)用Western Blot檢測(cè)咖啡醇誘導(dǎo)的LC3B-II的表達(dá)變化情況,最后使用實(shí)時(shí)熒光定量PCR檢測(cè)不同自噬通路上的關(guān)鍵靶基因。[結(jié)果]1.MTT的結(jié)果表明,隨著咖啡醇濃度的不斷增加,細(xì)胞活性受到了抑制,即咖啡醇能夠抑制結(jié)腸癌細(xì)胞的增殖。2.將HCT116細(xì)胞用不同濃度咖啡醇(OμM、20μM、40μM)處理24h后,用40μM咖啡醇孵育的細(xì)胞在電鏡下觀察可見線粒體發(fā)生變形,有雙層膜環(huán)繞的自噬體,這意味著咖啡醇誘導(dǎo)HCT116細(xì)胞發(fā)生了細(xì)胞自噬。3.Western印跡檢測(cè)自噬標(biāo)志物L(fēng)C3B-Ⅱ。研究結(jié)果顯示,隨著咖啡醇濃度的不斷增加,LC3B-Ⅰ條帶痕跡逐漸減淡,而LC3B-Ⅱ則逐漸加強(qiáng),推測(cè)咖啡醇到達(dá)一定濃度時(shí)會(huì)引起結(jié)腸癌細(xì)胞發(fā)生細(xì)胞自噬。4.隨著咖啡醇濃度逐漸增加,Beclinl和mTOR的表達(dá)變化水平無統(tǒng)計(jì)學(xué)意義,推測(cè)結(jié)腸癌細(xì)胞HCT116的自噬發(fā)生可能不是通過這兩條通路,具體的關(guān)鍵靶點(diǎn)還需進(jìn)一步研究和探討。[結(jié)論]1.咖啡醇能抑制結(jié)腸癌細(xì)胞增殖。2.一定濃度的咖啡醇能誘導(dǎo)HCT116細(xì)胞發(fā)生自噬。3.不同咖啡醇濃度下Beclinl和mTOR的相對(duì)表達(dá)水平變化無統(tǒng)計(jì)學(xué)意義,提示咖啡醇誘導(dǎo)的細(xì)胞自噬可能不是通過這兩條通路,具體的關(guān)鍵靶點(diǎn)還需進(jìn)一步研究。
[Abstract]:[objective] to explore the effect of caffeine on the proliferation of colon cancer cell line HCT116 and its dose-response relationship, and to determine the autophagic death induced by caffeine by detecting autophagy markers. Furthermore, the key pathway and mechanism of caffeine induced autophagy were explored by detecting the key target genes in different autophagy pathways. By exploring the biological effects of caffeine on colon cancer, it is helpful to further develop the resources of coffee alcohol, to make caffeine play an important role in the prevention and control of colon cancer, and to promote the development of drug therapy. It is of great significance to promote basic research for the prevention and control of chronic diseases. [methods] cultured cells were incubated with HCT116, 10 渭 MU 10 渭 MU 10 渭 MU (10 渭 M) and 40 渭 M (40 渭 M), 80 渭 MU (160 渭 M) for 48 h. The effects of caffeine on the proliferation of HCT116 and its dose-effect relationship were detected by MTT assay, and the autophagy markers were detected by transmission electron microscopy (TEM). Western Blot was used to detect the changes of LC3B-II expression induced by caffeine, and real-time quantitative PCR was used to detect the key target genes in different autophagy pathways. [results] the results of 1.MTT showed that with the increasing of caffeine concentration, cell activity was inhibited, that is, caffeine could inhibit the proliferation of colon cancer cells. After HCT116 cells were treated with different concentrations of caffeine O 渭 M (20 渭 M) 40 渭 M for 24 h, the cells incubated with 40 渭 M caffeine showed that mitochondria deformed and there was a double layer membrane surrounded autophagy under electron microscope. This means that caffeine induces autophagy in HCT116 cells. 3. Western blot detection of autophagy marker LC3B- 鈪，

本文編號(hào)：1908562