發(fā)布時(shí)間：2018-03-06 12:55

  本文選題：喉腫瘤　切入點(diǎn)：免疫球蛋白M　出處：《瀘州醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：免疫球蛋白(immunoglobulin, Ig)是機(jī)體免疫系統(tǒng)中非常重要的免疫分子,其功能主要是介導(dǎo)體液免疫,傳統(tǒng)的免疫學(xué)理論認(rèn)為,Ig是B淋巴細(xì)胞特有產(chǎn)物,機(jī)體其它細(xì)胞不產(chǎn)生Ig。然而,近年來人們發(fā)現(xiàn)Ig異位表達(dá)于多種上皮源性的惡性腫瘤組織和細(xì)胞系,初步研究表明異位表達(dá)的Ig具有生長(zhǎng)因子樣作用。我們研究小組已對(duì)IgM在多種上皮來源腫瘤組織和細(xì)胞系中的表達(dá)進(jìn)行了報(bào)道,本實(shí)驗(yàn)旨在探討喉鱗癌Hep-2細(xì)胞中IgM蛋白的表達(dá)情況,并對(duì)喉鱗癌Hep-2細(xì)胞表達(dá)IgM蛋白的生物學(xué)活性進(jìn)行初步研究。方法：進(jìn)行喉鱗癌Hep-2細(xì)胞體外培養(yǎng),分別通過免疫細(xì)胞化學(xué)技術(shù)(鏈霉親和素生物素法,SABC法)、免疫印記法(Western blot)以及流式細(xì)胞術(shù)(FCM)檢測(cè)Hep-2細(xì)胞中IgM蛋白的表達(dá)情況；應(yīng)用抗人IgM抗體,設(shè)置時(shí)間梯度和濃度梯度,通過細(xì)胞增殖抑制試驗(yàn)(MTS法)研究抗人IgM抗體對(duì)喉鱗癌Hep-2細(xì)胞生長(zhǎng)增殖活性的影響,并對(duì)細(xì)胞形態(tài)進(jìn)行觀察；通過軟瓊脂細(xì)胞集落形成實(shí)驗(yàn)檢測(cè)不同濃度抗人IgM抗體對(duì)Hep-2細(xì)胞增殖能力的影響；通過流式細(xì)胞儀(Annexin V/PI染色)和Hoechst染色方法,研究抗人IgM抗體對(duì)喉鱗癌Hep-2細(xì)胞凋亡的影響,并觀察凋亡細(xì)胞形態(tài)；應(yīng)用流式細(xì)胞儀PI染色法觀察抗人IgM抗體對(duì)喉鱗癌Hep-2細(xì)胞細(xì)胞周期的影響。結(jié)果：免疫細(xì)胞化學(xué)SABC法顯示Hep-2細(xì)胞存在IgM蛋白表達(dá)且主要表達(dá)于細(xì)胞漿中；將Hep-2細(xì)胞的蛋白提取液進(jìn)行免疫印記法檢測(cè),在分子量75KD處檢出了陽性信號(hào),提示Hep-2細(xì)胞存在IgM蛋白的表達(dá)；應(yīng)用流式細(xì)胞儀間接免疫熒光法檢測(cè)出Hep-2細(xì)胞的熒光強(qiáng)度為12.4±2.55,陽性對(duì)照組Raji細(xì)胞熒光強(qiáng)度為17.2±2.30,分別與陰性對(duì)照組熒光強(qiáng)度2.29±0.50比較差異顯著(P0.05),說明IgM在喉鱗癌Hep-2細(xì)胞中有表達(dá)；MTS顯示喉鱗癌Hep-2細(xì)胞的抑制率隨時(shí)間延長(zhǎng)而增高,在第三天開始出現(xiàn)明顯抑制作用,同時(shí)喉鱗癌Hep-2細(xì)胞的抑制率隨濃度增加而增高,在50μg/ml濃度開始出現(xiàn)明顯抑制作用,說明抗人IgM抗體具有時(shí)間和濃度依賴性地抑制喉鱗癌Hep-2細(xì)胞增殖生長(zhǎng)的作用(P0.05),并使細(xì)胞出現(xiàn)相應(yīng)形態(tài)學(xué)改變；軟瓊脂細(xì)胞集落形成實(shí)驗(yàn)顯示抗人IgM抗體能明顯抑制喉鱗癌Hep-2細(xì)胞的增殖能力,并具有濃度依賴性,與對(duì)照組比較有顯著性差異(P0.05)；抗人IgM抗體能明顯促進(jìn)喉鱗癌Hep-2細(xì)胞凋亡,且使喉鱗癌Hep-2細(xì)胞呈現(xiàn)出典型凋亡形態(tài)改變,流式細(xì)胞儀Annexin V/PI染色和Hoechst染色方法檢測(cè)Hep-2細(xì)胞凋亡率分別為(20.33±1.93)%和(20.45±3.45)%,與對(duì)照組比較差異顯著(均P0.05)；流式細(xì)胞術(shù)顯示抗人IgM抗體還能影響喉鱗癌Hep-2細(xì)胞的細(xì)胞周期,導(dǎo)致G1期細(xì)胞減少,S期細(xì)胞增多,從而使細(xì)胞周期阻滯于S期(P0.05)。結(jié)論：喉鱗癌Hep-2細(xì)胞能夠表達(dá)IgM蛋白,且主要表達(dá)于Hep-2細(xì)胞漿中,抗人IgM抗體能夠抑制喉鱗癌Hep-2細(xì)胞增殖能力,誘導(dǎo)Hep-2細(xì)胞凋亡,并導(dǎo)致細(xì)胞周期阻滯,提示喉鱗癌細(xì)胞表達(dá)IgM具有生長(zhǎng)因子樣活性,這可能為喉鱗癌治療提供一個(gè)新的靶點(diǎn)。
[Abstract]:Objective: immunoglobulin (immunoglobulin, Ig) is an important immune molecule in the body's immune system, its main function is to mediate humoral immunity, according to the traditional immunological theory, Ig is the specific product of B lymphocytes and other cells in the body does not produce Ig. however, in recent years it was found that malignant tumor of ectopic expression of Ig in epithelial tissue the source and cell lines, preliminary study showed that ectopic expression of Ig is a growth factor like role. Our research group has reported on the expression of IgM in Epitheliogenic Malignant tumor tissues and cell lines, to investigate the expression of IgM protein in laryngeal squamous cell carcinoma Hep-2 cells, and the expression of Hep-2 in laryngeal squamous cell carcinoma cells the biological activity of IgM protein was preliminarily studied. Methods: in vitro culture of laryngeal squamous cell carcinoma Hep-2 cells, respectively, by immunohistochemistry (streptavidin biotin method, SABC 娉，

本文編號(hào)：1574927