本文選題：MTA1 + 喉鱗癌　； 參考：《山西醫(yī)科大學(xué)》2012年博士論文

【摘要】：背景與目的：、 侵襲性與轉(zhuǎn)移性是惡性腫瘤的兩個(gè)主要特征,也是影響惡性腫瘤患者預(yù)后的關(guān)鍵因素,在腫瘤侵襲轉(zhuǎn)移的復(fù)雜過程中受到眾多基因的調(diào)控。腫瘤轉(zhuǎn)移相關(guān)基因1(MTA1)是新近發(fā)現(xiàn)的與惡性腫瘤轉(zhuǎn)移相關(guān)的基因,該基因在腫瘤轉(zhuǎn)移過程中表達(dá)明顯上調(diào)。MTA1具有組蛋白脫乙酰基酶活性,調(diào)控組蛋白脫乙�；�,通過影響染色質(zhì)的狀態(tài)來調(diào)整復(fù)制,從而發(fā)揮其生物學(xué)作用。基質(zhì)降解、細(xì)胞間的粘附及腫瘤的血管生成在腫瘤侵襲轉(zhuǎn)移的過程中均發(fā)揮著重要作用。本研究通過檢測喉鱗癌組織、喉部非典型增生組織及正常黏膜組織中MTA1、MMP-9、β-catenin及EGFR的表達(dá),以了解這些參與腫瘤侵襲轉(zhuǎn)移的基因表達(dá)在喉鱗癌發(fā)生發(fā)展的作用及其相關(guān)性。利用RNA干擾技術(shù)及質(zhì)粒轉(zhuǎn)染技術(shù)觀察MTA1對HEP-2細(xì)胞生物學(xué)特性變化的影響,進(jìn)一步認(rèn)識MTA1對喉癌細(xì)胞侵襲轉(zhuǎn)移的調(diào)控作用。 方法： 采用免疫組織化學(xué)法檢測MTA1蛋白在40例正常喉黏膜上皮、37例喉不典型增生組織及40例喉鱗癌組織中的表達(dá)水平,分析MTA1在喉鱗癌組織中的作用與臨床分期、分型、病理學(xué)分級及頸淋巴結(jié)轉(zhuǎn)移的關(guān)系；采用免疫組織化學(xué)法檢測MMP-9、EGFR及β-catenin蛋白在40例喉鱗癌組織與喉正常黏膜組織中的表達(dá),分析MMP-9、EGFR及β-catenin蛋白在喉鱗癌組織中的作用,并分析MTA1與MMP-9、EGFR及β-catenin表達(dá)的相關(guān)性。 在喉癌細(xì)胞株HEP-2細(xì)胞中,采用RNA干擾技術(shù)轉(zhuǎn)染針對MTA1的siRNA (MTA1-siRNA), RT-PCR法及western-blot法分別從轉(zhuǎn)錄水平與翻譯水平鑒定其基因沉默效果,并采用MTT法繪制細(xì)胞生長曲線、細(xì)胞侵襲及遷移實(shí)驗(yàn)、平板克隆形成實(shí)驗(yàn)、細(xì)胞黏附實(shí)驗(yàn)及細(xì)胞劃痕愈合實(shí)驗(yàn)等觀察細(xì)胞生物學(xué)特性的改變；穩(wěn)定轉(zhuǎn)染MTA1的表達(dá)質(zhì)粒及干擾質(zhì)粒,挑選陽性克隆,進(jìn)行MTT法繪制細(xì)胞生長曲線、細(xì)胞侵襲遷移實(shí)驗(yàn)、細(xì)胞黏附實(shí)驗(yàn)、平板克隆形成實(shí)驗(yàn)及劃痕愈合實(shí)驗(yàn),檢測MTA1基因表達(dá)水平的變化,對腫瘤細(xì)胞生物學(xué)特性的影響。 結(jié)果： MTA1蛋白表達(dá)于細(xì)胞核中。在喉正常黏膜組織中,MTA1蛋白呈低水平表達(dá),陽性表達(dá)率為5.0%,而在喉鱗癌組織中,MTA1蛋白陽性表達(dá)細(xì)胞數(shù)量及染色強(qiáng)度均明顯增高,陽性率達(dá)到67.5%,在不典型增生組織中,MTA1表達(dá)率為37.8%,介于正常組織與喉鱗癌組織之間。MTA1核表達(dá)水平在不典型增生組織與癌組織中的表達(dá)水平均高于喉正常鱗狀上皮組織(p0.01)；癌組織中MTA1表達(dá)水平高于不典型增生組織,且統(tǒng)計(jì)學(xué)意義顯著(p0.01)；MTA1蛋白在從喉正常黏膜組織、不典型增生組織到喉鱗癌組織中的表達(dá)呈進(jìn)行性增加。分析MTA1在喉鱗癌組織中的表達(dá)與臨床病理學(xué)參數(shù)的關(guān)系中顯示,在40例喉鱗癌組織標(biāo)本中,MTA1蛋白在低、中及高分化組織中陽性表達(dá)率分別為90.0%、72.2%及41.7%,表現(xiàn)為隨著分化程度的增高,其陽性表達(dá)率降低,有統(tǒng)計(jì)學(xué)差異(χ2=6.141,p0.05)；在喉鱗癌原發(fā)灶中MTA1蛋白在伴有頸淋巴結(jié)轉(zhuǎn)移的組織中陽性表達(dá)率為78.6%,而在不伴有頸淋巴結(jié)轉(zhuǎn)移組織中的表達(dá)率為41.7%,統(tǒng)計(jì)學(xué)分析結(jié)果顯示,MTA1蛋白的表達(dá)水平與喉鱗癌患者的頸淋巴結(jié)轉(zhuǎn)移相關(guān),MTA1蛋白的高表達(dá)能導(dǎo)致頸淋巴結(jié)轉(zhuǎn)移的發(fā)生(χ2=5.215,p0.05)；在與喉鱗癌臨床分期及分型的關(guān)系中,可見MTA1在Ⅰ、Ⅱ期的表達(dá)率較Ⅲ、Ⅳ期低,分別為53.8%、92.9%,但二者之間沒有差異(χ2=0.623,p0.05)；MTA1與喉鱗癌臨床分型也無關(guān)(χ2=1.138,p0.05)。MMP-9、EGFR及β-catenin在喉鱗癌組織中的表達(dá)率分別為82.5%、87.5%及87.5%,而在喉正常組織中的陽性表達(dá)率分別為35.0%、37.5%及35.0%,有統(tǒng)計(jì)學(xué)差異(p0.05)；經(jīng)過相關(guān)分析顯示,在喉鱗癌中,MTA1與MMP-9、EGFR及β-catenin的表達(dá)呈正相關(guān)(p0.05)。 在HEP-2細(xì)胞株,轉(zhuǎn)染靶向MTA1-siRNA后,MTA1基因蛋白及mRNA的表達(dá)水平明顯下降,與空白對照組及無義對照MTA1-NiRNA組相比,MTA1基因的表達(dá)被顯著抑制(p0.05)。在檢測隨后的細(xì)胞生物學(xué)表型中發(fā)現(xiàn),轉(zhuǎn)染MTA1-siRNA后,細(xì)胞的生長速度減慢、體外侵襲遷移能力下降、細(xì)胞的體外黏附及克隆形成能力均下降；穩(wěn)定轉(zhuǎn)染MTA1表達(dá)質(zhì)粒及干擾質(zhì)粒后,MTT法檢測細(xì)胞的生長速度,發(fā)現(xiàn)細(xì)胞的生長速度在表達(dá)質(zhì)粒組最快,而在于擾質(zhì)粒組最慢,有統(tǒng)計(jì)學(xué)差異；在細(xì)胞體外侵襲實(shí)驗(yàn)中,表達(dá)質(zhì)粒組、對照組及干擾質(zhì)粒組穿膜細(xì)胞數(shù)量分別為423.6±14.15,301.2±25.4和115.4±15.52；在遷移實(shí)驗(yàn)中的穿膜細(xì)胞數(shù)分別為549.2±21.51,352±25.03和120.8±17.28。在細(xì)胞遷移及侵襲實(shí)驗(yàn)中,穿膜細(xì)胞數(shù)量最多的是表達(dá)質(zhì)粒轉(zhuǎn)染組,明顯高于對照組及干擾質(zhì)粒組,干擾質(zhì)粒組中細(xì)胞的穿膜能力最弱,細(xì)胞數(shù)量最少；平板克隆形成實(shí)驗(yàn)中,表達(dá)質(zhì)粒組、對照組及干擾質(zhì)粒組中克隆形成數(shù)量分別為168.67±9.5、132.57±5.5和57.33±4.51,可見MTA1的高表達(dá)會(huì)促進(jìn)腫瘤細(xì)胞的克隆形成能力,而降低其表達(dá),則會(huì)導(dǎo)致克隆形成能力的下降；在細(xì)胞劃痕愈合實(shí)驗(yàn)及細(xì)胞黏附實(shí)驗(yàn)中,MTA1表達(dá)質(zhì)粒轉(zhuǎn)染組細(xì)胞的劃痕愈合時(shí)間縮短、黏附能力增強(qiáng),干擾質(zhì)粒組細(xì)胞劃痕愈合時(shí)間最長、黏附能力下降。 結(jié)論： 喉鱗癌組織中,MTA1的高表達(dá)在喉鱗癌的惡性進(jìn)展、侵襲轉(zhuǎn)移及頸淋巴結(jié)的轉(zhuǎn)移中起著重要的作用,或許可以作為判斷喉鱗癌惡性程度的一個(gè)分子生物學(xué)標(biāo)志；MMP-9、EGFR及(3-catenin蛋白在喉鱗癌的發(fā)生發(fā)展中起著重要的作用,MTA1的過度表達(dá)與MMP-9、EGFR及β-catenin成正相關(guān),MTA1過度表達(dá)導(dǎo)致腫瘤的浸潤轉(zhuǎn)移是與MMP-9、EGFR及β-catenin的異常表達(dá)共同作用的結(jié)果。在人喉癌細(xì)胞株HEP-2中,改變MTA1基因的表達(dá)水平,導(dǎo)致喉癌癌細(xì)胞侵襲轉(zhuǎn)移能力及其它惡性行為的變化。上調(diào)MTA1基因的表達(dá),會(huì)增強(qiáng)腫瘤細(xì)胞的惡性行為,導(dǎo)致腫瘤的侵襲轉(zhuǎn)移；而抑制其表達(dá),則可以降低腫瘤細(xì)胞的惡性表型。為研究MTA1基因在喉鱗癌中的作用及進(jìn)行以MTA1為靶點(diǎn)的基因治療研究打下了一定的基礎(chǔ)。
[Abstract]:Background and Purpose :

In this study , the expression of MTA1 , MMP - 9 , 尾 - catenin and EGFR in laryngeal squamous cell carcinoma ( SCC ) was investigated by means of RNA interference and plasmid transfection .

Method :

The expression levels of MTA1 protein in 40 normal laryngeal mucosa epithelial tissues , 37 laryngeal atypical hyperplasia tissues and 40 laryngeal squamous cell carcinoma tissues were detected by immunohistochemical method .
The expression of MMP - 9 , EGFR and 尾 - catenin in laryngeal squamous cell carcinoma was detected by immunohistochemical method . The role of MMP - 9 , EGFR and 尾 - catenin in laryngeal squamous cell carcinoma was analyzed , and the correlation between the expression of MMP - 9 , EGFR and 尾 - catenin in laryngeal squamous cell carcinoma was analyzed .

In the laryngeal cancer cell line HEP - 2 cells , the silencing effect of siRNA ( MTA1 - siRNA ) , reverse transcription polymerase chain reaction ( RT - PCR ) and western - blot ( RT - PCR ) were used to determine the gene silencing effect . The cell growth curve , cell invasion and migration experiment , plate clone formation experiment , cell adhesion experiment and cell scratch healing experiment were used to observe the changes of cell biological characteristics .
The expression plasmid and the interference plasmid of MTA1 were stably transfected . The positive clones were selected . Cell growth curve , cell invasion and migration experiment , cell adhesion experiment , plate clone formation experiment and scratch healing experiment were performed by MTT method .

Results :

The expression of MTA1 protein in laryngeal squamous cell carcinoma was significantly higher than that in normal tissue and laryngeal squamous cell carcinoma .
The expression level of MTA1 in cancer tissue was higher than that of atypical hyperplasia , and the statistical significance was significant ( p < 0.01 ) .
The expression of MTA1 protein in the tissues of laryngeal squamous cell carcinoma ( SCC ) was 92.0 % , 72.2 % and 41.7 % , respectively , and its positive rate of expression decreased with the degree of differentiation ( 蠂 ~ 2 = 6.141 , p < 0.05 ) .
In the primary foci of laryngeal squamous cell carcinoma , the positive expression rate was 78.6 % in the tissues with cervical lymph node metastasis , and the expression rate was 41.7 % in the tissues without cervical lymph node metastasis . The statistical analysis showed that the expression level of MTA1 protein was related to cervical lymph node metastasis in laryngeal squamous cell carcinoma .
In the relationship between clinical stage and typing of laryngeal squamous cell carcinoma , the expression rate of MTA1 in stage 鈪，

本文編號：2077882