本文選題：鼻咽癌 + LPLUNC1　； 參考：《中南大學(xué)》2012年博士論文

【摘要】：[研究背景] 慢性炎癥是腫瘤的第七大生物學(xué)特征,慢性炎癥在觸發(fā)腫瘤發(fā)生、促進(jìn)腫瘤發(fā)展中起著非常重要的作用；炎癥誘導(dǎo)的腫瘤稱為炎癥相關(guān)性腫瘤。鼻咽癌是一種具有明顯地區(qū)差異的惡性腫瘤。鼻咽由于生理和解剖位置的特性,鼻咽上皮細(xì)胞時(shí)刻受到各種外源病原微生物如EBV和有毒化學(xué)物質(zhì)等損害,使得鼻咽粘膜保護(hù)機(jī)制容易被破壞,極易導(dǎo)致鼻咽慢性炎癥,因而有利于誘導(dǎo)鼻咽癌發(fā)生發(fā)展,因此鼻咽癌也是一種炎癥相關(guān)性腫瘤。然而,鼻咽慢性炎癥如何誘發(fā)鼻咽癌發(fā)生發(fā)展,尤其是在此過(guò)程中激活的免疫細(xì)胞如巨噬細(xì)胞、鼻咽上皮細(xì)胞充當(dāng)何種角色,目前不是很清楚。LPLUNC1基因是我室克隆的鼻咽癌表達(dá)下調(diào)的基因�，F(xiàn)有研究表明LPLUNC1基因編碼的蛋白是一種分泌性蛋白,大量存在于鼻咽和呼吸道分泌物、灌洗液和痰液中；LPLUNC1蛋白可借助其BPI結(jié)構(gòu)域行使宿主防御功能,具有殺菌抗炎的作用,而B(niǎo)PI結(jié)構(gòu)域可以抑制腫瘤生長(zhǎng)與轉(zhuǎn)移。因此,我們對(duì)LPLUNC1基因在鼻咽慢性炎癥促進(jìn)鼻咽癌發(fā)生發(fā)展過(guò)程中的作用及其相關(guān)分子機(jī)制進(jìn)行了探討。 [炎癥因子刺激鼻咽癌上皮細(xì)胞參與炎癥反應(yīng)] 為盡可能的獲取炎癥時(shí)各種促炎因子,我們用低濃度LPS(10ng/ml,近似于細(xì)菌脂多糖的生理濃度,如細(xì)菌感染時(shí))刺激巨噬細(xì)胞,qRT-PCR檢測(cè)發(fā)現(xiàn)低濃度LPS可明顯上調(diào)促炎因子TNF-α、IL-6、IL-1β和IL-8基因表達(dá),ELISA檢測(cè)顯示在巨噬細(xì)胞上清培養(yǎng)液中含有大量的促炎因子TNF-α、IL-6、IL-1β和IL-8。因此,為能更好的在體外模擬炎癥環(huán)境,反映慢性炎癥時(shí)鼻咽癌細(xì)胞的狀態(tài),我們收集低濃度LPS刺激和未刺激巨噬細(xì)胞培養(yǎng)上清作為條件培養(yǎng)基(分別以D-THP-LPS, D-THP表示)處理鼻咽癌5-8F細(xì)胞,檢測(cè)促炎因子對(duì)5-8F細(xì)胞的影響。結(jié)果顯示,無(wú)論在mRNA水平還是蛋白水平,鼻咽癌5-8F細(xì)胞經(jīng)D-THP-LPS條件培養(yǎng)基處理24小時(shí)后,細(xì)胞中促炎因子TNF-α、IL-6、IL-1β和IL-8表達(dá)明顯增加,D-THP-LPS條件培養(yǎng)基可促進(jìn)鼻咽癌5-8F細(xì)胞增殖,激活NF-κB和Stat3信號(hào)通路。說(shuō)明炎癥微環(huán)境下鼻咽癌上皮細(xì)胞可因炎性因子的刺激而激活NF-κB和Stat3信號(hào)通路,自分泌促炎因子,維持和擴(kuò)大局部炎癥反應(yīng),從而促進(jìn)鼻咽癌發(fā)生發(fā)展。 [IL-6促進(jìn)鼻咽癌發(fā)生發(fā)展] 促炎因子IL-6是腫瘤相關(guān)巨噬細(xì)胞(TAMs)分泌的主要促炎因子之一,在炎癥促腫瘤發(fā)生發(fā)展過(guò)程中具有很重要的作用,而目前IL-6和腫瘤相關(guān)巨噬細(xì)胞(TAMs)對(duì)鼻咽癌發(fā)生發(fā)展的影響不是很清楚。因此我們檢測(cè)分析了鼻咽癌組織中IL-6蛋白表達(dá)情況和TAMs浸潤(rùn)程度,IL-6對(duì)鼻咽癌細(xì)胞生長(zhǎng)的影響。采用鼻咽癌組織芯片,免疫組化檢測(cè)發(fā)現(xiàn)鼻咽癌組織中IL-6和CD68(TAMs分子標(biāo)記物,表達(dá)強(qiáng)弱可以反映TAMs在腫瘤組織中浸潤(rùn)程度)均呈強(qiáng)陽(yáng)性表達(dá),IL-6蛋白表達(dá)與TAMs浸潤(rùn)密度正相關(guān),Kaplan-Meier生存分析顯示IL-6蛋白表達(dá)越高或者TAMs浸潤(rùn)密度越高,鼻咽癌患者預(yù)后越差；外源IL-6可促進(jìn)鼻咽癌細(xì)胞增殖、加快細(xì)胞周期演進(jìn),同時(shí)免疫熒光、熒光素酶報(bào)告系統(tǒng)及Western blot檢測(cè)顯示IL-6可激活NF-κBp65和p-Stat3蛋白。由此說(shuō)明鼻咽癌組織中TAMs浸潤(rùn)和IL-6蛋白表達(dá)與鼻咽癌發(fā)生發(fā)展密切相關(guān),IL-6可通過(guò)激活NF-κB和Stat3蛋白促進(jìn)鼻咽癌發(fā)生發(fā)展。 [LPLUNC1基因抑制鼻咽癌細(xì)胞生長(zhǎng)] 前期研究顯示LPLUNC1基因在鼻咽癌組織中低表達(dá),可能是鼻咽癌潛在的抑瘤基因。應(yīng)用鼻咽癌組織芯片,免疫組織化學(xué)檢測(cè)我們發(fā)現(xiàn)LPLUNC1蛋白在正常鼻咽上皮和腺體中高表達(dá),而在大多數(shù)鼻咽癌組織中低表達(dá)或陰性表達(dá),LPLUNC1蛋白表達(dá)下調(diào)與鼻咽癌臨床進(jìn)展分期正相關(guān),Kaplan-Meier生存顯示LPLUNC1蛋白陽(yáng)性表達(dá)的鼻咽癌患者臨床預(yù)后明顯好于陰性表達(dá)患者,生長(zhǎng)曲線繪制、MTT及裸鼠成瘤等實(shí)驗(yàn)均顯示LPLUNC1基因可明顯抑制鼻咽癌細(xì)胞生長(zhǎng)與增殖；流式細(xì)胞和凋亡檢測(cè)發(fā)現(xiàn)LPLUNC1基因可引起鼻咽癌細(xì)胞G0/G1期阻滯和誘導(dǎo)凋亡；進(jìn)一步研究顯示,LPLUNC1基因可通過(guò)抑制鼻咽癌細(xì)胞NF-κB和Stat3信號(hào)傳導(dǎo)通路,下調(diào)CDK4、cyclin D1、Bcl-2基因表達(dá),上調(diào)p21、Bax基因表達(dá)。隨后我們分別使用Jak2和NF-κB特異性抑制劑(AG490、BAY11-7082)分別阻斷HNE2/Vector、 HNE2/LPLUNC1細(xì)胞中Stat3和NF-κB信號(hào)通路,結(jié)果發(fā)現(xiàn)AG490、BAY11-7082均能明顯抑制鼻咽癌細(xì)胞增殖,而LPLUNC1基因與各抑制劑之間具有明顯的協(xié)同作用。上述結(jié)果表明LPLUNC1基因可抑制NF-κB和Stat3信號(hào)通路的激活,引起G0/G1期阻滯和誘導(dǎo)凋亡,從而抑制鼻咽癌細(xì)胞生長(zhǎng)。 [LPLUNC1抑制IL-6介導(dǎo)的NF-κB和Stat3信號(hào)通路的激活] 我們研究顯示IL-6可通過(guò)激活NF-κB和Stat3促進(jìn)鼻咽癌細(xì)胞生長(zhǎng)；組織芯片免疫組化結(jié)果分析發(fā)現(xiàn)鼻咽癌組織中LPLUNC1蛋白表達(dá)與IL-6蛋白表達(dá)和TAMs浸潤(rùn)密度明顯負(fù)相關(guān),同時(shí)應(yīng)用激光捕獲顯微切割技術(shù)分離獲得純化鼻咽癌和正常鼻咽上皮組織,qRT-PCR檢測(cè)也證實(shí)鼻咽癌組織中LPLUNC1mRNA表達(dá)與IL-6mRNA表達(dá)明顯負(fù)相關(guān),提示LPLUNC1可能參與炎癥反應(yīng),具有抑制IL-6分泌和TAMs浸潤(rùn)的作用。因此,我們探討了LPLUNC1對(duì)IL-6促進(jìn)鼻咽癌細(xì)胞增殖的影響。首先我們確證了LPLUNC1可明顯抑制LPS誘導(dǎo)的鼻咽癌細(xì)胞和巨噬細(xì)胞分泌促炎因子如TNF-α、IL-6、IL-1p和IL-8,參與抗炎反應(yīng)；隨后研究發(fā)現(xiàn),LPLUNC1基因可明顯抑制IL-6對(duì)鼻咽癌細(xì)胞的促增殖和加速細(xì)胞周期演進(jìn)的作用,熒光素酶報(bào)告基因檢測(cè)發(fā)現(xiàn)盡管HNE2細(xì)胞受到IL-6的刺激,LPLUNC1基因仍可明顯抑制NF-κB和Stat3的轉(zhuǎn)錄活性,免疫熒光檢測(cè)顯示LPLUNC1基因可明顯抑制IL-6誘導(dǎo)的NF-κBp65和Stat3舌化入核,Western blot檢測(cè)也顯示LPLUNC1基因可明顯抑制IL-6誘導(dǎo)的NF-κBp65和p-Stat3蛋白表達(dá),NF-κBp65蛋白表達(dá)減少的同時(shí)伴有IKKP蛋白表達(dá)減少和IκBα蛋白表達(dá)增加,p-Stat3蛋白表達(dá)減少的同時(shí)伴有p-Jak2蛋白表達(dá)減少,LPLUNC1基因還可通過(guò)抑制IL-6介導(dǎo)的NF-κB和Stat3信號(hào)通路的激活,下調(diào)cyclin D1、Bcl-2基因表達(dá),上調(diào)p21、Bax基因表達(dá)。因此,LPLUNC1基因可通過(guò)抑制IL-6介導(dǎo)的NF-κB和Stat3信號(hào)通路的激活,抵抗促炎因子IL-6對(duì)鼻咽癌細(xì)胞增殖的促進(jìn)作用。 [LPLUNC1對(duì)鼻咽癌5-8F細(xì)胞蛋白質(zhì)表達(dá)譜的影響] 蛋白質(zhì)是生命活動(dòng)中生物學(xué)功能的真正執(zhí)行者、生命現(xiàn)象的直接體現(xiàn)者,探討生命活動(dòng)中尤其是病理過(guò)程中發(fā)生改變的蛋白質(zhì)分子具有重大意義。因此,為進(jìn)一步了解LPLUNC1在鼻咽癌發(fā)生發(fā)展中的作用,我們采用熒光差異凝膠電泳(2D-DIGE)和質(zhì)譜分析技術(shù)獲得了鼻咽癌5-8F細(xì)胞過(guò)表達(dá)LPLUNC1基因前后的差異蛋白質(zhì)變化。共鑒定出44個(gè)差異表達(dá)蛋白質(zhì),包括上調(diào)19個(gè),下調(diào)25個(gè)。這些差異表達(dá)蛋白質(zhì)按其功能分類主要是(1)分子伴侶蛋白；(2)細(xì)胞骨架蛋白；(3)參與細(xì)胞新陳代謝的酶類或蛋白；(4)信號(hào)轉(zhuǎn)導(dǎo)分子；(5)其他蛋白。結(jié)果表明LPLUNC1基因可能通過(guò)調(diào)控上述蛋白分子表達(dá)參與細(xì)胞代謝、增殖、轉(zhuǎn)錄以及信號(hào)轉(zhuǎn)導(dǎo)等生物學(xué)過(guò)程,從而影響鼻咽癌的生長(zhǎng)。隨后,我們對(duì)上調(diào)差異蛋白Prohibitin (PHB)、下調(diào)差異蛋白hypoxia up-regulated1precursor (HYOU1)和calreticulin precursor variant (CALR)進(jìn)行了驗(yàn)證。結(jié)果顯示,LPLUNC1基因轉(zhuǎn)染5-8F細(xì)胞前后上述差異蛋白質(zhì)表達(dá)確實(shí)存在差異,與質(zhì)譜鑒定結(jié)果一致；對(duì)Prohibitin蛋白進(jìn)一步研究發(fā)現(xiàn),Prohibitin蛋白在正常鼻咽組織中呈高表達(dá),而在絕大部分鼻咽癌組織中呈低表達(dá),Prohibitin蛋白表達(dá)降低與鼻咽癌的臨床進(jìn)展分期和轉(zhuǎn)移相關(guān),Kaplan-Meier生存分析和多因素分析發(fā)現(xiàn)Prohibitin蛋白陰性表達(dá)與鼻咽癌患者預(yù)后差密切相關(guān)。提示Prohibitin可能是鼻咽癌的重要分子標(biāo)志。
[Abstract]:BACKGROUND OF THE STUDY

Chronic inflammation is the seventh major biological characteristic of the tumor , and chronic inflammation plays a very important role in triggering tumor development and promoting tumor development .
Nasopharyngeal carcinoma ( NPC ) is a kind of malignant tumor with obvious regional difference . Nasopharyngeal carcinoma is a kind of malignant tumor with obvious regional difference . The nasopharyngeal epithelial cell has been damaged by various exogenous pathogenic microorganisms such as EBV and toxic chemicals .
LPLUNC1 protein can inhibit the growth and metastasis of nasopharyngeal carcinoma by means of its function of host defense with the aid of its binding domain , and the role of LPLUNC1 gene in the development of nasopharyngeal carcinoma ( NPC ) and its related molecular mechanism are discussed .

Inflammatory factors stimulate the involvement of nasopharyngeal epithelial cells in inflammatory response

The expression of pro - inflammatory cytokines TNF - 偽 , IL - 6 , IL - 1尾 and IL - 8 in nasopharyngeal carcinoma ( NPC ) were measured with low concentration LPS ( 10 ng / ml , similar to the physiological concentration of bacterial lipopolysaccharide , such as bacterial infection ) .

Conclusion IL - 6 promotes the development of nasopharyngeal carcinoma .

IL - 6 and tumor - related macrophages ( TAMs ) have an important role in the development of nasopharyngeal carcinoma .
Exogenous IL - 6 can promote the proliferation of nasopharyngeal carcinoma cells , accelerate the development of cell cycle , and simultaneously immunofluorescence , luciferase reporter system and Western blot analysis show that IL - 6 can activate NF - 魏B and p - Stat3 protein . The expression of TAMs and IL - 6 in nasopharyngeal carcinoma is closely related to the development of nasopharyngeal carcinoma . IL - 6 can promote the development of nasopharyngeal carcinoma by activating NF - 魏B and Stat3 protein .

Inhibitory effect of recombinant LPLUNC1 gene on nasopharyngeal carcinoma cell growth

The low expression of LPLUNC1 gene in nasopharyngeal carcinoma ( NPC ) and the low expression of LPLUNC1 protein in normal nasopharyngeal epithelium and gland were detected by immunohistochemical method . The expression of LPLUNC1 was significantly correlated with the clinical progression of NPC .
LPLUNC1 gene can induce G0 / G1 arrest and induce apoptosis in nasopharyngeal carcinoma cells .
The results showed that AG490 , BAY11 - 7082 could inhibit the proliferation of nasopharyngeal carcinoma cells and inhibit the expression of Stat3 and NF - 魏B in HNE2 / Vector and HNE2 / LPLUNC1 cells .

Activation of IL - 6 - mediated NF - 魏B and Stat3 signaling pathways

Our study shows that IL - 6 can promote the growth of nasopharyngeal carcinoma cells by activating NF - 魏B and Stat3 ;
The expression of LPLUNC1 was negatively correlated with the expression of IL - 6 and the density of IL - 6 in nasopharyngeal carcinoma .
It was found that LPLUNC1 gene could significantly inhibit the expression of NF - 魏B and Stat3 in nasopharyngeal carcinoma cells . The expression of NF - 魏B and Stat3 was significantly inhibited by LPLUNC1 gene .

Effect of LPLUNC1 on protein expression profile of nasopharyngeal carcinoma ( NPC ) 5 - 8F cells

In order to further understand the role of LPLUNC1 in the development of nasopharyngeal carcinoma , we obtained 44 differentially expressed proteins , including up - regulation of 19 and down - regulation of 25 .
( 2 ) cytoskeletal proteins ;
( 3 ) enzymes or proteins involved in cell metabolism ;
( 4 ) signal transduction molecule ;
( 5 ) Other proteins . The results showed that the LPLUNC1 gene could regulate the biological processes of cell metabolism , proliferation , transcription and signal transduction by regulating the expression of the above - mentioned protein molecules , thus affecting the growth of nasopharyngeal carcinoma .
The results showed that the expression of bitin protein in nasopharyngeal carcinoma was significantly lower than that of nasopharyngeal carcinoma ( NPC ) . Kaplan - Meier survival analysis and multivariate analysis showed that the negative expression of bitin protein was closely related to the prognosis of NPC patients .
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.63

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本文編號(hào)：2015317