本文選題：下咽腫瘤 + 鱗狀細(xì)胞癌�。� 參考：《山東大學(xué)》2015年博士論文

【摘要】：第一部分TWIST誘導(dǎo)下咽癌FaDu細(xì)胞發(fā)生EMT并促進(jìn)其轉(zhuǎn)移的機(jī)制研究實(shí)驗(yàn)?zāi)康模阂环矫嫣接慣WIST的表達(dá)變化對(duì)下咽癌FaDu細(xì)胞發(fā)生上皮細(xì)胞-間充質(zhì)轉(zhuǎn)化(EMT)的影響及其促進(jìn)下咽癌FaDu細(xì)胞發(fā)生遷移、侵襲、轉(zhuǎn)移的內(nèi)在機(jī)制；另一方面探討TWIST在下咽癌組織中的表達(dá)及其與臨床病理資料的關(guān)系。實(shí)驗(yàn)方法：體外實(shí)驗(yàn)部分,實(shí)驗(yàn)分組為TWIST過(guò)表達(dá)載體組(pcDNA 3.1-TWIST)及其對(duì)照組,TWIST沉默載體組(MicroRNA-TWIST)及其對(duì)照組,利用脂質(zhì)體2000轉(zhuǎn)染下咽癌FaDu細(xì)胞,并利用Western blot的方法,驗(yàn)證TWIST蛋白在上述兩種載體及對(duì)照組中的表達(dá)情況；利用倒置相差顯微鏡,觀察TWIST過(guò)表達(dá)對(duì)下咽癌FaDu細(xì)胞形態(tài)的影響：利用Transwell小室實(shí)驗(yàn),觀察TWIST過(guò)表達(dá)對(duì)下咽癌FaDu細(xì)胞的遷移和侵襲能力的影響；Western blot檢測(cè)TWIST過(guò)表達(dá)和沉默時(shí)分別對(duì)E-cadherin和N-cadherin表達(dá)的影響；體內(nèi)實(shí)驗(yàn)部分,采用免疫組織化學(xué)的方法,檢測(cè)TWIST在下咽癌腫瘤組織和癌旁組織中的表達(dá)情況,并分析其表達(dá)與臨床病理資料(年齡,性別,腫瘤的大小、分化、淋巴結(jié)轉(zhuǎn)移)的關(guān)系。實(shí)驗(yàn)結(jié)果：Western blot結(jié)果顯示：TWIST在pcDNA3.1-TWIST組中較對(duì)照組表達(dá)升高,在miR-TWIST組中較對(duì)照組表達(dá)降低；倒置顯微鏡觀察Fadu細(xì)胞的形態(tài)改變,結(jié)果顯示：在pcDNA3.1-TWIST組中,細(xì)胞表現(xiàn)為長(zhǎng)條梭形,細(xì)胞處于松散狀,細(xì)胞間的極性丟失,細(xì)胞間的粘附性降低；TranswellTM小室實(shí)驗(yàn)發(fā)現(xiàn),遷移實(shí)驗(yàn)中,pcDNA3.1-TWIST組的細(xì)胞遷移數(shù)是對(duì)照組的1.8倍(P0.05),表明TWIST過(guò)表達(dá)能增強(qiáng)Fadu細(xì)胞遷移能力；侵襲實(shí)驗(yàn)中,pcDNA3.1-TWIST組中,細(xì)胞浸潤(rùn)數(shù)是對(duì)照組的1.6倍(P0.05),表明TWIST過(guò)表達(dá)同樣能增強(qiáng)Fadu細(xì)胞侵襲能力。檢測(cè)TWIST變化對(duì)E-cadherin和N-cadherin的影響,Western blot結(jié)果顯示：在pcDNA3.1-TWIST組中,TWIST表達(dá)升高時(shí),E-cadherin表達(dá)降低,N-cadherin升高；在miR-TWIST組表達(dá),TWIST表達(dá)降低時(shí),E-cadherin升高,N-cadherin表達(dá)降低,表明TWIST促進(jìn)Fadu細(xì)胞發(fā)生了EMT。利用免疫組織化學(xué)的方法,檢測(cè)下咽癌組織和癌旁組織中TWIST的表達(dá),結(jié)果顯示：TWIST在下咽癌組織中有表達(dá),癌旁組織中未見(jiàn)表達(dá)。TWIST在下咽癌組織的細(xì)胞核和細(xì)胞質(zhì)均有表達(dá),但細(xì)胞質(zhì)中表達(dá)更明顯；研究TWIST表達(dá)與臨床病理資料的關(guān)系,結(jié)果顯示：TWIST表達(dá)與下咽癌的分化(P=0.038),腫瘤大小(P=0.048),淋巴結(jié)轉(zhuǎn)移(P=0.044)有關(guān)；而TWIST表達(dá)與病人性別、年齡無(wú)關(guān)(P0.050)。實(shí)驗(yàn)結(jié)論：本實(shí)驗(yàn),體外實(shí)驗(yàn)首先證實(shí)了TWIST在pcDNA3.1-TWIST組中較對(duì)照組表達(dá)升高,在miR-TWIST組中較對(duì)照組表達(dá)降低,表明TWIST過(guò)表達(dá)載體和沉默載體構(gòu)建成功并在細(xì)胞內(nèi)發(fā)揮作用,其作為后續(xù)的基礎(chǔ)。TWIST的過(guò)表達(dá)能影響Fadu細(xì)胞的形態(tài)改變,并促進(jìn)Fadu細(xì)胞的遷移和侵襲能力；TWIST變化導(dǎo)致E-cadherin和N-cadherin的表達(dá)變化,表明TWIST誘導(dǎo)Fadu細(xì)胞發(fā)生EMT；體內(nèi)實(shí)驗(yàn)部分,研究發(fā)現(xiàn)TWIST在下咽癌組織中表達(dá),且其表達(dá)與下咽癌組織的分化、腫瘤大小、淋巴結(jié)轉(zhuǎn)移有關(guān),與年齡、性別無(wú)關(guān)。通過(guò)體內(nèi)外實(shí)驗(yàn)發(fā)現(xiàn),TWIST的表達(dá)與下咽癌組織的分化、腫瘤大小、淋巴結(jié)轉(zhuǎn)移有關(guān),且能促進(jìn)下咽癌Fadu細(xì)胞發(fā)生遷移、侵襲、轉(zhuǎn)移的內(nèi)在機(jī)制,是TWIST通過(guò)促進(jìn)下咽癌發(fā)生EMT和調(diào)控E-cadherin和N-cadherin表達(dá)實(shí)現(xiàn)的。第二部分TNFα通過(guò)NF-κB通路調(diào)控TWIST表達(dá)誘導(dǎo)下咽癌Fadu細(xì)胞發(fā)生EMT的機(jī)制研究實(shí)驗(yàn)?zāi)康模荷掀ぜ?xì)胞-間充質(zhì)轉(zhuǎn)化(EMT)的發(fā)生在腫瘤轉(zhuǎn)移中發(fā)揮重要作用,TNFa能促進(jìn)腫瘤的侵襲和轉(zhuǎn)移,其機(jī)制可能與EMT的發(fā)生有關(guān)。但是具體的機(jī)制并不清楚,所以,本實(shí)驗(yàn)研究意在探討TNFa對(duì)下咽癌細(xì)胞Fadu發(fā)生EMT及侵襲轉(zhuǎn)移能力的影響,并深入闡釋其內(nèi)在可能的機(jī)制。實(shí)驗(yàn)方法：以下咽癌Fadu細(xì)胞為實(shí)驗(yàn)材料,TNFa(10ng/ml)處理組為實(shí)驗(yàn)組,TNFa(10ng/ml)未處理組為對(duì)照組。TNFa(10ng/ml)作用Fadu細(xì)胞24 h,利用細(xì)胞劃痕-愈合實(shí)驗(yàn),檢測(cè)不同組細(xì)胞的運(yùn)動(dòng)能力,觀察TNFa(10 ng/ml)對(duì)FaDu細(xì)胞的運(yùn)動(dòng)能力的影響；采用Transwell實(shí)驗(yàn),檢測(cè)不同組細(xì)胞的遷移和侵襲能力,觀察TNFa(10 ng/ml)對(duì)下咽癌FaDu細(xì)胞遷移和侵襲能力的影響；利用倒置相差顯微鏡觀察細(xì)胞形態(tài)變化,探討TNFa(10 ng/ml)對(duì)下咽癌FaDu細(xì)胞形態(tài)的影響；利用細(xì)胞免疫熒光染色及共聚焦熒光顯微鏡檢測(cè)不同組中上皮間充質(zhì)標(biāo)志物E-cadherin, N-cadherin表達(dá),觀察TNFa(10 ng/ml)對(duì)E-cadherin, N-cadherin表達(dá)的影響；為了進(jìn)一步明確TNFa(10 ng/ml)對(duì)E-cadherin, Vimentin的影響,采用Western blot檢測(cè)NFa (10g/ml)處理FaDu細(xì)胞不同時(shí)間(0 h,1 h,3 h,5h)時(shí)E-cadherin, Vimentin的表達(dá)；為了進(jìn)一步探討TNFa (10 ng/ml)對(duì)E-cadherin, Vimentin影響的機(jī)制,采用了Western blot和免疫熒光染色及共聚焦熒光顯微鏡,檢測(cè)TNF (10 ng/ml)處理FaDu細(xì)胞不同時(shí)間(0h,1 h,3 h,5h)時(shí)TWIST,p65的表達(dá)變化；為了研究TNFa對(duì)p65的影響,同時(shí)檢測(cè)’TNFa(10 ng/ml)處理FaDu細(xì)胞不同時(shí)間(Oh,1 h,3 h,5h)時(shí)p-Ikk及p-IκBa表達(dá)變化；為了研究p65對(duì)TWIST的影響,利用NF-κB特異性抑制劑(BAY11-7082)和siRNA-p65質(zhì)粒,降低p65表達(dá),同時(shí)利用Western blot檢測(cè)各組中TWIST的表達(dá)變化。實(shí)驗(yàn)結(jié)果：TNFa作用細(xì)胞24 h后,TNFa處理組中,細(xì)胞的運(yùn)動(dòng)速度比對(duì)照組明顯加快；遷移實(shí)驗(yàn)中,處理組和對(duì)照組細(xì)胞數(shù)分別是276±38,124±15,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；浸潤(rùn)實(shí)驗(yàn)中,TNFa處理組和對(duì)照組細(xì)胞數(shù)分別是95±3,63±6,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；TNFa(10 ng/ml)作用后,Fadu細(xì)胞形態(tài)發(fā)生明顯變化,TNFa處理組的細(xì)胞極性丟失,細(xì)胞呈長(zhǎng)梭形,分散狀,細(xì)胞間的粘附性降低等間充質(zhì)細(xì)胞表型,而對(duì)照組細(xì)胞呈橢圓形,細(xì)胞間排列緊密,細(xì)胞間的粘附性高。此外,免疫熒光及Western blot檢測(cè)發(fā)現(xiàn)TNFa處理組中,上皮標(biāo)志物E-cadherin降低,間充質(zhì)標(biāo)志物Vimentin和N-cadherin表達(dá)升高；TNFa (10 ng/ml)作用Fadu細(xì)胞不同時(shí)間(0 h,1 h,3 h,5h)后,利用Western blot檢測(cè)作用不同時(shí)間的TWIST表達(dá),結(jié)果顯示隨著作用時(shí)間的延長(zhǎng),TWIST的表達(dá)升高；TNFa (10 ng/ml)作用Fadu細(xì)胞不同時(shí)間(0 h,1 h,3h,5h)后,利用Western blot檢測(cè)作用不同時(shí)間的p65, p-Ikk和p-IκBα的表達(dá),結(jié)果顯示：隨著作用時(shí)間的延長(zhǎng),其表達(dá)均升高；免疫熒光和Western blot檢測(cè)p65和TWIST表達(dá),結(jié)果顯示：與對(duì)照組比較,實(shí)驗(yàn)組核內(nèi)p65和TWIST表達(dá)升高；利用siRNA-p65和BAY11-7082降低p65表達(dá),Western blot檢測(cè)p65和TWIST表達(dá),結(jié)果顯示：siRNA-p65和BAY11-7082均能降低p65表達(dá),p65降低的同時(shí)TWIST表達(dá)降低。實(shí)驗(yàn)結(jié)論：fNFa能夠促進(jìn)FaDu田胞運(yùn)動(dòng),遷移及浸潤(rùn)能力；TNFa能誘導(dǎo)FaDu細(xì)胞形態(tài)由細(xì)胞間排列緊密、橢圓形,極性存在的上皮樣表型,向細(xì)胞松散、呈梭形、極性丟失的間充質(zhì)表型變化,同時(shí)伴有上皮標(biāo)志物E-cadherin降低,間充質(zhì)標(biāo)志物Vimentin和N-cadherin表達(dá)升高,表明TNFa誘導(dǎo)FaDu細(xì)胞發(fā)生具有較強(qiáng)遷移及侵襲能力的EMT。此外,TNFa誘導(dǎo)TWIST表達(dá)升高。這正是TNFa通過(guò)升高TWIST表達(dá),誘導(dǎo)FaDu細(xì)胞發(fā)生EMT,從而增強(qiáng)細(xì)胞的遷移及侵襲能力的機(jī)制。TNFa誘導(dǎo)TWIST表達(dá)升高的機(jī)制是TNFa促進(jìn)p-Ikk, p-IκBa的表達(dá),激活NF-κB通路中p65表達(dá),進(jìn)一步促進(jìn)TWIST表達(dá)升高。總之,TNFa通過(guò)NF-κB通路調(diào)控TWIST表達(dá),進(jìn)而促進(jìn)下咽癌發(fā)生EMT和增強(qiáng)細(xì)胞的轉(zhuǎn)移能力。
[Abstract]:The first part TWIST induces the mechanism of EMT in hypopharyngeal cancer FaDu cells and the mechanism to promote its metastasis. The objective of this study is to investigate the effect of TWIST expression on the epithelial cell mesenchymal transition (EMT) of hypopharyngeal carcinoma FaDu cells and the internal mechanism of promoting the migration, invasion and metastasis of the FaDu cells of hypopharynx cancer; on the other hand, TW is discussed. The expression of IST in hypopharyngeal carcinoma and its relationship with the clinicopathological data. Experimental methods: experimental part in vitro, the experimental group was divided into TWIST overexpression vector group (pcDNA 3.1-TWIST) and its control group, TWIST silencing carrier group (MicroRNA-TWIST) and its control group, using liposome 2000 to transfect the hypopharyngeal carcinoma FaDu cells, and use Western blot recipe. The expression of TWIST protein in the two carriers and control groups was tested, and the effect of TWIST overexpression on the morphology of FaDu cells in hypopharyngeal carcinoma was observed by inverted phase contrast microscope: the effect of TWIST overexpression on the migration and invasion ability of FaDu cells in hypopharyngeal carcinoma was observed by Transwell chamber test; Western blot detected TWIST over table. The effects of E-cadherin and N-cadherin on the expression of E-cadherin and N-cadherin respectively; in the experimental part of the body, immunohistochemistry was used to detect the expression of TWIST in the tumor tissues and para cancerous tissues of hypopharyngeal carcinoma, and to analyze the relationship between the expression and the clinicopathological data (age, sex, tumor size, differentiation, lymph node metastasis). Results: the results of Western blot showed that the expression of TWIST in the group pcDNA3.1-TWIST was higher than that in the control group, and the expression in the miR-TWIST group was lower than that in the control group. The morphological changes of Fadu cells were observed by the inverted microscope. The results showed that in the pcDNA3.1-TWIST group, the cells displayed a long shuttle shape, the cells were in the loose shape, the cell polarity was lost, and the cells were lost. In the TranswellTM lab experiment, the number of cell migration in group pcDNA3.1-TWIST was 1.8 times that of the control group (P0.05), indicating that TWIST overexpression could enhance the migration ability of Fadu cells. In the invasive experiment, the number of cell infiltration in the pcDNA3.1-TWIST group was 1.6 times as much as that of the control group (P0.05), indicating that the over expression of TWIST was equally possible. The effects of TWIST changes on E-cadherin and N-cadherin were enhanced. The results of Western blot showed that the expression of E-cadherin decreased and N-cadherin increased when the expression of TWIST increased in the pcDNA3.1-TWIST group, and in miR-TWIST group, when the TWIST expression decreased, the expression decreased. The expression of TWIST in hypopharyngeal and paracancerous tissues was detected by EMT. in U cells. The results showed that TWIST was expressed in the hypopharyngeal carcinoma tissue. The expression of.TWIST in the nucleus and cytoplasm of the hypopharyngeal carcinoma was not expressed in the para cancer tissues, but the expression of the cytoplasm in the cytoplasm was more obvious; the expression of TWIST was studied. The relationship with the clinicopathological data showed that the expression of TWIST was related to the differentiation of hypopharyngeal carcinoma (P=0.038), tumor size (P=0.048) and lymph node metastasis (P=0.044), and the expression of TWIST was not related to the sex of the patients and age (P0.050). Experimental conclusion: in this experiment, the expression of TWIST in the group of pcDNA3.1-TWIST was first confirmed in the pcDNA3.1-TWIST group than in the control group. The expression of TWIST over expression vector and silencing carrier was successfully constructed and played a role in the cells in the miR-TWIST group. The overexpression of.TWIST, as a follow-up basis, could affect the morphological changes of Fadu cells and promote the migration and invasion of Fadu cells, and the TWIST changes led to the expression of E-cadherin and N-cadherin. The results showed that TWIST induced the occurrence of EMT in Fadu cells. In the experimental part of the body, the expression of TWIST in hypopharyngeal carcinoma was found, and its expression was related to the differentiation of hypopharyngeal carcinoma, the size of the tumor, the metastasis of lymph nodes, and the age and sex. The expression of TWIST and the differentiation of the hypopharyngeal carcinoma, the size of the tumor, the lymph nodes, and the lymph nodes were found in the body and the body. Metastasis is related, and it can promote the migration, invasion and metastasis of hypopharyngeal carcinoma Fadu cells, which is realized by TWIST by promoting EMT and regulating E-cadherin and N-cadherin expression in hypopharyngeal carcinoma. The second part TNF alpha regulates TWIST expression by NF- kappa B pathway to regulate the EMT mechanism of Fadu cell occurrence in hypopharyngeal carcinoma: epithelial fine Cellular mesenchyme transformation (EMT) plays an important role in tumor metastasis. TNFa can promote tumor invasion and metastasis, and its mechanism may be related to the occurrence of EMT. However, the specific mechanism is not clear. Therefore, this study is intended to explore the effect of TNFa on the EMT and invasion and metastasis of hypopharynx cancer cells, and to explain it in depth. The possible mechanism. Experimental methods: the Fadu cells of the following pharynx cancer are the experimental material, the TNFa (10ng/ml) treatment group is the experimental group, the TNFa (10ng/ml) untreated group is 24 h of the.TNFa (10ng/ml) action of the control group, and the cell scratch healing experiment is used to detect the movement ability of the different groups of cells, and the movement ability of TNFa (10 ng/ml) on the FaDu cells is observed. The effect of Transwell test was used to detect the migration and invasion ability of different groups of cells and to observe the effect of TNFa (10 ng/ml) on the migration and invasion ability of FaDu cells in hypopharyngeal carcinoma. The morphological changes of the cells were observed by inverted phase contrast microscope and the effects of TNFa (10 ng/ml) on the morphology of the FaDu cells in hypopharyngeal carcinoma; and cell immunofluorescence staining was used. The effects of TNFa (10 ng/ml) on the expression of E-cadherin and N-cadherin were detected by TNFa (10 ng/ml), and the effects of TNFa (10 ng/ml) on E-cadherin and Vimentin were detected by confocal fluorescence microscopy, and the effects of TNFa (10 ng/ml) on E-cadherin and Vimentin were determined by Western blot (0, 1, 1). H, 3 h, 5H) the expression of E-cadherin, Vimentin; in order to further explore the mechanism of TNFa (10 ng/ml) on E-cadherin and Vimentin, Western blot and immunofluorescence staining and confocal fluorescence microscopy were used to detect the changes in the expression of TNF (1, 3, 3). In order to study the effect of TNFa (10 ng/ml) on the expression of p-Ikk and p-I kappa Ba at different times of FaDu cells (Oh, 1 h, 3 h, 5H), the expression of p65 and p-I kappa Ba were detected. Results: after 24 h of TNFa cells, the speed of cell movement in the TNFa treatment group was significantly faster than that in the control group, and the number of cells in the treatment group and the control group were 276 + 38124 + 15, respectively, and the difference was statistically significant (P0.05). In the infiltration experiment, the number of TNFa treatment group and the irradiated group was 95 + 3,63 + 6, and the difference was statistically significant. Meaning (P0.05); after the action of TNFa (10 ng/ml), the morphology of Fadu cells changed obviously. The cell polarity of the TNFa treatment group was lost, the cells showed long spindle shape, scattered, and the adhesion between cells decreased, while the cells in the control group were elliptical, the cells were arranged closely, and the adhesion between cells was high. In addition, immunofluorescence and Western Blot detection found that in the TNFa treatment group, the epithelial markers E-cadherin decreased and the expression of mesenchymal markers Vimentin and N-cadherin increased, and TNFa (10 ng/ml) was used to detect the expression of Fadu cells at different time (0 h, 1 h, 3 h, 5H). The results showed that the expression increased with the prolongation of action time. After TNFa (10 ng/ml) action of Fadu cells at different time (0 h, 1 h, 3h, 5H), Western blot was used to detect the expression of p65, p-Ikk and p-I kappa alpha. The results showed that the expression increased with the prolongation of action time, and the results showed that the experimental group was compared with the control group, and the experimental group was compared with the control group. The expression of p65 and TWIST in the nucleus was increased, p65 expression was reduced by siRNA-p65 and BAY11-7082, and Western blot was used to detect p65 and TWIST expression. The results showed that siRNA-p65 and BAY11-7082 could reduce the p65 expression and decrease the expression at the same time. The cell morphology is characterized by a compact, elliptical and polar epithelioid phenotype that is loose, spindle shaped, and an interstitial phenotypic change, accompanied by a decrease in the epithelial marker E-cadherin, and the increase in the expression of Vimentin and N-cadherin in the mesenchymal markers. It is indicated that TNFa induced FaDu cells have strong migration and invasion ability. In addition, TNFa induced the increase of TWIST expression. This is the mechanism that TNFa induces EMT in FaDu cells by increasing TWIST expression, thus enhancing the migration and invasion of cells. The mechanism.TNFa induces the increase of TWIST expression is TNFa promoting p-Ikk, p-I kappa expresses, and further promotes the increase of expression. TNFa regulates TWIST expression through NF- kappa B pathway, thereby promoting EMT and enhancing cell migration in hypopharyngeal carcinoma.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R739.6

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