本文選題：下咽腫瘤 + 鱗狀細胞癌　； 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細胞發(fā)生EMT并促進其轉(zhuǎn)移的機制研究實驗?zāi)康模阂环矫嫣接慣WIST的表達變化對下咽癌FaDu細胞發(fā)生上皮細胞-間充質(zhì)轉(zhuǎn)化(EMT)的影響及其促進下咽癌FaDu細胞發(fā)生遷移、侵襲、轉(zhuǎn)移的內(nèi)在機制；另一方面探討TWIST在下咽癌組織中的表達及其與臨床病理資料的關(guān)系。實驗方法：體外實驗部分,實驗分組為TWIST過表達載體組(pcDNA 3.1-TWIST)及其對照組,TWIST沉默載體組(MicroRNA-TWIST)及其對照組,利用脂質(zhì)體2000轉(zhuǎn)染下咽癌FaDu細胞,并利用Western blot的方法,驗證TWIST蛋白在上述兩種載體及對照組中的表達情況；利用倒置相差顯微鏡,觀察TWIST過表達對下咽癌FaDu細胞形態(tài)的影響：利用Transwell小室實驗,觀察TWIST過表達對下咽癌FaDu細胞的遷移和侵襲能力的影響；Western blot檢測TWIST過表達和沉默時分別對E-cadherin和N-cadherin表達的影響；體內(nèi)實驗部分,采用免疫組織化學(xué)的方法,檢測TWIST在下咽癌腫瘤組織和癌旁組織中的表達情況,并分析其表達與臨床病理資料(年齡,性別,腫瘤的大小、分化、淋巴結(jié)轉(zhuǎn)移)的關(guān)系。實驗結(jié)果：Western blot結(jié)果顯示：TWIST在pcDNA3.1-TWIST組中較對照組表達升高,在miR-TWIST組中較對照組表達降低；倒置顯微鏡觀察Fadu細胞的形態(tài)改變,結(jié)果顯示：在pcDNA3.1-TWIST組中,細胞表現(xiàn)為長條梭形,細胞處于松散狀,細胞間的極性丟失,細胞間的粘附性降低；TranswellTM小室實驗發(fā)現(xiàn),遷移實驗中,pcDNA3.1-TWIST組的細胞遷移數(shù)是對照組的1.8倍(P0.05),表明TWIST過表達能增強Fadu細胞遷移能力；侵襲實驗中,pcDNA3.1-TWIST組中,細胞浸潤數(shù)是對照組的1.6倍(P0.05),表明TWIST過表達同樣能增強Fadu細胞侵襲能力。檢測TWIST變化對E-cadherin和N-cadherin的影響,Western blot結(jié)果顯示：在pcDNA3.1-TWIST組中,TWIST表達升高時,E-cadherin表達降低,N-cadherin升高；在miR-TWIST組表達,TWIST表達降低時,E-cadherin升高,N-cadherin表達降低,表明TWIST促進Fadu細胞發(fā)生了EMT。利用免疫組織化學(xué)的方法,檢測下咽癌組織和癌旁組織中TWIST的表達,結(jié)果顯示：TWIST在下咽癌組織中有表達,癌旁組織中未見表達。TWIST在下咽癌組織的細胞核和細胞質(zhì)均有表達,但細胞質(zhì)中表達更明顯；研究TWIST表達與臨床病理資料的關(guān)系,結(jié)果顯示：TWIST表達與下咽癌的分化(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表達與病人性別、年齡無關(guān)(P0.050)。實驗結(jié)論：本實驗,體外實驗首先證實了TWIST在pcDNA3.1-TWIST組中較對照組表達升高,在miR-TWIST組中較對照組表達降低,表明TWIST過表達載體和沉默載體構(gòu)建成功并在細胞內(nèi)發(fā)揮作用,其作為后續(xù)的基礎(chǔ)。TWIST的過表達能影響Fadu細胞的形態(tài)改變,并促進Fadu細胞的遷移和侵襲能力；TWIST變化導(dǎo)致E-cadherin和N-cadherin的表達變化,表明TWIST誘導(dǎo)Fadu細胞發(fā)生EMT；體內(nèi)實驗部分,研究發(fā)現(xiàn)TWIST在下咽癌組織中表達,且其表達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、性別無關(guān)。通過體內(nèi)外實驗發(fā)現(xiàn),TWIST的表達與下咽癌組織的分化、腫瘤大小、淋巴結(jié)轉(zhuǎn)移有關(guān),且能促進下咽癌Fadu細胞發(fā)生遷移、侵襲、轉(zhuǎn)移的內(nèi)在機制,是TWIST通過促進下咽癌發(fā)生EMT和調(diào)控E-cadherin和N-cadherin表達實現(xiàn)的。第二部分TNFα通過NF-κB通路調(diào)控TWIST表達誘導(dǎo)下咽癌Fadu細胞發(fā)生EMT的機制研究實驗?zāi)康模荷掀ぜ毎?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能促進腫瘤的侵襲和轉(zhuǎn)移,其機制可能與EMT的發(fā)生有關(guān)。但是具體的機制并不清楚,所以,本實驗研究意在探討TNFa對下咽癌細胞Fadu發(fā)生EMT及侵襲轉(zhuǎn)移能力的影響,并深入闡釋其內(nèi)在可能的機制。實驗方法：以下咽癌Fadu細胞為實驗材料,TNFa(10ng/ml)處理組為實驗組,TNFa(10ng/ml)未處理組為對照組。TNFa(10ng/ml)作用Fadu細胞24 h,利用細胞劃痕-愈合實驗,檢測不同組細胞的運動能力,觀察TNFa(10 ng/ml)對FaDu細胞的運動能力的影響；采用Transwell實驗,檢測不同組細胞的遷移和侵襲能力,觀察TNFa(10 ng/ml)對下咽癌FaDu細胞遷移和侵襲能力的影響；利用倒置相差顯微鏡觀察細胞形態(tài)變化,探討TNFa(10 ng/ml)對下咽癌FaDu細胞形態(tài)的影響；利用細胞免疫熒光染色及共聚焦熒光顯微鏡檢測不同組中上皮間充質(zhì)標(biāo)志物E-cadherin, N-cadherin表達,觀察TNFa(10 ng/ml)對E-cadherin, N-cadherin表達的影響；為了進一步明確TNFa(10 ng/ml)對E-cadherin, Vimentin的影響,采用Western blot檢測NFa (10g/ml)處理FaDu細胞不同時間(0 h,1 h,3 h,5h)時E-cadherin, Vimentin的表達；為了進一步探討TNFa (10 ng/ml)對E-cadherin, Vimentin影響的機制,采用了Western blot和免疫熒光染色及共聚焦熒光顯微鏡,檢測TNF (10 ng/ml)處理FaDu細胞不同時間(0h,1 h,3 h,5h)時TWIST,p65的表達變化；為了研究TNFa對p65的影響,同時檢測’TNFa(10 ng/ml)處理FaDu細胞不同時間(Oh,1 h,3 h,5h)時p-Ikk及p-IκBa表達變化；為了研究p65對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,同時利用Western blot檢測各組中TWIST的表達變化。實驗結(jié)果：TNFa作用細胞24 h后,TNFa處理組中,細胞的運動速度比對照組明顯加快；遷移實驗中,處理組和對照組細胞數(shù)分別是276±38,124±15,差異具有統(tǒng)計學(xué)意義(P0.05)；浸潤實驗中,TNFa處理組和對照組細胞數(shù)分別是95±3,63±6,差異具有統(tǒng)計學(xué)意義(P0.05)；TNFa(10 ng/ml)作用后,Fadu細胞形態(tài)發(fā)生明顯變化,TNFa處理組的細胞極性丟失,細胞呈長梭形,分散狀,細胞間的粘附性降低等間充質(zhì)細胞表型,而對照組細胞呈橢圓形,細胞間排列緊密,細胞間的粘附性高。此外,免疫熒光及Western blot檢測發(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表達升高；TNFa (10 ng/ml)作用Fadu細胞不同時間(0 h,1 h,3 h,5h)后,利用Western blot檢測作用不同時間的TWIST表達,結(jié)果顯示隨著作用時間的延長,TWIST的表達升高；TNFa (10 ng/ml)作用Fadu細胞不同時間(0 h,1 h,3h,5h)后,利用Western blot檢測作用不同時間的p65, p-Ikk和p-IκBα的表達,結(jié)果顯示：隨著作用時間的延長,其表達均升高；免疫熒光和Western blot檢測p65和TWIST表達,結(jié)果顯示：與對照組比較,實驗組核內(nèi)p65和TWIST表達升高；利用siRNA-p65和BAY11-7082降低p65表達,Western blot檢測p65和TWIST表達,結(jié)果顯示：siRNA-p65和BAY11-7082均能降低p65表達,p65降低的同時TWIST表達降低。實驗結(jié)論：fNFa能夠促進FaDu田胞運動,遷移及浸潤能力；TNFa能誘導(dǎo)FaDu細胞形態(tài)由細胞間排列緊密、橢圓形,極性存在的上皮樣表型,向細胞松散、呈梭形、極性丟失的間充質(zhì)表型變化,同時伴有上皮標(biāo)志物E-cadherin降低,間充質(zhì)標(biāo)志物Vimentin和N-cadherin表達升高,表明TNFa誘導(dǎo)FaDu細胞發(fā)生具有較強遷移及侵襲能力的EMT。此外,TNFa誘導(dǎo)TWIST表達升高。這正是TNFa通過升高TWIST表達,誘導(dǎo)FaDu細胞發(fā)生EMT,從而增強細胞的遷移及侵襲能力的機制。TNFa誘導(dǎo)TWIST表達升高的機制是TNFa促進p-Ikk, p-IκBa的表達,激活NF-κB通路中p65表達,進一步促進TWIST表達升高�？傊�,TNFa通過NF-κB通路調(diào)控TWIST表達,進而促進下咽癌發(fā)生EMT和增強細胞的轉(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)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R739.6

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