發(fā)布時(shí)間：2018-04-27 20:23

  本文選題：甲狀腺癌 + 基質(zhì)細(xì)胞衍生因子-1�。� 參考：《吉林大學(xué)》2017年博士論文

【摘要】：背景和目的：甲狀腺癌的發(fā)病率逐年上升,包括:乳頭狀癌、濾泡狀癌、未分化癌和髓樣癌。盡管分化良好的甲狀腺癌占甲狀腺癌患者的90%以上,但甲狀腺癌轉(zhuǎn)移的發(fā)生率卻非常高,20%-90%的甲狀腺乳頭狀癌患者在首次確診時(shí)已發(fā)生局部淋巴結(jié)轉(zhuǎn)移,嚴(yán)重影響了疾病的預(yù)后[3]。目前,甲狀腺癌轉(zhuǎn)移的分子機(jī)制尚不明確,研究甲狀腺癌轉(zhuǎn)移的分子機(jī)制對(duì)于尋找甲狀腺癌新的治療靶點(diǎn)具有重要的理論和實(shí)際意義�；|(zhì)細(xì)胞衍生因子-1(SDF-1)是趨化因子蛋白家族主要成員之一,廣泛的表達(dá)于各種組織和細(xì)胞中。CXC族趨化因子受體4(CXCR4)是G蛋白偶聯(lián)受體家族的一員,是SDF-1的主要受體之一,SDF-1/CXCR4系統(tǒng)在腫瘤的發(fā)生、轉(zhuǎn)移過(guò)程中發(fā)揮重要作用。CXCR4在正常的甲狀腺細(xì)胞和多數(shù)良性的甲狀腺瘤細(xì)胞中低或不表達(dá),而在甲狀腺癌細(xì)胞中高表達(dá)CXCR4[6,7]。CXCR4受體密度的增加,可增強(qiáng)SDF-1引起的細(xì)胞侵襲活動(dòng),但對(duì)癌細(xì)胞增殖沒(méi)有影響[8]。提示SDF-1/CXC R4系統(tǒng)可能在甲狀腺癌細(xì)胞的遷移侵襲過(guò)程中發(fā)揮重要作用。本課題的目的是在細(xì)胞模型中研究SDF-1/CXCR4系統(tǒng)調(diào)節(jié)甲狀腺乳頭狀癌細(xì)胞遷移、侵襲的作用及相關(guān)分子機(jī)制。材料和方法:1.材料82例人甲狀腺乳頭狀癌組織為吉林大學(xué)中日聯(lián)誼醫(yī)院2014年12月至2016年5月手術(shù)切除標(biāo)本;B-CPAP購(gòu)于上海中喬新舟生物科技公司。2.方法(1)免疫組化檢測(cè)人甲狀腺乳頭狀癌組織標(biāo)本中CXCR4的表達(dá)。(2)體外培養(yǎng)人甲狀腺乳頭狀癌細(xì)胞株(B-CPAP)。設(shè)計(jì)并合成CXCR4基因CDS區(qū),構(gòu)建CXCR4過(guò)表達(dá)的質(zhì)粒。B-CPAP細(xì)胞轉(zhuǎn)染CXCR4過(guò)表達(dá)質(zhì)粒或?qū)?yīng)的vector(空載質(zhì)粒),瞬轉(zhuǎn)獲得B-CPAP-vector和B-CPAP-CXCR4細(xì)胞。轉(zhuǎn)染48小時(shí)后,免疫印跡和熒光定量實(shí)驗(yàn)檢測(cè)各組細(xì)胞中CXCR4的表達(dá)水平。(3)在B-CPAP細(xì)胞中轉(zhuǎn)染CXCR4過(guò)表達(dá)質(zhì)粒24小時(shí)后,用100ng/ml的SDF-1處理48小時(shí)。然后通過(guò)劃痕實(shí)驗(yàn)檢測(cè)各組細(xì)胞遷移能力;通過(guò)Transwel l實(shí)驗(yàn)檢測(cè)各組細(xì)胞侵襲能力。(4)在B-CPAP細(xì)胞中轉(zhuǎn)染CXCR4過(guò)表達(dá)質(zhì)粒24小時(shí)后,用100ng/ml的SDF-1處理48小時(shí)。然后免疫印跡檢測(cè)E-cadherin(上皮-鈣黏素),N-cadhe rin(神經(jīng)-鈣黏素),Vimentin(波形蛋白),NF-κB,磷酸化的IκB(P-IκB)以及細(xì)胞核內(nèi)NF-κB的表達(dá)水平。免疫熒光實(shí)驗(yàn)檢測(cè)E-cadherin的表達(dá)情況。(5)在B-CPAP細(xì)胞中轉(zhuǎn)染CXCR4過(guò)表達(dá)質(zhì)粒24小時(shí)后,用5μmol NF-κB抑制劑BAY11-7028(BAY)預(yù)處理1小時(shí),然后加入100ng/ml的SDF-1處理48小時(shí)。免疫印跡實(shí)驗(yàn)檢測(cè)NF-κB、P-IκB以及細(xì)胞核內(nèi)NF-κB的水平;劃痕實(shí)驗(yàn)檢測(cè)各組細(xì)胞遷移能力;Transwell實(shí)驗(yàn)檢測(cè)各組細(xì)胞侵襲能力。結(jié)果:(1)免疫組化檢測(cè)結(jié)果顯示CXCR4在甲狀腺乳頭狀癌組織中高表達(dá)(2)免疫印跡和熒光定量檢測(cè)結(jié)果顯示轉(zhuǎn)染CXCR4過(guò)表達(dá)質(zhì)粒后,B-CPA P-CXCR4細(xì)胞中CXCR4表達(dá)明顯增加。(3)在B-CPAP細(xì)胞中轉(zhuǎn)染CXCR4過(guò)表達(dá)質(zhì)粒后,用SDF-1處理,細(xì)胞的遷移性和侵襲性顯著增強(qiáng)。(4)SDF-1/CXCR4明顯減少了E-cadherin蛋白表達(dá)量,增加了N-cadhe rin蛋白、Vimentin蛋白的表達(dá)量。免疫熒光檢測(cè)E-cadherin蛋白發(fā)現(xiàn),SDF-1/CXCR4系統(tǒng)可以明顯抑制E-cadherin的表達(dá)量。表明SDF-1/CXCR4系統(tǒng)促進(jìn)B-CPAP的EMT過(guò)程。(5)SDF-1/CXCR4系統(tǒng)可以顯著性提高細(xì)胞核中NF-κB的表達(dá)量,降低細(xì)胞質(zhì)中NF-κB的表達(dá)量,降低細(xì)胞中P-IκB的表達(dá)量,激活NF-κB信號(hào)通路。(6)免疫印跡實(shí)驗(yàn)表明NF-κB抑制劑BAY可以提高E-cadherin表達(dá)量,降低N-cadherin、Vimentin表達(dá)量,抑制了SDF-1/CXCR4系統(tǒng)對(duì)B-CPAP的EM T過(guò)程的促進(jìn)作用;劃痕實(shí)驗(yàn)表明NF-κB抑制劑BAY可以減弱SDF-1/CXCR4系統(tǒng)對(duì)B-CPAP遷移能力的促進(jìn)作用;Transwell實(shí)驗(yàn)表明NF-κB抑制劑BAY可以減弱SDF-1/CXCR4系統(tǒng)對(duì)B-CPAP侵襲能力的促進(jìn)作用。結(jié)論:(1)CXCR4受體反應(yīng)系統(tǒng)可加強(qiáng)NF-κB通路的信號(hào)傳導(dǎo),促進(jìn)甲狀腺乳頭狀癌細(xì)胞的EMT過(guò)程,加強(qiáng)乳頭狀癌細(xì)胞的遷移侵襲。(2)NF-κB通路的抑制劑BAY可削弱CXCR4受體反應(yīng)系統(tǒng)對(duì)甲狀腺乳頭狀癌細(xì)胞EMT過(guò)程的促進(jìn)作用,減輕CXCR4受體反應(yīng)系統(tǒng)對(duì)甲狀腺乳頭狀癌細(xì)胞的促遷移侵襲作用。NF-κB通路可望成為抑制甲狀腺乳頭狀癌細(xì)胞遷移侵襲的潛在治療靶點(diǎn)。
[Abstract]:Background and objective: the incidence of thyroid cancer is increasing year by year, including papillary, follicular, undifferentiated and medullary cancer. Although well differentiated thyroid cancer accounts for more than 90% of thyroid cancer patients, the incidence of thyroid cancer metastasis is very high, and 20%-90% thyroid papillary carcinoma has been localized at the first diagnosis. The molecular mechanism of thyroid cancer metastasis is not clear. The molecular mechanism of thyroid cancer metastasis is of great theoretical and practical significance for finding new therapeutic targets for thyroid cancer. The matrix derived factor -1 (SDF-1) is one of the main members of the chemokine protein family, [3]. is a major member of the chemokine protein family. .CXC chemokine receptor 4 (CXCR4), a member of the G protein coupling receptor family, is widely expressed in various tissues and cells. It is one of the major receptors of SDF-1. The SDF-1/CXCR4 system plays an important role in the occurrence of tumor and in the metastasis process,.CXCR4 is low or non expression in normal thyroid cells and most benign thyroid tumor cells. The increase in the density of CXCR4[6,7].CXCR4 receptor in thyroid cancer cells enhances SDF-1 induced cell invasion, but does not affect the proliferation of cancer cells by [8]. suggesting that SDF-1/CXC R4 system may play an important role in the migration and invasion of thyroid cancer cells. The purpose of this topic is to study SDF-1/ in the cell model. CXCR4 system regulates the migration, invasion and related molecular mechanisms of thyroid papillary carcinoma cells. Materials and methods: 1. materials and 82 cases of human thyroid papillary carcinoma were resected from December 2014 to May 2016 in the Sino Japanese Friendship Hospital of Jilin University; B-CPAP was purchased in.2. method (1) of Shanghai Zhong Qiao new boat biotechnology company (1) immunization The expression of CXCR4 in human papillary thyroid carcinoma tissue was detected. (2) human papillary carcinoma cell line (B-CPAP) was cultured in vitro. The CDS region of CXCR4 gene was designed and synthesized, and CXCR4 overexpressed plasmid.B-CPAP cells were transfected into CXCR4 overexpressed plasmid or corresponding vector (empty plasmid), and B-CPAP-vector and B-CPAP-CXCR4 cells were transients. After 48 hours transfection, the expression level of CXCR4 in each group was detected by immunoblotting and fluorescence quantitative assay. (3) after transfecting CXCR4 overexpression plasmid in B-CPAP cells for 24 hours, 100ng/ml SDF-1 was treated for 48 hours. Then the migration ability of each group was detected by scratch test, and the invasive ability of each group was detected through Transwel l test. (4) After transfecting the CXCR4 overexpression plasmid in B-CPAP cells for 24 hours, SDF-1 was treated with 100ng/ml for 48 hours. Then, E-cadherin (epithelia calcinin), N-cadhe Rin (nerve calcin), Vimentin (vimentin), NF- kappa B, I kappa B (kappa B) and the expression level of nuclear kappa protein in the nucleus were detected by immunoblotting. (5) (5) after transfection of CXCR4 overexpression plasmid in B-CPAP cells for 24 hours, 1 hours were pretreated with BAY11-7028 (BAY) of 5 mol NF- kappa B inhibitor, and then 100ng/ml SDF-1 for 48 hours. Results: (1) immunohistochemical detection showed that CXCR4 was highly expressed in thyroid papillary carcinoma (2) the results of immunoblotting and fluorescence quantitative detection showed that after transfection of CXCR4 overexpressed plasmid, the expression of CXCR4 in B-CPA P-CXCR4 cells increased significantly. (3) transfection of CXCR4 overexpressed plasmid in B-CPAP cells. The mobility and invasiveness of the cells were significantly enhanced by SDF-1 treatment. (4) SDF-1/CXCR4 significantly reduced the expression of E-cadherin protein and increased the expression of N-cadhe Rin protein and Vimentin protein. The immunofluorescent detection of E-cadherin protein found that SDF-1/CXCR4 system could significantly inhibit the expression of E-cadherin. The SDF-1/CXCR4 system promoted B-C. The EMT process of PAP. (5) the SDF-1/CXCR4 system can significantly increase the expression of NF- kappa B in the nucleus, reduce the expression of NF- kappa B in the cytoplasm, reduce the expression of P-I kappa B in the cell, and activate the NF- kappa B signaling pathway. (6) immunoblotting experiments show that the expression of NF- kappa inhibitor can improve the expression, decrease the expression and suppress the expression. The effect of SDF-1/CXCR4 system on the EM T process of B-CPAP was made, and the scratch test showed that NF- kappa B inhibitor BAY could weaken the promoting effect of SDF-1/CXCR4 system on B-CPAP migration ability. Transwell experiment showed that NF- kappa B inhibitor could weaken the promoting effect of the system on the invasion energy. Conclusion: (1) the receptor reaction system The signal transduction of NF- kappa B pathway can be strengthened to promote the EMT process of papillary thyroid carcinoma cells and to strengthen the migration and invasion of papillary carcinoma cells. (2) the inhibitor BAY of the NF- kappa B pathway can weaken the EMT process of the thyroid papillary carcinoma cell by the CXCR4 receptor reaction system and reduce the CXCR4 receptor reaction system to the thyroid papillary carcinoma. Cell migration and invasion.NF- kappa B pathway is expected to be a potential therapeutic target to inhibit the migration and invasion of papillary thyroid carcinoma cells.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R736.1

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