CXCR4受体反应系统调控甲状腺乳头状癌细胞迁移、侵袭作用的机制研究

发布时间：2018-04-27 20:23

本文选题：甲状腺癌 + 基质细胞衍生因子-1　；参考：《吉林大学》2017年博士论文

【摘要】：背景和目的：甲状腺癌的发病率逐年上升,包括:乳头状癌、滤泡状癌、未分化癌和髓样癌。尽管分化良好的甲状腺癌占甲状腺癌患者的90%以上,但甲状腺癌转移的发生率却非常高,20%-90%的甲状腺乳头状癌患者在首次确诊时已发生局部淋巴结转移,严重影响了疾病的预后[3]。目前,甲状腺癌转移的分子机制尚不明确,研究甲状腺癌转移的分子机制对于寻找甲状腺癌新的治疗靶点具有重要的理论和实际意义。基质细胞衍生因子-1(SDF-1)是趋化因子蛋白家族主要成员之一,广泛的表达于各种组织和细胞中。CXC族趋化因子受体4(CXCR4)是G蛋白偶联受体家族的一员,是SDF-1的主要受体之一,SDF-1/CXCR4系统在肿瘤的发生、转移过程中发挥重要作用。CXCR4在正常的甲状腺细胞和多数良性的甲状腺瘤细胞中低或不表达,而在甲状腺癌细胞中高表达CXCR4[6,7]。CXCR4受体密度的增加,可增强SDF-1引起的细胞侵袭活动,但对癌细胞增殖没有影响[8]。提示SDF-1/CXC R4系统可能在甲状腺癌细胞的迁移侵袭过程中发挥重要作用。本课题的目的是在细胞模型中研究SDF-1/CXCR4系统调节甲状腺乳头状癌细胞迁移、侵袭的作用及相关分子机制。材料和方法:1.材料82例人甲状腺乳头状癌组织为吉林大学中日联谊医院2014年12月至2016年5月手术切除标本;B-CPAP购于上海中乔新舟生物科技公司。2.方法(1)免疫组化检测人甲状腺乳头状癌组织标本中CXCR4的表达。(2)体外培养人甲状腺乳头状癌细胞株(B-CPAP)。设计并合成CXCR4基因CDS区,构建CXCR4过表达的质粒。B-CPAP细胞转染CXCR4过表达质粒或对应的vector(空载质粒),瞬转获得B-CPAP-vector和B-CPAP-CXCR4细胞。转染48小时后,免疫印迹和荧光定量实验检测各组细胞中CXCR4的表达水平。(3)在B-CPAP细胞中转染CXCR4过表达质粒24小时后,用100ng/ml的SDF-1处理48小时。然后通过划痕实验检测各组细胞迁移能力;通过Transwel l实验检测各组细胞侵袭能力。(4)在B-CPAP细胞中转染CXCR4过表达质粒24小时后,用100ng/ml的SDF-1处理48小时。然后免疫印迹检测E-cadherin(上皮-钙黏素),N-cadhe rin(神经-钙黏素),Vimentin(波形蛋白),NF-κB,磷酸化的IκB(P-IκB)以及细胞核内NF-κB的表达水平。免疫荧光实验检测E-cadherin的表达情况。(5)在B-CPAP细胞中转染CXCR4过表达质粒24小时后,用5μmol NF-κB抑制剂BAY11-7028(BAY)预处理1小时,然后加入100ng/ml的SDF-1处理48小时。免疫印迹实验检测NF-κB、P-IκB以及细胞核内NF-κB的水平;划痕实验检测各组细胞迁移能力;Transwell实验检测各组细胞侵袭能力。结果:(1)免疫组化检测结果显示CXCR4在甲状腺乳头状癌组织中高表达(2)免疫印迹和荧光定量检测结果显示转染CXCR4过表达质粒后,B-CPA P-CXCR4细胞中CXCR4表达明显增加。(3)在B-CPAP细胞中转染CXCR4过表达质粒后,用SDF-1处理,细胞的迁移性和侵袭性显著增强。(4)SDF-1/CXCR4明显减少了E-cadherin蛋白表达量,增加了N-cadhe rin蛋白、Vimentin蛋白的表达量。免疫荧光检测E-cadherin蛋白发现,SDF-1/CXCR4系统可以明显抑制E-cadherin的表达量。表明SDF-1/CXCR4系统促进B-CPAP的EMT过程。(5)SDF-1/CXCR4系统可以显著性提高细胞核中NF-κB的表达量,降低细胞质中NF-κB的表达量,降低细胞中P-IκB的表达量,激活NF-κB信号通路。(6)免疫印迹实验表明NF-κB抑制剂BAY可以提高E-cadherin表达量,降低N-cadherin、Vimentin表达量,抑制了SDF-1/CXCR4系统对B-CPAP的EM T过程的促进作用;划痕实验表明NF-κB抑制剂BAY可以减弱SDF-1/CXCR4系统对B-CPAP迁移能力的促进作用;Transwell实验表明NF-κB抑制剂BAY可以减弱SDF-1/CXCR4系统对B-CPAP侵袭能力的促进作用。结论:(1)CXCR4受体反应系统可加强NF-κB通路的信号传导,促进甲状腺乳头状癌细胞的EMT过程,加强乳头状癌细胞的迁移侵袭。(2)NF-κB通路的抑制剂BAY可削弱CXCR4受体反应系统对甲状腺乳头状癌细胞EMT过程的促进作用,减轻CXCR4受体反应系统对甲状腺乳头状癌细胞的促迁移侵袭作用。NF-κB通路可望成为抑制甲状腺乳头状癌细胞迁移侵袭的潜在治疗靶点。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R736.1

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