【作者】 王萍；

【导师】 林洁；

【作者基本信息】 南方医科大学 ， 临床病理学（专业学位）， 2021， 硕士

【摘要】 研究背景:食管鳞癌是困扰人类健康的常见癌症之一。我国的食管鳞癌发病率和死亡率均排名世界首位。其中,临床上食管鳞癌病人预后不良的重要原因是淋巴结转移和化疗耐药。因此,寻找食管鳞癌发生发展的潜在分子靶点,对于食管鳞癌的早期确诊、及时治疗以及改善预后,进而降低食管鳞癌病人的病死率,提高生存率具有重要的科学、临床意义。课题组前期通过串联质谱标记技术筛选来源于食管上皮的癌组织与正常组织的差异蛋白,获得了在癌组织中高表达蛋白——潜在转化生长因子β结合蛋白1(Latent transforming growth factor beta binding protein 1,LTBP1),并已用western blot方法及GEPIA数据库进行验证[1]。LTBP1在食管癌中的作用未曾有过报道,因此本课题旨在研究LTBP1在食管鳞癌中的作用,并阐明其潜在的机制。研究方法及内容:1.利用免疫组织化学技术检测LTBP1在152例食管鳞癌组织与正常配对组织中的表达水平,分析LTBP1表达与临床病理各项指标间的关系。2.构建LTBP1表达抑制的食管鳞癌细胞株,用CCK-8实验、transwell迁移/侵袭实验,裸鼠尾静脉注射实验检测LTBP1对体内、外食管鳞癌细胞增殖、迁移侵袭和转移能力的影响。3.Western blot实验检测上皮间充质转化(EMT)标记物的表达。GEPIA数据库分析LTBP1与FN1的关系及WB、ELISA实验验证不同细胞中两者的表达。建立食管鳞癌细胞与成纤维细胞共培养体系。WB、免疫荧光实验检测肿瘤相关成纤维细胞(CAFs)标记物的表达,CCK8检测共培养后细胞增殖活性。免疫组化方法检测食管鳞癌组织中LTBP1和FN1的表达和定位。4.Western blot实验检测食管鳞癌耐药细胞株中LTBP1的表达水平。通过流式细胞术、CCK-8药敏实验检测LTBP1对食管鳞癌耐药细胞顺铂敏感性的影响。采用细胞黏附实验检测粘附性的变化。采用免疫组化方法和影像学比较化疗后不同反应评级的食管鳞癌病人其化疗前LTBP1表达情况。研究结果:1.LTBP1在食管鳞癌组织中高表达,与淋巴结有转移显著相关,可作为评价患者肿瘤进展程度的一个因素。2.LTBP1具有促进体内、外食管鳞癌细胞迁移、侵袭和转移的功能;而与增殖无关。3.LTBP1参与食管鳞癌EMT及诱导CAFs转化,肿瘤实质LTBP1的表达与间质FN1的表达呈正相关。4.LTBP1表达升高,增加细胞与基质的粘附性,诱导食管鳞癌顺铂耐药;食管鳞癌活检组织中LTBP1表达高,新辅助化疗效果差。结论:LTBP1促进食管鳞癌进展及顺铂耐药。更多还原

【Abstract】 BackgroundEsophageal squamous cell carcinoma(ESCC)is one of the most prevalent cancers plaguing human health.The incidence and mortality rate of ESCC in China ranks first in the world.Among them,the most important reasons for poor prognosis in patients with ESCC are lymph node metastasis and chemotherapy resistance.Therefore,the search for molecular targets for the occurrence and development of ESCC is of great scientific and clinical significance for the early diagnosis,timely treatment and improvement of prognosis of ESCC,so as to reduce the mortality and improve the survival rate of ESCC patients.The tandem mass tag(TMT)-based quantitative proteomic approach was applied to screen the differentially expressed proteins(DEPs)between 3 cases of ESCC tumor samples and paired normal tissues in our research group.We found protein—Latent transforming growth factor beta binding protein 1(LTBP1)that highly expressed in ESCC tissues,which was verified by Western blot method and GEPIA database[1].The role of LTBP1 in ESCC has not been reported,so this project aims to study the role of LTBP1 in ESCC and to clarify its underlying mechanism.Methods1.The expression level of LTBP1 was detected in 152 cases of ESCC tissues and paired normal tissues by IHC.We further analyze the relationship between LTBP1 expression and clinicopathological features of patients,such as age,gender,lymph node metastasis.2.Construction of esophageal cancer cell line inhibition of LTBP1.The proliferation ability of ESCC cells after LTBP1 inhibition was tested using the CCK-8 assay,in vivo.The metastatic ability of ESCC cells after LTBP1 inhibition was tested using transwell migration and invasion assay and tail vein metastasis assay in vivo and vitro.3.Western blot assay to detect the expression of epithelial mesenchymal transformation(EMT)markers.The relationship between LTBP1 and FN1 was analyzed by GEPIA database,WB and ELISA assayed to verify the expression of both in different cells.Indirect co-culture of ESCC cells and fibroblasts was used.The expression of markers in cancer-associated fibroblasts(CAFs)was detected by Western blot and immunofluorescence assays.CCK8 was used to detect cell proliferation activity after co-culture.The expression and localization of LTBP1 and FN1 in ESCC tissues were detected by immunohistochemistry.4.The expression level of LTBP1 protein in chemo-resistant ESCC cells was detected by western blot.The effect of LTBP1 on cisplatin sensitivity of ESCC cells was detected by flow cytometry and CCK-8 drug sensitivity assay.Cell adhesion test was used to detect the change of adhesion.LTBP1 expression in ESCC tissues with different regression grading after chemotherapy was compared by immunohistochemistry and CT image.Results1.LTBP1 highly expressed in ESCC.The expression level of LTBP1 was significantly related to lymph node metastasis.So it can be used as one of the factors to evaluate the tumor progression.2.LTBP1 promoted the migration,invasion and metastasis,of ESCC cells in vitro and vivo.It has nothing to do with proliferation.3.LTBP1 was involved in EMT and induced the transformation of CAFs in ESCC,and the expression of LTBP1 in tumor parenchyma was positively correlated with the expression of mesenchymal FN1.4.LTBP1 expression increased the cell adhesion and induced cisplatin resistance of ESCC.LTBP1 expression was high in the biopsy tissues of ESCC,then the treatment effect of neoadjuvant chemotherapy was poor.ConclusionsLTBP1 promotes tumor progression and cisplatin resistance in esophageal squamous cell carcinoma.更多还原