骨唾液酸蛋白通过整合素αvβ3调控乳腺癌细胞转移机制的初步研究

发布时间：2019-05-23 23:21

【摘要】：乳腺癌是女性中常见的一种恶性肿瘤,发病率很高,并且呈现出逐年上升的趋势。乳腺癌骨转移的发生率高且难以治疗,在尸检中发现有90%以上的乳腺癌死亡患者有骨转移。以手术为主的乳腺癌综合治疗方法虽然取得较好的疗效,但仍存在一定的局限性,转移仍是引起肿瘤术后复发的主要因素。因此深入研究乳腺癌骨转移的分子机制,寻找特异性靶向治疗位点具有重要意义。整合素αvβ3是一种能特异性识别RGD序列的膜受体蛋白,可特异性的与含RGD(Arg-Gly-Asp)基序的蛋白质结合,在肿瘤细胞的粘附、迁移、浸润及肿瘤血管新生中起重要作用。骨唾液酸蛋白(bone sialoprotein,BSP)是存在于细胞外基质中的一种高度磷酸化和糖基化的非胶原蛋白。BSP在亲骨转移乳腺癌、肺癌、前列腺癌细胞中异常表达,多项研究提示BSP在肿瘤靶向骨转移的进程中发挥重要作用。目前研究认为BSP通过与细胞膜αvβ3整合素受体结合可引发一系列细胞生物学效应。本课题组前期研究发现BSP通过整合素αvβ3调控亲骨转移乳腺癌细胞MDA-MB-231BO(231BO)的ILK信号通路,并影响细胞增殖;我们近期的实验结果还表明231BO细胞中BSP和αvβ3整合素表达水平之间存在一定的相关性,即BSP基因沉默的乳腺癌细胞(231BO-BSP27)中αvβ3整合素表达水平下降。但BSP和αvβ3整合素在临床乳腺癌组织中表达的相关性以及BSP是否依赖αvβ3整合素调控乳腺癌细胞转移并不清楚。本研究采用免疫组织化学方法分析BSP和αvβ3整合素在乳腺浸润性导管癌组织标本中表达的相关性;构建β3慢病毒表达载体,转染231BO-BSP27乳腺癌细胞,通过免疫细胞化学、流式细胞技术和Western Blot检测β3蛋白表达水平,筛选获得BSP-/β3+乳腺癌细胞株(231BO-BSP27/β3)。应用CCK8法、划痕实验、Transwell法体外检测细胞的增殖、迁移、侵袭能力,探究在BSP基因沉默的乳腺癌细胞中,回复表达β3整合素对乳腺癌细胞转移生物学性质的影响,以期初步揭示BSP通过调控β3整合素表达影响乳腺癌骨转移的分子机制。本实验的主要研究结果如下:1.通过61例乳腺浸润性导管癌组织的免疫组化检测,结果提示BSP和整合素αv、β3在癌组织中的表达呈正相关。2.采用β3慢病毒载体转染231BO-BSP27,经新霉素筛选,获得稳定表达β3整合素的231BO-BSP27/β3细胞系。3.与231BO-BSP27细胞相比,流式细胞检测结果表明231BO-BSP27/β3细胞中β3表达量增高(89.705±0.456,P0.05);Western Blot检测也证实231BO-BSP27/β3细胞的β3蛋白表达量(0.428±0.013)高于231BO-BSP27细胞(0.237±0.012)。免疫细胞化学检测231BO-BSP27/β3细胞显示β3呈强阳性表达,表达部位主要在胞膜和胞浆。4.体外细胞生物学实验结果提示231BO-BSP27/β3细胞生长速率、细胞迁移和侵袭能力均高于231BO-BSP27组细胞。
[Abstract]:Breast cancer is a common malignant tumor in women, the incidence is very high, and showing an upward trend year by year. The incidence of bone metastasis in breast cancer is high and difficult to treat. More than 90% of breast cancer patients died of bone metastasis during autopsies. Although the comprehensive treatment of breast cancer based on operation has achieved good curative effect, there are still some limitations, and metastasis is still the main factor leading to postoperative recurrence of breast cancer. Therefore, it is of great significance to study the molecular mechanism of bone metastasis of breast cancer and to find specific targeted therapeutic sites. Integrin 伪 v 尾 3 is a membrane receptor protein which can specifically recognize RGD sequence. It can specifically bind to proteins containing RGD (Arg-Gly-Asp) motif and play an important role in tumor cell adhesion, migration, invasion and tumor angiogenesis. Bone sialic acid protein (bone sialoprotein,BSP) is a highly phosphorylation and glycosylated non-collagen protein in extracellular matrix. BSP is abnormally expressed in breast cancer, lung cancer and prostate cancer cells. Many studies suggest that BSP plays an important role in the process of tumor targeted bone metastasis. At present, it is suggested that BSP can induce a series of cell biological effects by binding to 伪 v 尾 3 integrin receptor in cell membrane. Our previous study found that BSP regulated the ILK signaling pathway of osteophilic breast cancer cell line MDA-MB-231BO (231BO) through integrin 伪 v 尾 3, and affected the proliferation of breast cancer cells. Our recent results also showed that there was a certain correlation between the expression of BSP and 伪 v 尾 3 integrin in 231BO cells, that is, the expression of 伪 v 尾 3 integrin decreased in BSP gene silenced breast cancer cells (231BO-BSP27). However, the correlation between the expression of BSP and 伪 v 尾 3 integrin in clinical breast cancer and whether BSP depends on 伪 v 尾 3 integrin to regulate the metastasis of breast cancer cells is not clear. In this study, the correlation between the expression of BSP and 伪 v 尾 3 integrin in invasive ductal carcinoma of breast was analyzed by immunohistochemistry. The expression vector of 尾 3 lentivirus was constructed and transfected into 231BO-BSP27 breast cancer cells. BSP-/ 尾 3 breast cancer cell line (231BO-BSP27/ 尾 3) was screened by immunocytochemistry, flow cytometry and Western Blot. CCK8 assay, scratch test and Transwell assay were used to detect the proliferation, migration and invasion of breast cancer cells in vitro, and to explore the effect of 尾 3 integrin expression on the biological properties of breast cancer cell metastasis in breast cancer cells silenced by BSP gene. In order to reveal the molecular mechanism of BSP affecting bone metastasis of breast cancer by regulating the expression of 尾 3 integrin. The main results of this experiment are as follows: 1. The results showed that the expression of BSP and integrin 伪 v, 尾 3 in 61 cases of invasive ductal carcinoma of breast was positively correlated with the expression of integrin 伪 v, 尾 3 in breast invasive ductal carcinoma. 2. 231BO-BSP27/ 尾 3 cell line stably expressing 尾 3 integrin was obtained by transfection of 231BO / BSP27 with 尾 3 lentivirus vector. Compared with 231BO-BSP27 cells, flow cytometry showed that the expression of 尾 3 in 231BO-BSP27/ 尾 3 cells was higher than that in 231BO-BSP27/ 尾 3 cells (89.705 卤0.456, P 0.05). Western Blot also confirmed that the expression of 尾 3 protein in 231BO-BSP27/ 尾 3 cells (0.428 卤0.013) was higher than that in 231BO-BSP27 cells (0.237 卤0.012). The expression of 尾 3 in 231BO-BSP27/ 尾 3 cells was strongly positive by immunocytochemistry, and the expression site was mainly in the membrane and cytoplasm. 4. The results of cell biology in vitro suggested that the growth rate, cell migration and invasiveness of 231BO-BSP27/ 尾 3 cells were higher than those of 231BO-BSP27 group.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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