肿瘤细胞与血小板相互作用促进MMP-9分泌、Tenascin-C形成的分子机制
发布时间：2018-08-07 10:04
【摘要】：一、研究背景肿瘤细胞与微环境之间的相互作用越来越被认为是一个影响肿瘤恶性进展的重要因素,肿瘤细胞可以分泌一些生长因子和细胞因子激活细胞外基质,反过来,微环境提供的信号又促进肿瘤细胞侵袭和转移。目前研究认为,当肿瘤细胞存在于远处器官微血管系统时,它与血小板的聚集和微血栓的形成是密切相关的。一旦血小板被激活,血小板将释放特定生长因子,促进肿瘤转移形成。事实上,在许多试验中已经证实,在肿瘤发生血行转移过程中,血小板的作用是必不可少的。然而,肿瘤细胞与血小板相互作用的精确分子机制仍然是不清楚的。TN-C是一个复杂的多功能蛋白,它由一个N端tenascin适配区域,接着是14.5个类似表皮生长因子(EGF)重复结构域,可变的类似纤连蛋白III型(FN III)重复序列和C端类似纤连蛋白原的结构域组成。TN-C可以直接通过细胞表面受体或间接的结合其他基质蛋白影响肿瘤细胞行为,这将诱导血管生成和促进肿瘤细胞迁移。纤维性TN-C(fTN-C)主要表达于肿瘤细胞外基质(ECM),fTN-C基质的形成需要基质金属蛋白酶(MMPs)的参与,在促进肿瘤转移中可能发挥作用。MMPs是一群肽链内切酶,它能降解细胞外基质,调节ECM重塑。在以前的研究中已经证实,MMP-2和MMP-9的过表达可以极大的增强肿瘤的侵袭转移潜能。大多数研究表明,MMP-9和TN-C蛋白的过表达与肿瘤的进展和不良的预后是相关的,但MMP-9和TN-C在胰腺癌中的作用仍不清楚。血小板能促进肿瘤转移的形成,但是否是通过促进肿瘤细胞分泌MMP-9和TN-C来实现的,目前也是不清楚的。CD44是一种表达癌症表型的多功能细胞受体,CD44是一个单链,单程的跨膜糖蛋白,广泛表达于生理和病理系统。同时,CD44参与细胞粘附、肿瘤侵袭和转移。在以前的研究中,CD44已经被暗示能够以依赖透明质酸或不依赖透明质酸的方式调控基质金属蛋白酶的表达,主要是MMP-2和MMP-9。此外,在最近已有报道证实CD44在结肠癌细胞拥有selectin结合力,CD44是P-selectin配体。P-selectin是一个重要的粘附受体,表达于激活的内皮细胞上。与此同时,激活的血小板也表达P-selectin。所以我们可以假设肿瘤细胞与血小板之间的相互作用是通过cd44和p-selectin的粘附来完成的。因此,在这项研究中,我们调查tn-c和mmp-9在胰腺癌组织中的表达情况,分析mmp-9和tn-c表达和临床病理参数的相关性。与此同时,我们在肿瘤细胞与血小板共培养体系中研究人类胰腺癌细胞的侵袭能力。我们利用这个共培养系统,探测mmp-9和tn-c的表达水平。此外,我们进一步研究肿瘤细胞与血小板相互作用是否是通过cd44和p-selectin的结合来实现的。二、研究方法1.共收集了103例在第三军医大学西南医院肝胆外科研究所接受手术治疗的患者,从2007年1月至2010年6月的胰腺癌患者。所有患者接受根治性胰十二直肠切除术或保留幽门的胰十二直肠切除术,并做淋巴结清扫。没有一个病人接受了新辅助疗法和辅助放化疗。石蜡包埋切片样本用来做免疫组织化学分析。所有患者在术后3个月行超声波、放射x线及计算机断层扫描检查,探测到有新病变被认为是复发,平均随访期为13个月(范围3-49个月)。我们通过免疫组织化学的方法调查tn-c和mmp-9在胰腺癌组织的表达水平,分析单表达和共表达这两个分子和临床病理参数的相关性,并分析其与胰腺癌病人生存预后的相关性。2.人类胰腺癌细胞系aspc1和bxpc3。这些细胞系是购自美国atcc公司。这些细胞在含10%胎牛血清的rpmi1640或dmem培养基中培养,他们被保持在37°c的含有5%的二氧化碳环境中。与血小板共培养体系中,细胞被播种在六孔板中,孵育过夜,立即更换新鲜的无血清培养基。加入100μl1×108/毫升的纯血小板,培养至少48小时。为了探测血小板对肿瘤细胞侵袭的影响,我们在共培养系统进行transwell侵袭实验。条件培养基被收集来做明胶酶谱分析(gelatinzymography),提取培养细胞的蛋白质用于免疫印迹实验(westernblotting)。3.在细胞培养和共培养系统,我们使用特异性抗体封闭血小板p-selectin,并使用小干扰rna(sirna)敲掉bxpc3细胞的cd44,然后通过明胶酶谱分析(gelatinzymography)和免疫印迹实验(westernblotting)的方法探测mmp-9的表达和tn-c的表达。三、研究结果1.我们在103例胰腺癌组织中检测了tn-c和mmp-9的表达水平,分析了单表达和共表达这两个分子和胰腺癌患者的临床病理参数及生存预后的相关性。我们发现,在胰腺癌组织中,mmp-9和tn-c的表达水平是明显增加的。mmp-9和tn-c的共表达也是存在的。临床统计分析表明,MMP-9,TN-C和纤维TN-C(fTN-C)的表达水平与血管侵犯、淋巴结转移、肝转移和TNM分期是相关的。与此同时,我们发现MMP-9和TN-C的共表达与胰腺腺癌转移是显著相关的。生存分析显示,MMP-9或TN-C的单表达明显降低胰腺癌患者的总生存率,共表达MMP-9和TN-C的胰腺癌患者具有最低的总生存率。2.为了确定血小板对肿瘤细胞的影响,我们在血小板和胰腺癌细胞共培养体系中检测了MMP-9和TN-C的表达水平。我们发现,与单独的AsPc1和BxPc3细胞培养相比,在血小板与肿瘤细胞共培养体系中,肿瘤细胞穿透基底膜的能力是明显增加的,MMP-9和TN-C蛋白表达水平是增加的,尤其是小分质量TN-C片段。同时,MMP-9的活性也是明显增强的,尤其是BxPc3细胞。3.为了探索肿瘤细胞与血小板相互作用的分子机制,我们在血小板与BxPc3共培养体系中用特异性抗体封闭血小板P-selectin,我们发现MMP-9活性降低,MMP-9和TN-C蛋白表达水平也降低。我们用小干扰RNA(siRNA)敲掉BxPc3细胞的CD44,然后与血小板进行共培养,我们发现MMP-9活性也是降低的,MMP-9和TN-C蛋白表达水平也明显降低。四、讨论1.MMP-9和TN-C的共表达可能促进肿瘤转移,从而影响胰腺癌的进展。并且这两个分子的共表达可能暗示了胰腺癌患者的预后较差。2.肿瘤细胞和血小板的相互作用会诱导MMP-9分泌和TN-C形成,并促进胰腺癌细胞侵袭和转移。3.肿瘤细胞和血小板相互作用可能是通过CD44和P-selectin的结合实现的,这将为临床治疗提供新的靶点。
[Abstract]:First, the interaction between tumor cells and microenvironment is becoming more and more considered to be an important factor affecting the malignant progression of cancer. Tumor cells can secrete some growth factors and cytokines to activate the extracellular matrix. In turn, the signal provided by microenvironment promotes invasion and metastasis of tumor cells. When the tumor cells exist in the distant organ microvascular system, it is closely related to the aggregation of platelets and the formation of microthrombus. Once platelets are activated, platelets release specific growth factors and promote the formation of tumor metastasis. In fact, it has been confirmed in many trials that platelets are used in the process of hematogenous metastasis of the tumor. It is essential. However, the exact molecular mechanism of the interaction between tumor cells and platelets is still unclear..TN-C is a complex multifunctional protein, which consists of a N terminal tenascin adaptation region, followed by 14.5 similar epidermal growth factor (EGF) duplication domains, variable similar fibronectin III type (FN III) repeat sequences The domain of the fibronectin similar to the C terminal,.TN-C, can directly affect the tumor cell behavior through the cell surface receptor or the indirect binding of other matrix proteins, which will induce angiogenesis and promote tumor cell migration. The fibrous TN-C (fTN-C) is mainly expressed in the extracellular matrix of the tumor (ECM). The formation of fTN-C matrix requires matrix gold The involvement of protease (MMPs) may play a role in promoting tumor metastasis..MMPs is a group of peptide endonucleases, which degrade the extracellular matrix and regulate ECM remodeling. In previous studies, the overexpression of MMP-2 and MMP-9 could greatly enhance the invasion and transfer potential of the tumor. Most studies have shown that MMP-9 and TN-C proteins are over. Expression is related to the progression of tumors and poor prognosis, but the role of MMP-9 and TN-C in pancreatic cancer is still unclear. Platelets can promote the formation of tumor metastasis, but whether it is achieved by promoting the secretion of MMP-9 and TN-C by tumor cells, it is not clear that.CD44 is a multifunctional cell receptor that expresses the phenotype of cancer, CD44 It is a single chain, one-way transmembrane glycoprotein that is widely expressed in physiological and pathological systems. At the same time, CD44 is involved in cell adhesion, tumor invasion and metastasis. In previous studies, CD44 has been suggested to be able to regulate the expression of matrix metalloproteinases in a manner dependent on hyaluronic acid or without hyaluronic acid, mainly MMP-2 and MMP-9., Recently, it has been reported that CD44 has selectin binding force in colon cancer cells, and CD44 is a P-selectin ligand.P-selectin, an important adhesion receptor, expressed on activated endothelial cells. At the same time, activated platelets also express P-selectin. so that we can hypothesized that the interaction between tumor cells and platelets is common. In this study, we investigated the expression of TN-C and MMP-9 in pancreatic cancer tissues, and analyzed the correlation between the expression of MMP-9 and TN-C and the clinicopathological parameters of MMP-9 and TN-C. At the same time, we studied the invasiveness of human pancreatic cancer cells in the tumor cells and the platelets co culture system. We used this co culture system to detect the expression level of MMP-9 and TN-C. In addition, we further studied whether the interaction of tumor cells and platelets was achieved through the combination of CD44 and P-selectin. Two, study method 1. collected 103 cases of surgical treatment at the Department of hepatobiliary surgery, Southwest Hospital, Third Military Medical University. Patients with pancreatic cancer from January 2007 to June 2010. All patients received radical pancreatic resection or pylorus retention of the pancreas for twelve rectal excision and lymph node dissection. None of the patients received neoadjuvant therapy and adjuvant chemoradiotherapy. Paraffin embedded sections were used for immunohistochemical analysis. All patients were used for immunohistochemical analysis. All patients were used for immunohistochemical analysis. Ultrasound, radiography and computed tomography were performed 3 months after the operation to detect the recurrence of new lesions. The average follow-up period was 13 months (range 3-49 months). We investigated the expression level of TN-C and MMP-9 in the pancreatic cancer tissue by immunohistochemical method, and analyzed the single expression and co expression of the two molecules and clinical manifestations. Correlation of pathological parameters and its correlation with survival prognosis of pancreatic cancer patients.2. human pancreatic cancer cell lines aspc1 and bxpc3. were purchased from American ATCC company. These cells were cultured in RPMI1640 or DMEM medium containing 10% fetal bovine serum, and they were kept in the 37 degree C containing 5% carbon dioxide environment. In the plate co culture system, the cells were seeded in the six pore plate, incubated for the night, and replaced the fresh serum-free medium immediately. 100 mu L1 x 108/ ml of pure platelets were added for at least 48 hours. In order to detect the impact of platelets on the invasion of the tumor cells, we carried out the Transwell invasion experiment in the co culture system. Gelatinase spectrum analysis (gelatinzymography) was used to extract the protein of culture cells for immunoblotting experiment (westernblotting).3. in cell culture and co culture system. We used specific antibodies to seal platelet P-selectin, and use small interference RNA (siRNA) to knock off CD44 of bxpc3 cells, and then analyze (gelatinzymogr) by gelatinase spectrum (gelatinzymogr). Aphy) and Western blot assay (westernblotting) method to detect the expression of MMP-9 and the expression of TN-C. Three. Results 1. we detected the expression of TN-C and MMP-9 in 103 cases of pancreatic cancer, and analyzed the correlation between the single expression and co expression of the two molecules and the clinicopathological parameters and survival prognosis of the patients with pancreatic cancer. In pancreatic cancer, the expression level of MMP-9 and TN-C is significantly increased by.Mmp-9 and TN-C. Clinical statistical analysis shows that the expression level of MMP-9, TN-C and fibrous TN-C (fTN-C) is associated with vascular invasion, lymph node metastasis, liver metastasis and TNM staging. At the same time, we found co expression of MMP-9 and TN-C. The survival analysis showed that the single expression of MMP-9 or TN-C significantly reduced the total survival rate of the patients with pancreatic cancer, and the total survival rate of the patients with MMP-9 and TN-C had the lowest total survival rate.2. to determine the effect of platelets on the tumor cells, and we examined the platelet and pancreatic cancer cell co culture system. The expression level of MMP-9 and TN-C was measured. We found that the ability of the tumor cells to penetrate the basement membrane was significantly increased in the co culture system of platelets and tumor cells compared with the individual AsPc1 and BxPc3 cell cultures, and the expression level of MMP-9 and TN-C protein was increased, especially the small mass TN-C fragment. At the same time, the activity of MMP-9 was also clear. Enhanced, especially BxPc3 cell.3., in order to explore the molecular mechanism of the interaction between tumor cells and platelets, we closed platelet P-selectin with specific antibodies in the platelets and BxPc3 co culture system. We found that MMP-9 activity decreased and the expression of MMP-9 and TN-C protein was flat and decreased. We knocked off BxPc3 with small interference RNA (siRNA). CD44, and then co culture with platelets, we found that MMP-9 activity was also reduced, and the expression level of MMP-9 and TN-C protein was also significantly reduced. Four. Co expression of 1.MMP-9 and TN-C may promote tumor metastasis and affect the progression of pancreatic cancer. Co expression of these two points may imply the prognosis of pancreatic cancer patients. The interaction of poor.2. tumor cells and platelets induces MMP-9 secretion and TN-C formation, and promotes the invasion and metastasis of pancreatic cancer cells and the interaction of.3. tumor cells and platelets, which may be achieved through the combination of CD44 and P-selectin, which will provide new targets for clinical treatment.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.9
本文编号：2169684
[Abstract]:First, the interaction between tumor cells and microenvironment is becoming more and more considered to be an important factor affecting the malignant progression of cancer. Tumor cells can secrete some growth factors and cytokines to activate the extracellular matrix. In turn, the signal provided by microenvironment promotes invasion and metastasis of tumor cells. When the tumor cells exist in the distant organ microvascular system, it is closely related to the aggregation of platelets and the formation of microthrombus. Once platelets are activated, platelets release specific growth factors and promote the formation of tumor metastasis. In fact, it has been confirmed in many trials that platelets are used in the process of hematogenous metastasis of the tumor. It is essential. However, the exact molecular mechanism of the interaction between tumor cells and platelets is still unclear..TN-C is a complex multifunctional protein, which consists of a N terminal tenascin adaptation region, followed by 14.5 similar epidermal growth factor (EGF) duplication domains, variable similar fibronectin III type (FN III) repeat sequences The domain of the fibronectin similar to the C terminal,.TN-C, can directly affect the tumor cell behavior through the cell surface receptor or the indirect binding of other matrix proteins, which will induce angiogenesis and promote tumor cell migration. The fibrous TN-C (fTN-C) is mainly expressed in the extracellular matrix of the tumor (ECM). The formation of fTN-C matrix requires matrix gold The involvement of protease (MMPs) may play a role in promoting tumor metastasis..MMPs is a group of peptide endonucleases, which degrade the extracellular matrix and regulate ECM remodeling. In previous studies, the overexpression of MMP-2 and MMP-9 could greatly enhance the invasion and transfer potential of the tumor. Most studies have shown that MMP-9 and TN-C proteins are over. Expression is related to the progression of tumors and poor prognosis, but the role of MMP-9 and TN-C in pancreatic cancer is still unclear. Platelets can promote the formation of tumor metastasis, but whether it is achieved by promoting the secretion of MMP-9 and TN-C by tumor cells, it is not clear that.CD44 is a multifunctional cell receptor that expresses the phenotype of cancer, CD44 It is a single chain, one-way transmembrane glycoprotein that is widely expressed in physiological and pathological systems. At the same time, CD44 is involved in cell adhesion, tumor invasion and metastasis. In previous studies, CD44 has been suggested to be able to regulate the expression of matrix metalloproteinases in a manner dependent on hyaluronic acid or without hyaluronic acid, mainly MMP-2 and MMP-9., Recently, it has been reported that CD44 has selectin binding force in colon cancer cells, and CD44 is a P-selectin ligand.P-selectin, an important adhesion receptor, expressed on activated endothelial cells. At the same time, activated platelets also express P-selectin. so that we can hypothesized that the interaction between tumor cells and platelets is common. In this study, we investigated the expression of TN-C and MMP-9 in pancreatic cancer tissues, and analyzed the correlation between the expression of MMP-9 and TN-C and the clinicopathological parameters of MMP-9 and TN-C. At the same time, we studied the invasiveness of human pancreatic cancer cells in the tumor cells and the platelets co culture system. We used this co culture system to detect the expression level of MMP-9 and TN-C. In addition, we further studied whether the interaction of tumor cells and platelets was achieved through the combination of CD44 and P-selectin. Two, study method 1. collected 103 cases of surgical treatment at the Department of hepatobiliary surgery, Southwest Hospital, Third Military Medical University. Patients with pancreatic cancer from January 2007 to June 2010. All patients received radical pancreatic resection or pylorus retention of the pancreas for twelve rectal excision and lymph node dissection. None of the patients received neoadjuvant therapy and adjuvant chemoradiotherapy. Paraffin embedded sections were used for immunohistochemical analysis. All patients were used for immunohistochemical analysis. All patients were used for immunohistochemical analysis. Ultrasound, radiography and computed tomography were performed 3 months after the operation to detect the recurrence of new lesions. The average follow-up period was 13 months (range 3-49 months). We investigated the expression level of TN-C and MMP-9 in the pancreatic cancer tissue by immunohistochemical method, and analyzed the single expression and co expression of the two molecules and clinical manifestations. Correlation of pathological parameters and its correlation with survival prognosis of pancreatic cancer patients.2. human pancreatic cancer cell lines aspc1 and bxpc3. were purchased from American ATCC company. These cells were cultured in RPMI1640 or DMEM medium containing 10% fetal bovine serum, and they were kept in the 37 degree C containing 5% carbon dioxide environment. In the plate co culture system, the cells were seeded in the six pore plate, incubated for the night, and replaced the fresh serum-free medium immediately. 100 mu L1 x 108/ ml of pure platelets were added for at least 48 hours. In order to detect the impact of platelets on the invasion of the tumor cells, we carried out the Transwell invasion experiment in the co culture system. Gelatinase spectrum analysis (gelatinzymography) was used to extract the protein of culture cells for immunoblotting experiment (westernblotting).3. in cell culture and co culture system. We used specific antibodies to seal platelet P-selectin, and use small interference RNA (siRNA) to knock off CD44 of bxpc3 cells, and then analyze (gelatinzymogr) by gelatinase spectrum (gelatinzymogr). Aphy) and Western blot assay (westernblotting) method to detect the expression of MMP-9 and the expression of TN-C. Three. Results 1. we detected the expression of TN-C and MMP-9 in 103 cases of pancreatic cancer, and analyzed the correlation between the single expression and co expression of the two molecules and the clinicopathological parameters and survival prognosis of the patients with pancreatic cancer. In pancreatic cancer, the expression level of MMP-9 and TN-C is significantly increased by.Mmp-9 and TN-C. Clinical statistical analysis shows that the expression level of MMP-9, TN-C and fibrous TN-C (fTN-C) is associated with vascular invasion, lymph node metastasis, liver metastasis and TNM staging. At the same time, we found co expression of MMP-9 and TN-C. The survival analysis showed that the single expression of MMP-9 or TN-C significantly reduced the total survival rate of the patients with pancreatic cancer, and the total survival rate of the patients with MMP-9 and TN-C had the lowest total survival rate.2. to determine the effect of platelets on the tumor cells, and we examined the platelet and pancreatic cancer cell co culture system. The expression level of MMP-9 and TN-C was measured. We found that the ability of the tumor cells to penetrate the basement membrane was significantly increased in the co culture system of platelets and tumor cells compared with the individual AsPc1 and BxPc3 cell cultures, and the expression level of MMP-9 and TN-C protein was increased, especially the small mass TN-C fragment. At the same time, the activity of MMP-9 was also clear. Enhanced, especially BxPc3 cell.3., in order to explore the molecular mechanism of the interaction between tumor cells and platelets, we closed platelet P-selectin with specific antibodies in the platelets and BxPc3 co culture system. We found that MMP-9 activity decreased and the expression of MMP-9 and TN-C protein was flat and decreased. We knocked off BxPc3 with small interference RNA (siRNA). CD44, and then co culture with platelets, we found that MMP-9 activity was also reduced, and the expression level of MMP-9 and TN-C protein was also significantly reduced. Four. Co expression of 1.MMP-9 and TN-C may promote tumor metastasis and affect the progression of pancreatic cancer. Co expression of these two points may imply the prognosis of pancreatic cancer patients. The interaction of poor.2. tumor cells and platelets induces MMP-9 secretion and TN-C formation, and promotes the invasion and metastasis of pancreatic cancer cells and the interaction of.3. tumor cells and platelets, which may be achieved through the combination of CD44 and P-selectin, which will provide new targets for clinical treatment.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.9
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