【摘要】：一、研究背景腫瘤細(xì)胞與微環(huán)境之間的相互作用越來(lái)越被認(rèn)為是一個(gè)影響腫瘤惡性進(jìn)展的重要因素,腫瘤細(xì)胞可以分泌一些生長(zhǎng)因子和細(xì)胞因子激活細(xì)胞外基質(zhì),反過(guò)來(lái),微環(huán)境提供的信號(hào)又促進(jìn)腫瘤細(xì)胞侵襲和轉(zhuǎn)移。目前研究認(rèn)為,當(dāng)腫瘤細(xì)胞存在于遠(yuǎn)處器官微血管系統(tǒng)時(shí),它與血小板的聚集和微血栓的形成是密切相關(guān)的。一旦血小板被激活,血小板將釋放特定生長(zhǎng)因子,促進(jìn)腫瘤轉(zhuǎn)移形成。事實(shí)上,在許多試驗(yàn)中已經(jīng)證實(shí),在腫瘤發(fā)生血行轉(zhuǎn)移過(guò)程中,血小板的作用是必不可少的。然而,腫瘤細(xì)胞與血小板相互作用的精確分子機(jī)制仍然是不清楚的。TN-C是一個(gè)復(fù)雜的多功能蛋白,它由一個(gè)N端tenascin適配區(qū)域,接著是14.5個(gè)類(lèi)似表皮生長(zhǎng)因子(EGF)重復(fù)結(jié)構(gòu)域,可變的類(lèi)似纖連蛋白III型(FN III)重復(fù)序列和C端類(lèi)似纖連蛋白原的結(jié)構(gòu)域組成。TN-C可以直接通過(guò)細(xì)胞表面受體或間接的結(jié)合其他基質(zhì)蛋白影響腫瘤細(xì)胞行為,這將誘導(dǎo)血管生成和促進(jìn)腫瘤細(xì)胞遷移。纖維性TN-C(fTN-C)主要表達(dá)于腫瘤細(xì)胞外基質(zhì)(ECM),fTN-C基質(zhì)的形成需要基質(zhì)金屬蛋白酶(MMPs)的參與,在促進(jìn)腫瘤轉(zhuǎn)移中可能發(fā)揮作用。MMPs是一群肽鏈內(nèi)切酶,它能降解細(xì)胞外基質(zhì),調(diào)節(jié)ECM重塑。在以前的研究中已經(jīng)證實(shí),MMP-2和MMP-9的過(guò)表達(dá)可以極大的增強(qiáng)腫瘤的侵襲轉(zhuǎn)移潛能。大多數(shù)研究表明,MMP-9和TN-C蛋白的過(guò)表達(dá)與腫瘤的進(jìn)展和不良的預(yù)后是相關(guān)的,但MMP-9和TN-C在胰腺癌中的作用仍不清楚。血小板能促進(jìn)腫瘤轉(zhuǎn)移的形成,但是否是通過(guò)促進(jìn)腫瘤細(xì)胞分泌MMP-9和TN-C來(lái)實(shí)現(xiàn)的,目前也是不清楚的。CD44是一種表達(dá)癌癥表型的多功能細(xì)胞受體,CD44是一個(gè)單鏈,單程的跨膜糖蛋白,廣泛表達(dá)于生理和病理系統(tǒng)。同時(shí),CD44參與細(xì)胞粘附、腫瘤侵襲和轉(zhuǎn)移。在以前的研究中,CD44已經(jīng)被暗示能夠以依賴(lài)透明質(zhì)酸或不依賴(lài)透明質(zhì)酸的方式調(diào)控基質(zhì)金屬蛋白酶的表達(dá),主要是MMP-2和MMP-9。此外,在最近已有報(bào)道證實(shí)CD44在結(jié)腸癌細(xì)胞擁有selectin結(jié)合力,CD44是P-selectin配體。P-selectin是一個(gè)重要的粘附受體,表達(dá)于激活的內(nèi)皮細(xì)胞上。與此同時(shí),激活的血小板也表達(dá)P-selectin。所以我們可以假設(shè)腫瘤細(xì)胞與血小板之間的相互作用是通過(guò)cd44和p-selectin的粘附來(lái)完成的。因此,在這項(xiàng)研究中,我們調(diào)查tn-c和mmp-9在胰腺癌組織中的表達(dá)情況,分析mmp-9和tn-c表達(dá)和臨床病理參數(shù)的相關(guān)性。與此同時(shí),我們?cè)谀[瘤細(xì)胞與血小板共培養(yǎng)體系中研究人類(lèi)胰腺癌細(xì)胞的侵襲能力。我們利用這個(gè)共培養(yǎng)系統(tǒng),探測(cè)mmp-9和tn-c的表達(dá)水平。此外,我們進(jìn)一步研究腫瘤細(xì)胞與血小板相互作用是否是通過(guò)cd44和p-selectin的結(jié)合來(lái)實(shí)現(xiàn)的。二、研究方法1.共收集了103例在第三軍醫(yī)大學(xué)西南醫(yī)院肝膽外科研究所接受手術(shù)治療的患者,從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腸切除術(shù)或保留幽門(mén)的胰十二直腸切除術(shù),并做淋巴結(jié)清掃。沒(méi)有一個(gè)病人接受了新輔助療法和輔助放化療。石蠟包埋切片樣本用來(lái)做免疫組織化學(xué)分析。所有患者在術(shù)后3個(gè)月行超聲波、放射x線及計(jì)算機(jī)斷層掃描檢查,探測(cè)到有新病變被認(rèn)為是復(fù)發(fā),平均隨訪期為13個(gè)月(范圍3-49個(gè)月)。我們通過(guò)免疫組織化學(xué)的方法調(diào)查tn-c和mmp-9在胰腺癌組織的表達(dá)水平,分析單表達(dá)和共表達(dá)這兩個(gè)分子和臨床病理參數(shù)的相關(guān)性,并分析其與胰腺癌病人生存預(yù)后的相關(guān)性。2.人類(lèi)胰腺癌細(xì)胞系aspc1和bxpc3。這些細(xì)胞系是購(gòu)自美國(guó)atcc公司。這些細(xì)胞在含10%胎牛血清的rpmi1640或dmem培養(yǎng)基中培養(yǎng),他們被保持在37°c的含有5%的二氧化碳環(huán)境中。與血小板共培養(yǎng)體系中,細(xì)胞被播種在六孔板中,孵育過(guò)夜,立即更換新鮮的無(wú)血清培養(yǎng)基。加入100μl1×108/毫升的純血小板,培養(yǎng)至少48小時(shí)。為了探測(cè)血小板對(duì)腫瘤細(xì)胞侵襲的影響,我們?cè)诠才囵B(yǎng)系統(tǒng)進(jìn)行transwell侵襲實(shí)驗(yàn)。條件培養(yǎng)基被收集來(lái)做明膠酶譜分析(gelatinzymography),提取培養(yǎng)細(xì)胞的蛋白質(zhì)用于免疫印跡實(shí)驗(yàn)(westernblotting)。3.在細(xì)胞培養(yǎng)和共培養(yǎng)系統(tǒng),我們使用特異性抗體封閉血小板p-selectin,并使用小干擾rna(sirna)敲掉bxpc3細(xì)胞的cd44,然后通過(guò)明膠酶譜分析(gelatinzymography)和免疫印跡實(shí)驗(yàn)(westernblotting)的方法探測(cè)mmp-9的表達(dá)和tn-c的表達(dá)。三、研究結(jié)果1.我們?cè)?03例胰腺癌組織中檢測(cè)了tn-c和mmp-9的表達(dá)水平,分析了單表達(dá)和共表達(dá)這兩個(gè)分子和胰腺癌患者的臨床病理參數(shù)及生存預(yù)后的相關(guān)性。我們發(fā)現(xiàn),在胰腺癌組織中,mmp-9和tn-c的表達(dá)水平是明顯增加的。mmp-9和tn-c的共表達(dá)也是存在的。臨床統(tǒng)計(jì)分析表明,MMP-9,TN-C和纖維TN-C(fTN-C)的表達(dá)水平與血管侵犯、淋巴結(jié)轉(zhuǎn)移、肝轉(zhuǎn)移和TNM分期是相關(guān)的。與此同時(shí),我們發(fā)現(xiàn)MMP-9和TN-C的共表達(dá)與胰腺腺癌轉(zhuǎn)移是顯著相關(guān)的。生存分析顯示,MMP-9或TN-C的單表達(dá)明顯降低胰腺癌患者的總生存率,共表達(dá)MMP-9和TN-C的胰腺癌患者具有最低的總生存率。2.為了確定血小板對(duì)腫瘤細(xì)胞的影響,我們?cè)谘“搴鸵认侔┘?xì)胞共培養(yǎng)體系中檢測(cè)了MMP-9和TN-C的表達(dá)水平。我們發(fā)現(xiàn),與單獨(dú)的AsPc1和BxPc3細(xì)胞培養(yǎng)相比,在血小板與腫瘤細(xì)胞共培養(yǎng)體系中,腫瘤細(xì)胞穿透基底膜的能力是明顯增加的,MMP-9和TN-C蛋白表達(dá)水平是增加的,尤其是小分質(zhì)量TN-C片段。同時(shí),MMP-9的活性也是明顯增強(qiáng)的,尤其是BxPc3細(xì)胞。3.為了探索腫瘤細(xì)胞與血小板相互作用的分子機(jī)制,我們?cè)谘“迮cBxPc3共培養(yǎng)體系中用特異性抗體封閉血小板P-selectin,我們發(fā)現(xiàn)MMP-9活性降低,MMP-9和TN-C蛋白表達(dá)水平也降低。我們用小干擾RNA(siRNA)敲掉BxPc3細(xì)胞的CD44,然后與血小板進(jìn)行共培養(yǎng),我們發(fā)現(xiàn)MMP-9活性也是降低的,MMP-9和TN-C蛋白表達(dá)水平也明顯降低。四、討論1.MMP-9和TN-C的共表達(dá)可能促進(jìn)腫瘤轉(zhuǎn)移,從而影響胰腺癌的進(jìn)展。并且這兩個(gè)分子的共表達(dá)可能暗示了胰腺癌患者的預(yù)后較差。2.腫瘤細(xì)胞和血小板的相互作用會(huì)誘導(dǎo)MMP-9分泌和TN-C形成,并促進(jìn)胰腺癌細(xì)胞侵襲和轉(zhuǎn)移。3.腫瘤細(xì)胞和血小板相互作用可能是通過(guò)CD44和P-selectin的結(jié)合實(shí)現(xiàn)的,這將為臨床治療提供新的靶點(diǎn)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R735.9

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