本文選題：組織因子 + 剪切體��； 參考：《蘇州大學(xué)》2016年博士論文

【摘要】：組織因子(tissue factor,TF)是一種分子量為47kDa的膜性糖蛋白,不僅作為凝血激活因子,在多種生物學(xué)過程尤其在腫瘤生長與轉(zhuǎn)移中具有重要意義。TF表達(dá)為兩種自然發(fā)生的蛋白亞型,膜結(jié)合的全長TF(flTF)和選擇性剪切亞型組織因子(asTF)。實(shí)驗(yàn)和臨床證據(jù)均表明,在腫瘤中有異常的TF表達(dá),并促進(jìn)腫瘤生長與轉(zhuǎn)移。TF及其剪切亞型(剪切體)的表達(dá)和功能的調(diào)節(jié)受多種因素影響。腫瘤相關(guān)刺激信號可調(diào)節(jié)TF的表達(dá)。作為TF轉(zhuǎn)錄后調(diào)節(jié)機(jī)制,microRNA直接或間接控制TF的表達(dá)和功能。胃癌是最常見的惡性腫瘤之一。目前證實(shí)胃癌中有TF表達(dá)異常,并且其表達(dá)水平與腫瘤的多種生物學(xué)行為有關(guān)。但asTF在胃癌中的臨床意義及調(diào)節(jié)機(jī)制尚不完全明了。本實(shí)驗(yàn)通過自行設(shè)計(jì)asTF的引物和探針應(yīng)用qRT-PCR方法檢測人胃癌組織中flTF、asTF mRNA的表達(dá)水平并分析與臨床病理的相關(guān)性,應(yīng)用Cox模型探討其在患者生存時間等預(yù)后因素中的價值。體外研究了促炎因子TNF-α刺激胃癌MKN28細(xì)胞后flTF、asTF mRNA表達(dá)水平的變化。由于長鏈非編碼RNA(lncRNAs)可以作為一種競爭性內(nèi)源性RNA(ceRNA)與miRNA相互作用,參與靶基因的表達(dá)調(diào)控,并在腫瘤的發(fā)生發(fā)展中發(fā)揮重要的作用。我們通過構(gòu)建慢病毒pLVX-H19 lncRNA-shRNA下調(diào)載體系統(tǒng),TF過表達(dá)載體,qRT-PCR法檢測基因表達(dá)水平,WesternBlot檢測TF蛋白表達(dá)水平,CCK8法測定細(xì)胞生長活力,Transwell檢測細(xì)胞侵襲能力,探討lncRNA H19與miRNA19a(miR-19a)相互作用通路在胃癌細(xì)胞中對flTF、asTF基因表達(dá)的調(diào)控機(jī)制,為TF的轉(zhuǎn)錄后調(diào)節(jié)途徑的靶向治療提供理論依據(jù)。本課題結(jié)果提示:(1)meta分析結(jié)果表明,TF陽性表達(dá)與腫瘤區(qū)域淋巴結(jié)轉(zhuǎn)移之間存在一定的相關(guān)性(P=0.001),單變量與多變量相關(guān)生存分析TF陽性表達(dá)與腫瘤生存、預(yù)后之間均存在明顯的相關(guān)性(P值均0.0001);我們未發(fā)現(xiàn)TF陽性表達(dá)與腫瘤瘤體大小、生長之間有相關(guān)性;關(guān)于asTF在腫瘤中表達(dá)及臨床意義的研究報(bào)道較少,檢索到的文獻(xiàn)原始數(shù)據(jù)不足,無法完成meta分析。(2)本研究通過自行設(shè)計(jì)的檢測astf的引物和taqman探針將上游引物設(shè)計(jì)在第四、第六外顯子的連接處,達(dá)到了避免同時擴(kuò)增出fltf基因片段的目的,可高效、準(zhǔn)確地檢測fltf及astf的基因表達(dá)。(3)胃癌組織中fltfmrna相對定量[7.45(0.34~33.68)]顯著高于正常胃組織[3.00(1.36~5.02)],胃癌組織中astfmrna相對定量[0.88(0.07~26.00)]顯著高于正常胃組織[0.33(0.03~0.97)],差異均有統(tǒng)計(jì)學(xué)意義(p0.01)。fltf與astf基因表達(dá)水平與性別、年齡、tnm分期、腫瘤大小、組織學(xué)分型和化療敏感性等相關(guān)因素的關(guān)系差異均無統(tǒng)計(jì)學(xué)意義(p0.05)。log-rank生存分析顯示fltfmrna低表達(dá)組[33.83(25.24~42.43)]比高表達(dá)組[18.18(13.32~23.04)]平均術(shù)后生存時間顯著延長15.66個月(χ2=6.185,p=0.013)。astfmrna低表達(dá)組[25.32(21.19~29.45)]比高表達(dá)組[20.50(12.51~28.48)]平均術(shù)后生存時間顯著延長4.82個月(χ2=4.604,p=0.032)。fltfmrna與astfmrna都是低表達(dá)組與fltfmrna或astfmrna有一組高表達(dá)組比較,平均生存時間(月)及95%ci分別為28.96(24.90~33.00)、23.40(16.27~30.51),患者術(shù)后平均生存時間顯著延長5.56個月(χ2=4.548,p=0.033)。fltfmrna與astfmrna同時低表達(dá)組與fltfmrna與astfmrna同時高表達(dá)組比較,平均生存時間(月)及95%ci分別為31.07(23.21~38.92)、11.13(6.90~15.36),患者術(shù)后平均生存時間顯著延長19.94個月(χ2=13.005,p=0.000)。fltfmrna與astfmrna有一組高表達(dá)組較同時高表達(dá)組胃癌患者平均生存時間顯著延長16.92個月(χ2=6.346,p=0.012)。多因素cox模型分析顯示,fltf及astf均是胃癌獨(dú)立的預(yù)后風(fēng)險(xiǎn)因素(hr=6.03,95%ci=1.02~35.71,p0.05)及(hr=10.74,95%ci=2.32~49.59,p0.01)。(4)體外實(shí)驗(yàn)發(fā)現(xiàn)tnf-α刺激下胃癌mkn28細(xì)胞的lncrnah19和fltf及astfmrna表達(dá)水平增高而mir-19a表達(dá)水平降低。mir-19a可與tf3’utr結(jié)合抑制其表達(dá),mir-19a可抑制胃癌細(xì)胞的tf蛋白水平的表達(dá),fltf/astf可促進(jìn)胃癌細(xì)胞的增殖和侵襲能力,而mir-19a可抑制其促增殖和侵襲作用。下調(diào)lncrnah19可促進(jìn)mir-19a表達(dá)水平增高,降低astf/fltf表達(dá)水平,抑制胃癌細(xì)胞的增殖和侵襲能力。綜上所述,我們自行設(shè)計(jì)的astf引物和taqman探針可較準(zhǔn)確地檢測astf的基因表達(dá)。研究發(fā)現(xiàn)astfmrna的表達(dá)與fltfmrna一樣,在胃癌組織中高表達(dá),且與臨床病理無相關(guān)性,但與預(yù)后密切相關(guān),其表達(dá)水平與胃癌患者術(shù)后生存時間負(fù)相關(guān),聯(lián)合fltf的基因表達(dá)水平預(yù)測患者生存時間更有意義。證實(shí)tnf-α可體外促進(jìn)胃癌MKN28細(xì)胞TF表達(dá)增加,且受lncRNA H19與miR-19a通路的轉(zhuǎn)錄后調(diào)控,參與促進(jìn)胃癌的發(fā)生與發(fā)展。asTF與flTF以及l(fā)ncRNA H19可作為胃癌預(yù)后判斷的重要生物學(xué)指標(biāo)。本研究不僅發(fā)現(xiàn)了胃癌發(fā)生發(fā)展過程中TF相關(guān)的lncRNA H19與mi R-19a的轉(zhuǎn)錄后調(diào)節(jié)通路,而且為asTF與fl TF以及l(fā)ncRNA H19在胃癌的靶向免疫治療提供了實(shí)驗(yàn)基礎(chǔ),具有一定的臨床應(yīng)用價值。
[Abstract]:Tissue factor (TF) is a membranous glycoprotein with a molecular weight of 47kDa, not only as a coagulation activator, but also in a variety of biological processes, especially in tumor growth and metastasis,.TF is expressed as two naturally occurring protein subtypes, full long TF (flTF) and selective shear subtype tissue factor (asTF) in membrane binding. Both the clinical and clinical evidence show that abnormal TF expression in the tumor and the regulation of the expression and function of tumor growth and metastasis.TF and its shear subtype (shear body) are influenced by many factors. The tumor related stimulus signals can regulate the expression of TF. As a post transcriptional regulation mechanism of TF, microRNA directly or indirectly controls the expression and function of TF. Gastric cancer is one of the most common malignant tumors. It is confirmed that the expression of TF is abnormal in gastric cancer and its expression level is related to a variety of biological behavior of the tumor. But the clinical significance and regulation mechanism of asTF in gastric cancer are not completely clear. This experiment was used to detect human gastric cancer tissue by using qRT-PCR method by self designed asTF primers and probes. The expression level of flTF, asTF mRNA and the correlation with clinicopathological analysis and the value of the Cox model in the prognosis factors such as the patient's survival time. In vitro, the changes of flTF, asTF mRNA expression level after stimulating TNF- alpha in gastric cancer cells were studied. Long chain non coded RNA (lncRNAs) could be a competitive factor. Endogenous RNA (ceRNA) interacts with miRNA, participates in the regulation of target gene expression, and plays an important role in the development of tumor. We constructed the lentivirus pLVX-H19 lncRNA-shRNA down regulation system, TF overexpression vector, qRT-PCR method to detect the gene expression level, WesternBlot to detect the expression level of TF protein, CCK8 method for the determination of fine. Cell growth activity, Transwell detection of cell invasiveness, and explore the regulatory mechanism of lncRNA H19 and miRNA19a (miR-19a) interaction pathway in the expression of flTF and asTF in gastric cancer cells, providing a theoretical basis for the targeting therapy of TF's post transcriptional regulation pathway. The results of this study show that (1) meta analysis results show that TF positive expression and tumor area There was a certain correlation between regional lymph node metastasis (P=0.001). There was a significant correlation between the positive expression of TF and the survival of the tumor and the prognosis (P value 0.0001). We did not find the correlation between the positive expression of TF and the tumor size and growth, and the expression and clinical meaning of asTF in the tumor. The original data of the retrieved literature are insufficient and the meta analysis can not be completed. (2) this study designed the primers of the ASTF and the TaqMan probe to design the upstream primers at the junction of fourth and sixth exons, thus achieving the purpose of avoiding the simultaneous amplification of the fltf gene fragment, which can detect fltf efficiently and accurately. The gene expression of ASTF. (3) fltfmrna relative quantitative [7.45 (0.34~33.68) in gastric carcinoma was significantly higher than that of normal gastric tissue [3.00 (1.36~5.02)], astfmrna relative quantitative [0.88 (0.07~26.00) in gastric cancer tissues was significantly higher than normal gastric tissue [0.33 (0.03~0.97)], and the difference was statistically significant (P0.01) and sex, age and age. There was no significant difference in the relationship between TNM staging, tumor size, histological type and chemosensitivity (P0.05).Log-rank survival analysis showed that [33.83 (25.24~42.43) in fltfmrna low expression group ([18.18 (13.32~23.04)) was significantly longer than the high expression group [18.18 (13.32~23.04)] for 15.66 months (x 2=6.185, p=0.013).Astfmrna low table The average postoperative survival time of the group [25.32 (21.19~29.45) was significantly longer than the high expression group [20.50 (12.51~28.48)] (x 2=4.604, p=0.032).Fltfmrna and astfmrna were both low expression group and fltfmrna or astfmrna group with a group of high expression groups, the average survival time (month) and 95%ci were 28.96 (24.90~33.00), 23.40, patients, respectively. The average survival time was significantly longer after 5.56 months (x 2=4.548, p=0.033).Fltfmrna and astfmrna, compared with fltfmrna and astfmrna high expression groups, the average survival time (month) and 95%ci were 31.07 (23.21~38.92) and 11.13 (6.90~15.36), and the average survival time of the patients was significantly longer after 19.94 months (x 2=13.005, p=0.000). The average survival time of gastric cancer patients with high expression group of.Fltfmrna and astfmrna was significantly prolonged by 16.92 months (x 2=6.346, p=0.012). The multivariate Cox model analysis showed that fltf and ASTF were both independent prognostic risk factors of gastric cancer (hr=6.03,95%ci=1.02~35.71, P0.05) and (hr=10.74,95%ci=2.32~49.59, P0.01). (4) in vitro reality Tnf- alpha stimulated the expression of lncrnah19, fltf and astfmrna in gastric cancer MKN28 cells, while mir-19a expression level decreased.Mir-19a can inhibit the expression of.Mir-19a with TF3 'UTR. Mir-19a can inhibit the expression of TF protein level in gastric cancer cells. Fltf/astf can promote the proliferation and invasion of gastric cancer cells. Proliferation and invasion. Down regulation of lncrnah19 can increase the level of mir-19a expression, reduce the expression level of astf/fltf, inhibit the proliferation and invasion of gastric cancer cells. In summary, our self designed ASTF primers and TaqMan probes can accurately detect the gene expression of ASTF. The study found that the expression of astfmrna is like fltfmrna in gastric cancer. The high expression in the tissue is not related to the clinicopathological correlation, but it is closely related to the prognosis. The expression level is negatively related to the survival time of the patients with gastric cancer. It is more meaningful to predict the survival time of the patients with fltf gene expression. It is confirmed that tnf- alpha can promote the expression of TF in gastric cancer MKN28 cells in vitro, and be transferred to the lncRNA H19 and miR-19a pathway. .asTF and flTF, as well as lncRNA H19, are important biological indicators for predicting the prognosis of gastric cancer. This study not only found the post transcriptional regulation pathway of TF related lncRNA H19 and MI R-19a in the development of gastric cancer, but also for asTF and FL TF, as well as the targeted immunization of TF in gastric cancer. The treatment provides experimental basis and has certain clinical application value.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.2
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本文編號：2105044