本文選題：胃癌 + 新輔助化療　； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：背景：胃癌是常見的消化道惡性腫瘤,以手術(shù)和化療為主的綜合治療是胃癌的標(biāo)準(zhǔn)治療方式。其中,新輔助化療是進(jìn)展期胃癌綜合治療的重要組成部分。但是目前在進(jìn)展期胃癌的診斷和治療過程中,仍缺乏對腫瘤情況及新輔助化療的療效進(jìn)行實(shí)時(shí)準(zhǔn)確評價(jià)的有效評估手段。多數(shù)患者在治療過程中出現(xiàn)化療耐藥,甚至在術(shù)前新輔助化療期間即出現(xiàn)原發(fā)耐藥,從而導(dǎo)致腫瘤進(jìn)展,這是進(jìn)展期胃癌預(yù)后不良的主要原因。因此,探索敏感性和特異性更高的胃癌診療相關(guān)標(biāo)志物,對于準(zhǔn)確判斷腫瘤進(jìn)展、實(shí)時(shí)評價(jià)療效有重要意義,也是進(jìn)一步探索胃癌發(fā)生發(fā)展機(jī)制,尋找有效治療靶點(diǎn)的重要手段。MicroRNA (miRN A)是一類內(nèi)源性非編碼小分子單鏈RNA,通過與靶基因mRNA的3’UTR堿基互補(bǔ)結(jié)合,促進(jìn)靶基因mRNA的降解或抑制其翻譯,導(dǎo)致靶基因表達(dá)下調(diào),從而影響細(xì)胞的增殖、分化和凋亡等過程。miRNA參與腫瘤細(xì)胞的活動及相鄰微環(huán)境的變化,在多種腫瘤中異常表達(dá),與腫瘤的發(fā)生發(fā)展密切相關(guān)。外周循環(huán)血中也存在miRNA,其含量可在一定程度上反映腫瘤大小、浸潤深度等情況；同時(shí),腫瘤組織中往往存在特異性miRNA表達(dá)量的上調(diào)或下調(diào)。因此,miRNA可以作為腫瘤診斷和治療的靶標(biāo)。MiR-301a在結(jié)直腸癌、胰腺癌等腫瘤中呈現(xiàn)異常高表達(dá),與多種惡性生物學(xué)行為及預(yù)后密切相關(guān)。研究表明,miR-301a在胃癌組織中的表達(dá)較配對的癌旁組織明顯升高,與胃癌的分化程度、增殖、侵襲、轉(zhuǎn)移密切相關(guān),在胃癌的發(fā)生、發(fā)展過程中具有重要作用。但是miR-301a在胃癌發(fā)生發(fā)展中的作用及機(jī)制尚不明確,其與胃癌新輔助化療的相關(guān)性也不明確。深入研究其作用機(jī)制,對于探索胃癌新輔助化療的療效評價(jià)相關(guān)標(biāo)志物具有重要意義。在胃癌的發(fā)生發(fā)展過程中有許多因素參與,其中microRNA也發(fā)揮了重要作用。miR-32在許多惡性腫瘤中表現(xiàn)為“癌基因”的作用,但在胃癌中的作用尚不清楚。我們發(fā)現(xiàn)miR-32在胃癌患者的外周血和胃癌組織中呈異常高表達(dá),可能促進(jìn)了胃癌的發(fā)生發(fā)展,miR-32通過調(diào)控其靶基因Kruppel樣因子-4(KLF-4)促進(jìn)胃癌細(xì)胞的增殖、侵襲和遷移。探索miR-32在胃癌發(fā)生發(fā)展中的作用機(jī)制,為胃癌的臨床診治提供新的理論依據(jù)和潛在靶點(diǎn)。胃印戒細(xì)胞癌是胃癌的一種特殊病理類型,具有獨(dú)特的臨床病理特征。例如,早期診斷率低、對化療不敏感、易于向胃壁發(fā)生浸潤性生長等。目前對于胃印戒細(xì)胞癌尚無針對性的個(gè)體化治療方案,患者預(yù)后極差。利用microRNA芯片發(fā)現(xiàn)miR-935在胃印戒細(xì)胞癌中較非印戒細(xì)胞癌表達(dá)明顯降低,miR-935可能通過調(diào)控靶基因Notch1在胃印戒細(xì)胞癌中發(fā)揮作用。研究miR-935在胃印戒細(xì)胞癌中的作用機(jī)制,利于我們更好地了解這種特殊類型的胃癌,為提高此類患者的診療效果提供新的思路。目的：(1)研究新輔助化療及營養(yǎng)支持治療對進(jìn)展期胃癌患者腫瘤緩解及預(yù)后的意義;(2)研究miR-301a對進(jìn)展期胃癌新輔助化療療效評價(jià)的意義,明確miR-301a及其靶基因在胃癌新輔助化療中的作用機(jī)制；(3)研究miR-32及其靶基因在胃癌發(fā)生發(fā)展中的作用機(jī)制；(4)研究miR-935及其靶基因在胃印戒細(xì)胞癌中的作用機(jī)制。方法：(1)收集在我院接受新輔助化療及手術(shù)治療的147例進(jìn)展期胃癌患者的臨床病理資料,分析患者在接受新輔助化療及營養(yǎng)支持治療后的腫瘤緩解情況及預(yù)后。(2)通過實(shí)時(shí)定量PCR法檢測miR-301a在新輔助化療患者胃癌組織及外周血中的表達(dá)水平,并對新輔助化療前后CA199、CEA、CA724的表達(dá)變化、腫瘤的緩解情況進(jìn)行對比；在胃癌細(xì)胞AGS和MGC8-03中發(fā)現(xiàn)miR-301a抑制順鉑、5-氟尿嘧啶誘導(dǎo)的細(xì)胞凋亡；預(yù)測并確認(rèn)谷氨酰胺合成酶(GLUL)為miR-301a的靶基因?yàn)�；采用qPCR檢測胃癌組織和配對癌旁中GLUL的表達(dá),并分析其與miR-301a的相關(guān)性。(3)采用實(shí)時(shí)定量PCR檢測miR-32在胃癌組織及其配對的癌旁組織中的表達(dá)情況；在胃癌細(xì)胞系HGC-27和AGS中瞬時(shí)轉(zhuǎn)染miR-32 mimic, MGC8-03MKN-45中瞬時(shí)轉(zhuǎn)染miR-32 inhibitor,通過xCELLigence RTCA MP系統(tǒng)檢測miR-32對胃癌細(xì)胞增殖能力的影響,通過transwe11實(shí)驗(yàn)檢測miR-32對胃癌細(xì)胞侵襲和遷移能力的影響;通過Targetscan7.0軟件預(yù)測、雙熒光酶報(bào)告基因確認(rèn)miR-32與KLF4的3’UTR結(jié)合;觀察KLF4基因?qū)ξ赴┘?xì)胞增殖、侵襲和遷移的作用；采用qPCR檢測胃癌組織和配對癌旁中KLF4的表達(dá)情況,并分析其與miR-32的相關(guān)性。(4)在非印戒細(xì)胞癌(MGC8-03, SGC-7901, AGS)和印戒細(xì)胞癌(MKN-45和KATO-Ⅲ)中進(jìn)行Affymetrix miRNA芯片分析,篩選出功能尚未被報(bào)道的miR-935；在健康人胃組織、永生化的正常胃粘膜上皮細(xì)胞株GES-1及9株胃癌細(xì)胞系中檢測miR-935的表達(dá)情況；在MKN-45和KATO-Ⅲ中瞬時(shí)轉(zhuǎn)染miR-935 mimic, MGC8-03和HGC-27中瞬時(shí)轉(zhuǎn)染miR-935 inhibitor:通過xCELLigence RTCA MP系統(tǒng)檢測miR-935對胃癌細(xì)胞增殖能力的影響；通過transwell實(shí)驗(yàn)檢測miR-935對胃癌細(xì)胞侵襲和遷移能力的影響；通過Targetscan7.0軟件預(yù)測、雙熒光酶報(bào)告基因驗(yàn)證Notch 1是miR-935的靶基因；采用qPCR檢測胃癌組織和配對癌旁中Notch 1的表達(dá)情況,并分析其與miR-935的相關(guān)性。結(jié)果：(1)147例進(jìn)展期胃癌患者中,男性占66%(97/147),女性占34%(50/147)。治療前病變部位位于胃上部者占23%(34/147),胃體中部者占31%(46/147),胃竇部者占46%(67/147)。R0切除者135例,占91.8%�；颊咂骄邮�3.7周期的新輔助化療。術(shù)中清掃淋巴結(jié)15-77枚,平均28枚,淋巴結(jié)陽性者占68.7%(101/147)。147例患者新輔助化療后評價(jià)為CR(完全緩解)者2例,PR(部分緩解)者74例,SD(穩(wěn)定)者55例,PD(進(jìn)展)者16例,化療有效率(CR+PR)為51.7%(76/147),疾病控制率為89.1%(131/147)。45.5%(67/147)的患者N分期發(fā)生降期：55.6%(5/9)的T2期患者T分期發(fā)生降期；42.6%(29/68)的T3期患者T分期發(fā)生降期；58.6%(41/70)的T4期患者T分期發(fā)生降期。新輔助化療后的病理TNM分期與預(yù)后顯著相關(guān)(P＜0.01)。(2)miR-301a在胃癌組織及外周血中的表達(dá)較正常人明顯升高。相比于CA199、CEA、 CA724, miR-301a在新輔助化療前后的表達(dá)水平變化更加符合腫瘤緩解情況。在胃癌細(xì)胞中過表達(dá)miR-301a可降低細(xì)胞的化療敏感性。谷氨酰胺合成酶(GLUL)是miR-301a的靶基因。在胃癌組織中miR-301a與GLUL的表達(dá)呈負(fù)相關(guān)。(3)miR-32在胃癌組織、胃癌患者血漿以及胃癌細(xì)胞中呈高表達(dá)。過表達(dá)miR-32可促進(jìn)胃癌細(xì)胞的增殖、侵襲和遷移。KLF4是miR-32的靶基因。在胃癌組織中,miR-32與KLF4的mRNA水平呈負(fù)相關(guān)。(4)與非印戒細(xì)胞癌及正常胃癌組織相比,miR-935在胃印戒細(xì)胞癌細(xì)胞系、胃印戒細(xì)胞癌組織中呈低表達(dá)。miR-935可抑制胃印戒細(xì)胞癌細(xì)胞的增殖、侵襲和遷移。Notch 1是miR-935的靶基因,miR-935通過Notch 1在胃癌中發(fā)揮類似抑癌基因的作用。在胃癌組織中,miR-935與Notch 1的mRNA水平呈負(fù)相關(guān)。結(jié)論：(1)新輔助化療對進(jìn)展期胃癌患者具有良好的化療有效率和疾病控制率,腫瘤分期與新輔助化療患者的預(yù)后具有相關(guān)性,新輔助化療后TNM分期較早的患者預(yù)后較好。營養(yǎng)支持治療可改善患者的營養(yǎng)狀況,維持體重,降低化療不良反應(yīng)發(fā)生率。(2) miR-301a比傳統(tǒng)腫瘤標(biāo)志物更有效地評價(jià)胃癌新輔助化療的療效,可作為胃癌診斷和化療療效評價(jià)的標(biāo)志物；miR-30la可通過調(diào)控靶基因GLUL降低胃癌細(xì)胞對化療藥物的敏感性。(3)miR-32在胃癌患者的血漿、組織及胃癌細(xì)胞系中高表達(dá)；miR-32可通過靶基因KLF4促進(jìn)胃癌細(xì)胞的增殖、侵襲和遷移,發(fā)揮癌基因的作用。(4)miR-935在胃印戒細(xì)胞癌細(xì)胞系、胃印戒細(xì)胞癌組織中呈低表達(dá)。miR-935可通過靶基因Notch 1抑制胃印戒細(xì)胞癌細(xì)胞的增殖、侵襲和遷移,在胃癌中發(fā)揮類似抑癌基因的作用。
[Abstract]:Background: gastric cancer is a common malignant tumor of the digestive tract. Comprehensive treatment based on surgery and chemotherapy is the standard treatment for gastric cancer. Among them, neoadjuvant chemotherapy is an important part of the comprehensive treatment of advanced gastric cancer. However, in the diagnosis and treatment of advanced gastric cancer, there is still a lack of treatment for tumor and neoadjuvant chemotherapy. Most patients have chemoresistance in the course of treatment, even in the course of neoadjuvant chemotherapy, which may lead to the development of the tumor, which is the main cause of poor prognosis in advanced gastric cancer. Therefore, to explore the related markers of sensitivity and specificity for the diagnosis and treatment of gastric cancer. It is of great significance to accurately judge the progression of tumor and evaluate the curative effect in real time. It is an important means to further explore the development mechanism of gastric cancer and to find effective therapeutic targets,.MicroRNA (miRN A) is a class of endogenous non coding small molecule single strand RNA, which can promote the degradation of target gene mRNA by combining with the 3 'UTR base of the target gene mRNA. Or inhibition of its translation, which leads to down regulation of target gene expression, which affects cell proliferation, differentiation and apoptosis, and.MiRNA participates in the activity of tumor cells and changes in adjacent microenvironment. Abnormal expression in various tumors is closely related to the occurrence and development of tumors. There are also miRNA in peripheral circulation blood, which can be reflected to a certain extent. Tumor size, depth of infiltration and so on. At the same time, specific miRNA expression is often up or down in tumor tissue. Therefore, miRNA can be used as a target for diagnosis and treatment of cancer,.MiR-301a is abnormal high expression in colorectal cancer, pancreatic cancer and other tumors, and is closely related to many malignant biological behavior and prognosis. The expression of miR-301a in gastric cancer tissue is significantly higher than that of para cancerous tissue, which is closely related to the differentiation, proliferation, invasion and metastasis of gastric cancer, and plays an important role in the occurrence and development of gastric cancer. However, the role and mechanism of miR-301a in the development of gastric cancer are not clear, and the correlation with the neoadjuvant chemotherapy of gastric cancer is not It is of great significance to explore the mechanism of the action of the new adjuvant chemotherapy for gastric cancer. There are many factors involved in the development of gastric cancer, and microRNA also plays an important role in the role of "oncogene" in many malignant tumors, but the role of.MiR-32 in gastric cancer. It is not clear that miR-32 is highly expressed in the peripheral blood and gastric cancer tissues of the patients with gastric cancer, which may promote the development of gastric cancer. MiR-32 can promote the proliferation, invasion and migration of gastric cancer cells by regulating the target gene Kruppel like factor -4 (KLF-4), and explore the mechanism of miR-32 in the development of gastric cancer for gastric cancer. The diagnosis and treatment of the bed provides a new theoretical basis and potential targets. Gastric signet ring cell carcinoma is a special pathological type of gastric cancer and has unique clinicopathological features. For example, early diagnosis rate is low, chemotherapy is insensitive to chemotherapy, and the invasive growth of gastric wall is easy to occur. At present, there is no targeted individualized treatment for gastric signet ring cell carcinoma, patients The prognosis is very poor. The expression of miR-935 in gastric signet ring cell carcinoma is obviously lower than that of non signet ring cell carcinoma by microRNA chip. MiR-935 may play a role in the cancer of gastric signet ring cell by regulating target gene Notch1. The mechanism of miR-935 in gastric signet ring cell carcinoma is helpful for us to better understand this special type of gastric cancer, To improve the diagnosis and treatment effect of such patients. Objective: (1) to study the significance of neoadjuvant chemotherapy and nutrition support therapy on the tumor remission and prognosis of advanced gastric cancer patients; (2) to study the significance of miR-301a on the evaluation of the therapeutic effect of neoadjuvant chemotherapy on advanced gastric cancer, and to clarify the role of miR-301a and its target genes in neoadjuvant chemotherapy for gastric cancer. Mechanism; (3) study the mechanism of miR-32 and its target gene in the development of gastric cancer; (4) study the mechanism of miR-935 and its target gene in gastric signet ring cell carcinoma. Methods: (1) collect the clinicopathological data of 147 cases of gastric cancer patients receiving neoadjuvant chemotherapy and surgical treatment in our hospital, and analyze the patients receiving new AIDS Tumor remission and prognosis after chemotherapy and nutritional support. (2) the expression of miR-301a in gastric cancer tissue and peripheral blood in neoadjuvant chemotherapy patients was detected by real-time quantitative PCR method, and the expression changes of CA199, CEA, CA724 before and after neoadjuvant chemotherapy were compared, and the tumor remission situation was compared, and in AGS and MGC8-03 of gastric cancer cells. MiR-301a inhibits the apoptosis induced by cisplatin and 5- fluorouracil; predicts and confirms that glutamine synthetase (GLUL) is a target for miR-301a; qPCR is used to detect the expression of GLUL in gastric cancer tissue and para cancerous, and to analyze its correlation with miR-301a. (3) detection of miR-32 in gastric cancer tissue and its paired cancer by real time quantitative PCR Transient transfection of miR-32 mimic in gastric cancer cell lines HGC-27 and AGS, transient transfection of miR-32 inhibitor in MGC8-03MKN-45, and the effect of miR-32 on the proliferation of gastric cancer cells through xCELLigence RTCA MP system, and the effect of transwe11 experiments on the invasion and migration of gastric cancer cells; Targetscan7.0 software predicted that the double fluorescent enzyme reporter gene confirmed the combination of miR-32 with 3 'UTR of KLF4, observed the effect of KLF4 gene on the proliferation, invasion and migration of gastric cancer cells, and used qPCR to detect the expression of KLF4 in gastric cancer tissues and paired cancerous tissues, and to analyze its correlation with miR-32. (4) in non signet ring cell carcinoma (MGC8-03, SGC-7901, AGS). The analysis of Affymetrix miRNA chip in MKN-45 and KATO- III of signet ring cell carcinoma (MKN-45 and KATO- III) screened the miR-935 that had not been reported. The expression of miR-935 was detected in healthy human gastric tissue, GES-1 of immortalized normal gastric mucosa epithelial cell strain and 9 lines of gastric cancer cell lines; miR-935 mimic was transfected in MKN-45 and KATO- III, MGC8-0. The effect of miR-935 on the proliferation of gastric cancer cells was detected by transient transfection of miR-935 inhibitor: in 3 and HGC-27 through xCELLigence RTCA MP system, and the effect of miR-935 on the invasion and migration of gastric cancer cells was detected by Transwell test. The target gene of Notch 1 was verified by the Targetscan7.0 software, and the double fluorescent enzyme reported that Notch 1 was the target gene of miR-935. QPCR was used to detect the expression of Notch 1 in gastric cancer tissue and paracanal para cancer, and the correlation with miR-935 was analyzed. Results: (1) among 147 patients with gastric cancer, 66% (97/147) and 34% (50/147) were male, 23% (34/147), 31% (46/147) in the middle of the stomach, and 46% in the gastric antrum (67/1). 47) 135 cases of.R0 resection, accounting for 3.7 cycles of neoadjuvant chemotherapy in patients with 91.8%., 15-77 of lymph nodes, 28, 68.7% (101/147), 2 cases of CR (complete remission), 74 cases of PR (partial remission), 55 cases of SD (stable), 16 cases of PD (progressing), and effective rate of chemotherapy (CR) +PR) for 51.7% (76/147), the disease control rate was 89.1% (131/147).45.5% (67/147). The stage of N stage occurred: 55.6% (5/9) of T2 stage patients with T staging; 42.6% (29/68) T3 phase of the stage of T; 58.6% (2) (0.01) (2) the expression of miR-301a in gastric cancer tissue and peripheral blood was significantly higher than that of normal people. Compared to CA199, CEA, CA724, miR-301a was more in line with the tumor remission situation before and after the neoadjuvant chemotherapy. The overexpression of miR-301a in gastric cancer cells could reduce the chemosensitivity of the cell cell. The glutamine synthetase (GLUL) was miR-3 The target gene of 01A is negatively correlated with the expression of miR-301a and GLUL in gastric cancer. (3) miR-32 is highly expressed in gastric cancer tissue, gastric cancer plasma and gastric cancer cells. Overexpression of miR-32 can promote the proliferation of gastric cancer cells, invasion and migration of.KLF4 are the target genes of miR-32. In gastric cancer, miR-32 and KLF4 mRNA levels are negatively correlated. (4) Compared with non signet ring cell carcinoma and normal gastric cancer tissue, miR-935 in gastric signet ring cell carcinoma cell line and low expression of.MiR-935 in gastric signet ring cell carcinoma can inhibit the proliferation of gastric signet ring cell carcinoma cells, invasion and migration of.Notch 1 is the target gene of miR-935, and miR-935 plays a role similar to tumor suppressor gene in gastric cancer through Notch 1. In the tissue, miR-935 was negatively correlated with the mRNA level of Notch 1. Conclusion: (1) neoadjuvant chemotherapy has good Chemotherapy Effectiveness and disease control rate for advanced gastric cancer patients. Tumor staging is related to the prognosis of neoadjuvant chemotherapy patients. After neoadjuvant chemotherapy, the prognosis of patients with early TNM staging is better. Nutrition support therapy can improve the patient's prognosis. (2) miR-301a is more effective than traditional tumor markers to evaluate the curative effect of neoadjuvant chemotherapy for gastric cancer, which can be used as a marker for the evaluation of gastric cancer diagnosis and chemotherapy, and miR-30la can reduce the sensitivity of gastric cancer cells to chemotherapeutic drugs by regulating target gene GLUL. (3) miR-32 MiR-32 can promote the proliferation, invasion and migration of gastric cancer cells and play the role of oncogene through target gene KLF4. (4) miR-935 in gastric signet ring cell carcinoma cell line, low expression of.MiR-935 in gastric signet ring cell carcinoma can inhibit gastric signet ring cell carcinoma by target gene Notch 1 Cell proliferation, invasion and migration play a similar role as tumor suppressor genes in gastric cancer.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.2

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