【摘要】：研究背景：早期診斷困難、化療耐藥、缺乏有效的預(yù)后指標(biāo)是目前胃癌研究領(lǐng)域的重要難題,限制了胃癌患者昀總體預(yù)后。尋找新的診斷標(biāo)記物,深入了解胃癌發(fā)生、發(fā)展機(jī)制,逆轉(zhuǎn)化療耐藥是改善胃癌患者預(yù)后的重要手段。MicroRNAs (miRNAs)是一類長(zhǎng)約22個(gè)核苷酸的非編碼單鏈小分子RNA,在腫瘤發(fā)生、發(fā)展、增殖、侵襲、轉(zhuǎn)移及化療耐藥等多個(gè)方面發(fā)揮重要作用。MiR-30b在結(jié)直腸癌、宮頸癌、乳腺癌等腫瘤中表達(dá)降低,參與多種腫瘤生物學(xué)表型的調(diào)控。但是其在胃癌中的表達(dá)水平、調(diào)控作用及機(jī)制尚不明確。因此,檢測(cè)胃癌中miR-30b的表達(dá)水平,深入研究miR-30b的調(diào)控作用及機(jī)制,對(duì)于明確其在胃癌診斷、判斷預(yù)后等方面具有重要的意義。新輔助化療作為腫瘤綜合治療的一部分,已經(jīng)逐漸被引用至局部進(jìn)展期胃癌的治療上。新輔助化療應(yīng)用于胃癌的手術(shù)之前,具有以下優(yōu)點(diǎn)：提高切除率,降低腫瘤的分期,相比于單純手術(shù),還可以減少淋巴結(jié)和血管的轉(zhuǎn)移。術(shù)前患者的營(yíng)養(yǎng)狀況良好,可以更好的耐受化療的副反應(yīng)。近年來(lái),國(guó)內(nèi)外關(guān)于胃癌新輔助化療的研究都顯示新輔助化療有明顯的效果。但是,新輔助化療方案沒(méi)有統(tǒng)一的標(biāo)準(zhǔn),化療藥物、化療周期的選擇及療效評(píng)價(jià)的時(shí)間都存在爭(zhēng)議。目的：(1)探討血清癌胚抗原(CEA)、糖類抗原19-9(CA19-9)、糖類抗原242(CA242)及糖類抗原50(CA50)聯(lián)合檢測(cè)在胃癌患者診斷、臨床病理因素及預(yù)后判斷的價(jià)值。(2)探索miR-30b對(duì)胃癌細(xì)胞株增殖、凋亡、侵襲及遷移等惡性表型的調(diào)控作用。明確miR-30b的調(diào)控的靶基因及信號(hào)通路。(3)評(píng)估進(jìn)展期胃癌患者行新輔助治療后的臨床病理特征及效果。評(píng)估空腸穿刺置管早期腸內(nèi)營(yíng)養(yǎng)支持治療對(duì)接受新輔助化療并手術(shù)胃癌患者的營(yíng)養(yǎng)狀況和并發(fā)癥的影響。方法：(1)收集18I例于我院行手術(shù)治療的胃癌患者臨床資料,回顧性分析其術(shù)前血清CEA、CA19-9, CA242及CA50的水平。通過(guò)ROC曲線,對(duì)四項(xiàng)腫瘤標(biāo)志物單獨(dú)和不同組合方式診斷胃癌的敏感性進(jìn)行比較分析。單因素及多因素生存分析探討腫瘤標(biāo)志物與患者術(shù)后病理及生存預(yù)后的關(guān)系。(2)通過(guò)實(shí)時(shí)定量PCR法檢測(cè)miR-30b的表達(dá)水平；CCK8法檢測(cè)細(xì)胞增殖；流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡；transwell法檢測(cè)細(xì)胞侵襲和遷移能力；western blot檢測(cè)蛋白表達(dá)水平；雙螢光素酶報(bào)告基因?qū)嶒?yàn)驗(yàn)證miR-30b的靶基因。(3)分析2007年2月至2015年3月于北京協(xié)和醫(yī)院收治接受術(shù)前化療和手術(shù)的115例胃癌患者的臨床病例資料,所有患者采用Xelox化療方案,奧沙利鉑130mg/m2靜脈滴入2h,第一天；口服卡培他濱1000mg/m2,第1-2周,后休息一周。三周為一療程,患者至少接受2周期的術(shù)前化療。評(píng)估新輔助化療的療效、毒副反應(yīng)及與患者預(yù)后的關(guān)系。通過(guò)檢測(cè)臨床及實(shí)驗(yàn)室指標(biāo),評(píng)估空腸穿刺置管腸內(nèi)營(yíng)養(yǎng)對(duì)胃癌患者術(shù)后營(yíng)養(yǎng)狀況及并發(fā)癥的影響。結(jié)果(1)腫瘤標(biāo)志物CEA、CA19-9、CA242及CA50檢測(cè)胃癌的陽(yáng)性率分別為17.67,17.12,20.44和13.81%。四項(xiàng)腫瘤標(biāo)志物聯(lián)合檢測(cè)的陽(yáng)性率為36.57%。術(shù)前血清CEA、CA19-9、CA242和CA50升高的胃癌患者,其腫瘤臨床分期也較晚,并且有較差的總生存率。術(shù)前血清CEA、CA19-9、CA242和CA50升高的胃癌患者,5年生存率分別為28.12,25.83,27.02和24.05%,而術(shù)前此四項(xiàng)腫瘤標(biāo)志物正常的患者5年生存率則分別為55.01,55.37,56.36,54.51%(P0.01)。Cox比例風(fēng)險(xiǎn)模型行多因素分析示升高的CA242可作為判斷胃癌預(yù)后差的獨(dú)立因素。(2)miR-30b表達(dá)水平在胃癌組織中明顯下降。miR-30b低表達(dá)與淋巴結(jié)轉(zhuǎn)移密切相關(guān),miR-30b在23對(duì)胃癌組織中的表達(dá)與患者的年齡、性別和Lauren分型無(wú)明顯相關(guān)性。(3)CCK-8實(shí)驗(yàn)結(jié)果顯示,過(guò)表達(dá)miR-30b可明顯抑制胃癌細(xì)胞的生長(zhǎng)。相反,將miR-30b inhibitor轉(zhuǎn)染至胃癌細(xì)胞降低miR-30b的表達(dá),結(jié)果顯示明顯促進(jìn)了胃癌細(xì)胞生長(zhǎng)。流式細(xì)胞儀檢測(cè)了miR-30b對(duì)于胃癌細(xì)胞凋亡的影響,表明過(guò)表達(dá)miR-30b可以誘導(dǎo)胃癌細(xì)胞的凋亡。在胃癌細(xì)胞中過(guò)表達(dá)miR-30b,可以顯著的抑制穿過(guò)小室孔膜的細(xì)胞數(shù),抑制胃癌細(xì)胞侵襲遷移能力。相反,轉(zhuǎn)染inhibitor下調(diào)miR-30b在胃癌細(xì)胞的表達(dá)水平,則促進(jìn)了胃癌細(xì)胞的侵襲轉(zhuǎn)移。(4)通過(guò)構(gòu)建包含種子區(qū)的野生型和突變型雙螢光素酶報(bào)告基因載體,并分別與miR-30b mimics或mimics control共轉(zhuǎn)染293T細(xì)胞,發(fā)現(xiàn)共轉(zhuǎn)染mimics與野生型載體的細(xì)胞的熒光素酶活性顯著低于共轉(zhuǎn)染mimics與突變型載體,mimics control與野生型載體,提示EIF5A2是miR-30b的直接靶基因。(5)miR-30b可能通過(guò)下調(diào)EIF5A2進(jìn)而激活上皮間質(zhì)轉(zhuǎn)化的相關(guān)指標(biāo)E-cadherin口β-catenin,抑制了腫瘤細(xì)胞的侵襲轉(zhuǎn)移。(6)對(duì)新輔助化療并行手術(shù)治療的患者進(jìn)行分析,完全緩解(CR)2例,部分緩解(PR)55例,穩(wěn)定(SD)47例,進(jìn)展(PD)有11例,化療有效率為49.6%,疾病控制率為90.4%。術(shù)前T分期的降期率為48.7%�；熀笞畛Ｒ�(jiàn)的不良反應(yīng)為白細(xì)胞減少(16.5%)和胃腸道反應(yīng)(12.2%),多為I-II度。經(jīng)過(guò)對(duì)癥治療后均可得到緩解。接受新輔助化療和手術(shù)的患者中位生存期48個(gè)月,5年生存率42.4%�；煰熜镻R的患者生存期明顯優(yōu)于SD和PD的患者,差異具有統(tǒng)計(jì)學(xué)意義(P0.001)。GPS評(píng)分(Glasgow prognostic score)較低的患者對(duì)于術(shù)前化療有較好的緩解(P=0.028)。多因素回歸比例模型分析顯示化療反應(yīng)性(P=0.026)、腫瘤pTNM分期(P=0.037)、Lauren分型(P=0.033)和GPS評(píng)分(P=0.002)為新輔助化療胃癌患者預(yù)后的獨(dú)立因素。(7)術(shù)后第3、7天,置管的腸內(nèi)營(yíng)養(yǎng)組白蛋白水平要高于不置管的腸外營(yíng)養(yǎng)組患者；術(shù)后第3天,前白蛋白水平在置管組要高于不置管組。經(jīng)空腸穿刺管營(yíng)養(yǎng)可以促進(jìn)患者早日排氣,并且對(duì)肝功能具有保護(hù)作用。兩組患者的生存期尚未觀察到明顯差別。結(jié)論：(1)聯(lián)合檢測(cè)CEA、CA19-9、CA242%和CA50可以提高胃癌診斷的陽(yáng)性率。CA242有較高的診斷價(jià)值。在可切除的胃癌患者中,術(shù)前的CA242與胃癌的分期有關(guān),其是胃癌患者預(yù)后的獨(dú)立預(yù)測(cè)因子；(2)miR-30b在胃癌組織中及胃癌細(xì)胞中低表達(dá)；過(guò)表達(dá)miR-30b可以抑制胃癌細(xì)胞增殖、促進(jìn)細(xì)胞凋亡,抑制胃癌細(xì)胞的侵襲轉(zhuǎn)移；(3) EIF5A2是miR-30b的直接靶基因,miR-30b可能通過(guò)下調(diào)EIF5A2進(jìn)而激活上皮間質(zhì)轉(zhuǎn)化的相關(guān)指標(biāo)E-cadherin和β-catenin,抑制了腫瘤細(xì)胞的侵襲轉(zhuǎn)移；(4) Xelox新輔助化療方案對(duì)于進(jìn)展期胃癌患者有較好的有效率,化療毒副反應(yīng)不重。新輔助化療后取得明顯臨床緩解的患者預(yù)后要好�；煼磻�(yīng)性、術(shù)后病理分期、Lauren分型和GPS評(píng)分可以作為新輔助化療并手術(shù)治療的胃癌患者預(yù)后的獨(dú)立因素；(5)接受新輔助化療手術(shù)的患者經(jīng)空腸營(yíng)養(yǎng)管早期腸內(nèi)營(yíng)養(yǎng)安全方便,可以改善患者術(shù)后早期的營(yíng)養(yǎng)狀況。
[Abstract]:Background: The early diagnosis is difficult, the resistance of chemotherapy and the lack of effective prognostic index are important problems in the research field of gastric cancer, and the overall prognosis of patients with gastric cancer is limited. It is an important means to find a new diagnostic marker for deep understanding of the occurrence and development mechanism of gastric cancer and to reverse the resistance of chemotherapy to the prognosis of patients with gastric cancer. MicroRNAs (miRNAs) are a class of non-coding single-chain small-molecule RNA with a length of about 22 nucleotides, which play an important role in the aspects of tumorigenesis, development, proliferation, invasion, metastasis and chemotherapy resistance. The expression of MiR-30b in the tumors, such as colorectal cancer, cervical cancer, and breast cancer, is reduced and involved in the control of various tumor biological phenotypes. But its expression level, regulation and mechanism in gastric cancer are not clear. Therefore, it is of great significance to study the expression level of miR-30b in gastric cancer and to study the regulation and mechanism of miR-30b. Neoadjuvant chemotherapy, as part of the comprehensive treatment of the tumor, has been gradually referred to the treatment of local progressive gastric cancer. The new adjuvant chemotherapy is applied to the operation of gastric cancer, and has the following advantages of improving the resection rate and reducing the stage of the tumor, and can also reduce the metastasis of the lymph node and the blood vessel compared with the simple operation. The nutritional status of the pre-operation patients is good, and the side effects of the chemotherapy can be better tolerated. In recent years, the research on neoadjuvant chemotherapy of gastric cancer both at home and abroad has shown that the new adjuvant chemotherapy has obvious effect. However, there is no uniform standard for the new adjuvant chemotherapy regimen, and there is a dispute between the selection of the chemotherapy drug and the chemotherapy period and the time of the evaluation of the curative effect. Objective: (1) To study the value of serum carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 242 (CA242) and carbohydrate antigen 50 (CA50) in the diagnosis, clinical pathology and prognosis of gastric cancer. (2) To explore the effects of miR-30b on the proliferation, apoptosis, invasion and migration of gastric cancer cell line. And a target gene and a signal path for determining the regulation of the miR-30b. (3) To evaluate the clinical and pathological features and effects of the new adjuvant therapy for patients with advanced gastric cancer. To evaluate the effect of early enteral nutrition support on the nutritional status and complications of patients undergoing neoadjuvant chemotherapy and operation of gastric cancer. Methods: (1) The clinical data of 18 patients with gastric cancer treated by operation in our hospital were collected, and the levels of serum CEA, CA19-9, CA242 and CA50 were analyzed retrospectively. The sensitivity of four tumor markers to the diagnosis of gastric cancer was compared and analyzed by ROC curve. The relationship between tumor markers and postoperative pathology and survival was discussed by single factor and multi-factor survival analysis. (2) the expression level of miR-30b was detected by real-time quantitative PCR; the cell proliferation was detected by the method of CCK8; the cell apoptosis was detected by flow cytometry; the cell invasion and the migration ability were detected by transwell method; the expression level of the protein was detected by western blot; and the target gene of the miR-30b was verified by the double luciferase reporter gene. (3) To analyze the clinical data of 115 patients with gastric cancer who received pre-operative chemotherapy and operation from February 2007 to March 2015. All patients received the Xelerox chemotherapy, oxaliplatin 130 mg/ m2 for 2 hours, the first day, oral capecitabine 1000 mg/ m2, week 1-2, The rest of the week. Three weeks for a course of treatment, the patient received at least 2 cycles of pre-operative chemotherapy. To evaluate the effect of neoadjuvant chemotherapy, the side-side reaction and the relationship with the prognosis of the patients. To evaluate the effect of enteral nutrition on the nutritional status and complications of patients with gastric cancer by detecting the clinical and laboratory parameters. Results (1) The positive rates of CEA, CA19-9, CA242 and CA50 in the detection of gastric cancer were 17.67, 17.12, 20.44 and 13.81%, respectively. The positive rate of the four tumor markers was 36.57%. Pre-operative serum CEA, CA19-9, CA242 and CA50 increased patients with gastric cancer, and their clinical stages were later, and there was a poor overall survival rate. The 5-year survival rates of the pre-operative serum CEA, CA19-9, CA242 and CA50 were 28.12, 25.83, 27.02, and 24.05%, respectively, while the 5-year survival rate of the four tumor markers was 55.01, 55.37, 56.36, 54.51% (P0.01). (2) The expression level of miR-30b was significantly decreased in gastric cancer tissue. The low expression of miR-30b is closely related to lymph node metastasis, and the expression of miR-30b in the tissue of gastric cancer has no significant correlation with the age, sex and Lauren type of the patient. (3) The results of CCK-8 show that the expression of miR-30b can significantly inhibit the growth of gastric cancer cells. In contrast, the transfection of miR-30b in human gastric cancer cells to the expression of miR-30b in gastric cancer cells revealed a significant increase in the growth of gastric cancer cells. The effect of miR-30b on the apoptosis of gastric cancer cells was detected by flow cytometry. It was shown that the expression of miR-30b could induce the apoptosis of gastric cancer cells. The expression of the miR-30b in the gastric cancer cell can significantly inhibit the number of cells passing through the cell membrane and inhibit the invasion and migration of the gastric cancer cells. In contrast, down-regulation of the expression level of miR-30b in gastric cancer cells by transfectitor promoted the invasion and metastasis of gastric cancer cells. (4) By constructing the wild-type and mutant double-luciferase reporter gene vector containing the seed region and co-transfecting the 293T cells with the miR-30b misitics or the mimics control, the luciferase activity of the cells of the co-transfected mimics and the wild-type vector was found to be significantly lower than that of the co-transfected mimetics and the mutant vectors, The micionics control is similar to the wild type vector, suggesting that EIF5A2 is a direct target gene of miR-30b. (5) miR-30b may inhibit the invasion and metastasis of tumor cells by down-regulating EIF5A2 and then activating the relevant index E-cadherin of EIF5A2. (6) The patients who were treated with the new adjuvant chemotherapy were analyzed, the complete response (CR) was 2, the partial response (PR) was 55, the stability (SD) was 47, the progress (PD) was 11, the effective rate of the chemotherapy was 49.6%, and the disease control rate was 90.4%. The pre-operative T staging was 48.7%. The most common adverse reactions after chemotherapy were leukopenia (16.5%) and gastrointestinal (12.2%), mostly I-II. Can be relieved after symptomatic treatment. The median survival in patients receiving neoadjuvant chemotherapy and surgery was 48 months and the 5-year survival rate was 42.4%. The survival time of patients with PR was significantly better than that of SD and PD (P = 0.001). The patients with low level of Glasgow prognostic score had a better response to preoperative chemotherapy (P = 0.028). The multi-factor regression model analysis showed that the response of chemotherapy (P = 0.026), tumor pTNM stage (P = 0.037), Lauren type (P = 0.033) and GPS (P = 0.002) were independent factors for the prognosis of patients with gastric cancer with neoadjuvant chemotherapy. (7) In the third and seventh day of the operation, the level of the intestinal nutrition group of the tube was higher than that of the parenteral nutrition group without the tube, and the pre-albumin level was higher than that of the non-set tube group on the third day after the operation. The nutrition of the jejunal puncture tube can promote the early exhaust of the patient, and has a protective effect on the liver function. There was no significant difference in the survival of the two groups. Conclusion: (1) The positive rate of CEA, CA19-9, CA242% and CA50 in the diagnosis of gastric cancer can be increased. CA242 has high diagnostic value. In the patients with gastric cancer, the preoperative CA242 is related to the stage of gastric cancer, which is an independent predictor of the prognosis of the gastric cancer. (2) The miR-30b is expressed in the gastric cancer tissues and the gastric cancer cells, and the overexpression of the miR-30b can inhibit the proliferation of the gastric cancer cells and promote the cell apoptosis. (3) EIF5A2 is a direct target gene of miR-30b, and miR-30b may further activate the relevant indexes E-cadherin and antigen-catenin of epithelial-mesenchymal transition by down-regulating EIF5A2, and the invasion and metastasis of tumor cells are inhibited; (4) Xelerox neoadjuvant chemotherapy is effective in the patients with advanced gastric cancer. The prognosis of patients with significant clinical remission after neoadjuvant chemotherapy is good. the chemotherapy, the postoperative pathological stage, the Lauren type and the GPS score can be used as independent factors for the prognosis of the gastric cancer patients with neoadjuvant chemotherapy and operation treatment; and (5) the patients receiving the neoadjuvant chemotherapy operation are safe and convenient to the early enteral nutrition of the jejunal nutrition tube, Can improve the nutritional status of the early postoperative patients.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.2

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中國(guó)期刊全文數(shù)據(jù)庫(kù) 前8條

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