發(fā)布時間：2018-07-25 20:00

【摘要】：背景和研究目的胃腸胰神經(jīng)內(nèi)分泌腫瘤(GEP-NENs)是最常見的NENs類型,近年來其發(fā)病率呈現(xiàn)上升趨勢。但其發(fā)生機(jī)制不清,且缺乏有效的診斷和治療標(biāo)志物。本課題組前期研究發(fā)現(xiàn),CD147在正常胰島中不表達(dá),在絕大部分胰腺神經(jīng)內(nèi)分泌腫瘤(pNENs)中表達(dá),且隨著腫瘤惡性程度的增高,CD147的表達(dá)水平增高。提示CD147可能參與了 pNENs的腫瘤發(fā)生,且表現(xiàn)為促癌作用,但對CD147在胃腸NENs中的表達(dá)情況,在pNENs中的作用及其機(jī)制,以及在正常胰島中不表達(dá)的分子機(jī)制尚需進(jìn)一步研究。因此本研究主要包括以下幾個部分:CD147抗體驗(yàn)證;檢測CD147在胃腸NENs中的表達(dá)表達(dá)研究;檢測CD147、MCT-1、MCT-4慢性胰腺炎中的表達(dá),MCT-1、MCT-4、PDX1、nkx6.1、Pax-6在pNENs中的表達(dá)并分析其與CD147表達(dá)的相關(guān)性研究;pNENs中CD147基因的序列變異研究。方法1.構(gòu)建CD147真核過表達(dá)載體,轉(zhuǎn)染HEK293T細(xì)胞,提取蛋白行去糖基化處理后,CD147抗體和抗His抗體進(jìn)行western blot驗(yàn)證。若CD147抗體和抗His(C端)抗體能得到同樣分子量的融合蛋白目的條帶,證明抗體識別的是CD147分子。2.采用免疫組化方法檢測109例胃腸NENs中CD147的表達(dá),檢測36例慢性胰腺炎中 CD147、MCT-1、MCT-4 的表達(dá),及 42 例 pNENs 中 MCT-1、MCT-4、PDX1、nkx6.1、Pax-6的表達(dá),并與CD147表達(dá)進(jìn)行相關(guān)性分析。3.選取29例pNENs患者的正常組織和腫瘤組織配對石蠟樣本共60個,對CD147基因進(jìn)行測序檢測。結(jié)果第一部分CD147抗體驗(yàn)證:證實(shí)前期實(shí)驗(yàn)所用CD147單克隆抗體特異性識別地為CD147分子,抗體可用。第二部分CD147在胃腸NENs中的表達(dá)研究:1.CD147在胃、十二指腸、結(jié)直腸NENs中均有表達(dá),免疫表型可表現(xiàn)為漿型、膜型、膜漿型,其陽性表達(dá)率分別為65.7%、76.5%、19.3%。2.CD147在胃腸NENs中的表達(dá)水平與組織學(xué)分級呈顯著正相關(guān),在胃NENs中CD147的免疫表型與其表達(dá)水平有關(guān),表達(dá)水平越高,膜型的比例越高。第三部分CD147、MCT-1、MCT-4在慢性胰腺炎及pNENs瘤旁正常胰島中表達(dá)的研究:CD147、MCT-1、MCT-4在36例慢性胰腺炎中的胰島中均呈陰性表達(dá)。42例pNENs病例中,MCT-1、MCT-4在瘤旁正常腺泡和導(dǎo)管及正常胰島中均呈陰性表達(dá)。第四部分MCT-1、MCT-4、PDX1、nkx6.1、Pax-6在pNENs中的表達(dá)及其與CD147 表達(dá)的相關(guān)性研究:1.MCT-1、MCT-4、Pax-6、nkx6.1、PDX1 在 pNENs中均呈陽性表達(dá)。2.MCT-1、MCT-4的表達(dá)水平與腫瘤的大小、組織學(xué)分級、pTNM分期呈顯著正相關(guān),而Pax-6、nkx6.1、PDX1的表達(dá)水平與腫瘤的大小、組織學(xué)分級、pTNM分期呈顯著負(fù)相關(guān)。3.發(fā)生淋巴結(jié)轉(zhuǎn)移或遠(yuǎn)處轉(zhuǎn)移的腫瘤中的MCT-1、MCT-4表達(dá)水平要高于未發(fā)生淋巴結(jié)轉(zhuǎn)移或遠(yuǎn)處轉(zhuǎn)移的;而未發(fā)生局部浸潤和/或淋巴結(jié)轉(zhuǎn)移的腫瘤中Pax-6的表達(dá)水平比發(fā)生局部浸潤和/或淋巴結(jié)轉(zhuǎn)移的高,無遠(yuǎn)處轉(zhuǎn)移的比有遠(yuǎn)處轉(zhuǎn)移的腫瘤Pax-6、PDX1表達(dá)水平高。4.CD147與MCT-1的表達(dá)水平呈顯著正相關(guān),與MCT-4的表達(dá)水平無相關(guān)性,CD147與PDX1、nkx6.1、Pax-6的表達(dá)水平呈顯著負(fù)相關(guān)。第五部分pNENs中CD147基因的序列變異研究:29例pNENs患者的60個組織樣本中共檢測出47個CD147基因變異位點(diǎn)。其中直徑2cm的腫瘤中19:580388CG(rs2229664)CG 雜合基因型、19:582775AG GG 純合基因型、19:582927TA(rs8259)AA純合基因型型比"f2cm的多,差異有統(tǒng)計(jì)學(xué)意義。19:579627CT(rs1803202)在腫瘤組織或正常與腫瘤組織同時存在的T純合基因型比C/T雜合基因型多,差異有統(tǒng)計(jì)學(xué)意義。19:582927TA在正常組織和腫瘤組織中的變異(AA基因型和A/T雜合基因型)在不同的腫瘤組織學(xué)分級間存在顯著差異,組織學(xué)分級越高,腫瘤組織中存在的19:582927TA變異(AA基因型和A/T雜合基因型)越多。結(jié)論1.CD147在GEP-NENs不同部位中的表達(dá)有所差異。2.CD147在胃腸NENs中的表達(dá)水平與組織學(xué)分級呈顯著正相關(guān),其表達(dá)水平高可能提示腫瘤的分化差、惡性程度高,可能能作為評估胃腸NENs嚴(yán)重程度的一個指標(biāo)。3.CD147、MCT-1、MCT-4在慢性胰腺炎和pNENs瘤旁胰島中均呈陰性表達(dá)。4.MCT-1、MCT-4的表達(dá)水平與腫瘤的大小、組織學(xué)分級、pTNM分期呈顯著正相關(guān);Pax-6、nkx6.1、PDX1的表達(dá)水平與腫瘤的大小、組織學(xué)分級、pTNM分期呈顯著負(fù)相關(guān),其表達(dá)水平高可能能提示腫瘤的分化好、惡性程度低。5.pNENs中CD147與MCT-1的表達(dá)水平呈顯著正相關(guān),與PDX1、nkx6.1、Pax-6的表達(dá)水平呈顯著負(fù)相關(guān)。提示MCT-1、PDX1、nkx6.1、Pax-6可能參與了 CD147在pNENs中的作用。6.在pNENs組織中共檢測出4個與臨床病理特征相關(guān)的CD147基因變異位點(diǎn)rs2229664CG、19:582775AG、rs8259TA、rs1803202CT,可能具有一定臨床意義,尚需進(jìn)一步分析驗(yàn)證。
[Abstract]:Background and research objective gastrointestinal pancreatic neuroendocrine tumor (GEP-NENs) is the most common type of NENs. In recent years, its incidence is rising. However, its pathogenesis is not clear, and it lacks effective diagnostic and therapeutic markers. The previous study in our group found that CD147 was not expressed in normal pancreatic islets, and most of the pancreatic neuroendocrine swelling was in the pancreas. The expression of the tumor (pNENs) is increased with the increase of the malignancy of the tumor, and the expression of CD147 increases. It suggests that CD147 may be involved in the carcinogenesis of pNENs, and it is a cancer promoting effect. However, the expression of CD147 in the gastrointestinal NENs, the role and mechanism in pNENs, and the molecular mechanism of non expression in the normal islets still need to be further studied. This study mainly includes the following parts: CD147 antibody test; detection of expression and expression of CD147 in gastrointestinal NENs; detection of expression in CD147, MCT-1, MCT-4 chronic pancreatitis; expression of MCT-1, MCT-4, PDX1, Nkx6.1, Pax-6 in pNENs. Method 1. CD147 eukaryotic overexpression vector was constructed, HEK293T cells were transfected and the extracted protein was treated with glycosylation. CD147 antibody and anti His antibody were verified by Western blot. If CD147 antibody and anti His (C terminal) antibody can get the same molecular weight fusion protein target band, it is identified that the CD147 molecule.2. adopts the immunohistochemical method. The expression of CD147 in 109 cases of gastrointestinal NENs was detected, the expression of CD147, MCT-1, MCT-4 in 36 cases of chronic pancreatitis, and the expression of MCT-1, MCT-4, PDX1, Nkx6.1, Pax-6 in 42 cases of pNENs were detected, and the correlation analysis between the normal tissues and tumor tissues of 29 patients was selected and 60 samples were paired. The results of CD147 antibody test in part 1 confirmed that the CD147 monoclonal antibody was specifically identified as CD147 molecule and the antibody was available. The expression of second part of CD147 in the NENs of the gastrointestinal tract: 1.CD147 in the stomach, duodenum, and colorectal NENs, the immunophenotype could be expressed as plasma, membrane and membranous type. The positive expression rate was 65.7%, 76.5%, and the expression level of 19.3%.2.CD147 in the gastrointestinal NENs was positively correlated with the histological grade. In the gastric NENs, the immunophenotype of CD147 was related to the expression level, the higher the expression level, the higher the proportion of the membrane type. The third part of CD147, MCT-1, MCT-4 was expressed in the normal pancreatic islets by the chronic pancreatitis and pNENs tumor. CD147, MCT-1, MCT-4 in 36 cases of chronic pancreatitis were negative expression of.42 cases in pNENs, MCT-1, MCT-4 was negative in normal acinus and ducts and normal islets in the paracarma. Fourth part MCT-1, MCT-4, PDX1, Nkx6.1. The expression level of X-6, Nkx6.1 and PDX1 in pNENs was.2.MCT-1, and the expression level of MCT-4 was positively correlated with the size of tumor, histological grade and pTNM stage, while the expression level of Pax-6, Nkx6.1, PDX1 was significantly negatively correlated with lymph node metastasis or distant metastasis in tumor size, histological grade and pTNM stage. The expression level of T-4 was higher than that of no lymph node metastasis or distant metastasis, while the expression level of Pax-6 in tumor without local infiltration and / or lymph node metastasis was higher than that of local infiltration and / or lymph node metastasis, and there was no distant metastasis of tumor Pax-6 with distant metastasis, and the expression level of.4.CD147 and MCT-1 was high in PDX1 expression level. Significant positive correlation was not related to the expression level of MCT-4, CD147 was negatively correlated with the expression level of PDX1, Nkx6.1 and Pax-6. Fifth part of the sequence variation of the CD147 gene in pNENs: 47 CD147 gene mutation sites were detected in 60 tissues of pNENs patients. The 19:580388CG (rs2229664) heterozygosity in the tumor of the diameter 2cm was mixed. Genotypes, 19:582775AG GG homozygous genotypes, 19:582927TA (rs8259) AA homozygous genotypes more than "f2cm", the difference is statistically significant.19:579627CT (rs1803202) in the tumor tissue or normal and tumor tissues, the T homozygous genotype is more than the C/T heterozygous genotype, the difference is statistically significant.19:582927TA in normal tissue and swelling. The variation in the tumor tissue (AA genotype and A/T heterozygous genotype) was significantly different between different tumor histological grades. The higher the histological grade, the more 19:582927TA variation (AA genotype and A/T heterozygous genotype) existed in the tumor tissue. Conclusion the expression of 1.CD147 in the different parts of GEP-NENs is different from that of.2.CD147 in gastrointestinal NENs. There is a significant positive correlation between the level of expression and histological grade. The high level of expression may indicate that the differentiation of tumor and the high degree of malignancy may be an indicator of the severity of NENs in the gastrointestinal tract. MCT-1 and MCT-4 are negative in the expression of.4.MCT-1 in chronic pancreatitis and in the pancreatic islets of pNENs, and the expression level of MCT-4 is larger than that of the tumor. Small, histological grade, pTNM staging showed significant positive correlation, Pax-6, Nkx6.1, PDX1 expression level was negatively correlated with tumor size, histological grade and pTNM staging. The high expression level may indicate the good differentiation of tumor. The expression level of CD147 and MCT-1 in.5.pNENs with low degree of malignancy is positively correlated with the expression of PDX1, Nkx6.1, Pax-6. MCT-1, PDX1, Nkx6.1, Pax-6 may be involved in the role of CD147 in pNENs..6. can detect 4 CD147 gene variants associated with clinicopathological features in pNENs tissue, rs2229664CG, 19:582775AG, rs8259TA, and may have certain clinical significance, and further analysis and verification are needed.
【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】：2017
【分類號】：R735

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2 爾麗綿;IMP3對胃腸胰神經(jīng)內(nèi)分泌腫瘤增殖侵襲影響及機(jī)制研究[D];河北醫(yī)科大學(xué);2016年

3 周波;胰腺神經(jīng)內(nèi)分泌腫瘤的臨床病理特征及預(yù)后分析的研究[D];浙江大學(xué);2016年

4 邱偉;針對多發(fā)性內(nèi)分泌腫瘤-1型中的胰腺神經(jīng)內(nèi)分泌腫瘤的系統(tǒng)臨床研究[D];吉林大學(xué);2017年

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6 徐瑩;胰腺、直腸神經(jīng)內(nèi)分泌腫瘤臨床特征及預(yù)后分析[D];上海交通大學(xué);2014年

7 李e，

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