CD147在胃肠胰神经内分泌肿瘤中的表达模式及其影响机制初探
发布时间：2018-07-25 20:00
【摘要】：背景和研究目的胃肠胰神经内分泌肿瘤(GEP-NENs)是最常见的NENs类型,近年来其发病率呈现上升趋势。但其发生机制不清,且缺乏有效的诊断和治疗标志物。本课题组前期研究发现,CD147在正常胰岛中不表达,在绝大部分胰腺神经内分泌肿瘤(pNENs)中表达,且随着肿瘤恶性程度的增高,CD147的表达水平增高。提示CD147可能参与了 pNENs的肿瘤发生,且表现为促癌作用,但对CD147在胃肠NENs中的表达情况,在pNENs中的作用及其机制,以及在正常胰岛中不表达的分子机制尚需进一步研究。因此本研究主要包括以下几个部分:CD147抗体验证;检测CD147在胃肠NENs中的表达表达研究;检测CD147、MCT-1、MCT-4慢性胰腺炎中的表达,MCT-1、MCT-4、PDX1、nkx6.1、Pax-6在pNENs中的表达并分析其与CD147表达的相关性研究;pNENs中CD147基因的序列变异研究。方法1.构建CD147真核过表达载体,转染HEK293T细胞,提取蛋白行去糖基化处理后,CD147抗体和抗His抗体进行western blot验证。若CD147抗体和抗His(C端)抗体能得到同样分子量的融合蛋白目的条带,证明抗体识别的是CD147分子。2.采用免疫组化方法检测109例胃肠NENs中CD147的表达,检测36例慢性胰腺炎中 CD147、MCT-1、MCT-4 的表达,及 42 例 pNENs 中 MCT-1、MCT-4、PDX1、nkx6.1、Pax-6的表达,并与CD147表达进行相关性分析。3.选取29例pNENs患者的正常组织和肿瘤组织配对石蜡样本共60个,对CD147基因进行测序检测。结果第一部分CD147抗体验证:证实前期实验所用CD147单克隆抗体特异性识别地为CD147分子,抗体可用。第二部分CD147在胃肠NENs中的表达研究:1.CD147在胃、十二指肠、结直肠NENs中均有表达,免疫表型可表现为浆型、膜型、膜浆型,其阳性表达率分别为65.7%、76.5%、19.3%。2.CD147在胃肠NENs中的表达水平与组织学分级呈显著正相关,在胃NENs中CD147的免疫表型与其表达水平有关,表达水平越高,膜型的比例越高。第三部分CD147、MCT-1、MCT-4在慢性胰腺炎及pNENs瘤旁正常胰岛中表达的研究:CD147、MCT-1、MCT-4在36例慢性胰腺炎中的胰岛中均呈阴性表达。42例pNENs病例中,MCT-1、MCT-4在瘤旁正常腺泡和导管及正常胰岛中均呈阴性表达。第四部分MCT-1、MCT-4、PDX1、nkx6.1、Pax-6在pNENs中的表达及其与CD147 表达的相关性研究:1.MCT-1、MCT-4、Pax-6、nkx6.1、PDX1 在 pNENs中均呈阳性表达。2.MCT-1、MCT-4的表达水平与肿瘤的大小、组织学分级、pTNM分期呈显著正相关,而Pax-6、nkx6.1、PDX1的表达水平与肿瘤的大小、组织学分级、pTNM分期呈显著负相关。3.发生淋巴结转移或远处转移的肿瘤中的MCT-1、MCT-4表达水平要高于未发生淋巴结转移或远处转移的;而未发生局部浸润和/或淋巴结转移的肿瘤中Pax-6的表达水平比发生局部浸润和/或淋巴结转移的高,无远处转移的比有远处转移的肿瘤Pax-6、PDX1表达水平高。4.CD147与MCT-1的表达水平呈显著正相关,与MCT-4的表达水平无相关性,CD147与PDX1、nkx6.1、Pax-6的表达水平呈显著负相关。第五部分pNENs中CD147基因的序列变异研究:29例pNENs患者的60个组织样本中共检测出47个CD147基因变异位点。其中直径2cm的肿瘤中19:580388CG(rs2229664)CG 杂合基因型、19:582775AG GG 纯合基因型、19:582927TA(rs8259)AA纯合基因型型比"f2cm的多,差异有统计学意义。19:579627CT(rs1803202)在肿瘤组织或正常与肿瘤组织同时存在的T纯合基因型比C/T杂合基因型多,差异有统计学意义。19:582927TA在正常组织和肿瘤组织中的变异(AA基因型和A/T杂合基因型)在不同的肿瘤组织学分级间存在显著差异,组织学分级越高,肿瘤组织中存在的19:582927TA变异(AA基因型和A/T杂合基因型)越多。结论1.CD147在GEP-NENs不同部位中的表达有所差异。2.CD147在胃肠NENs中的表达水平与组织学分级呈显著正相关,其表达水平高可能提示肿瘤的分化差、恶性程度高,可能能作为评估胃肠NENs严重程度的一个指标。3.CD147、MCT-1、MCT-4在慢性胰腺炎和pNENs瘤旁胰岛中均呈阴性表达。4.MCT-1、MCT-4的表达水平与肿瘤的大小、组织学分级、pTNM分期呈显著正相关;Pax-6、nkx6.1、PDX1的表达水平与肿瘤的大小、组织学分级、pTNM分期呈显著负相关,其表达水平高可能能提示肿瘤的分化好、恶性程度低。5.pNENs中CD147与MCT-1的表达水平呈显著正相关,与PDX1、nkx6.1、Pax-6的表达水平呈显著负相关。提示MCT-1、PDX1、nkx6.1、Pax-6可能参与了 CD147在pNENs中的作用。6.在pNENs组织中共检测出4个与临床病理特征相关的CD147基因变异位点rs2229664CG、19:582775AG、rs8259TA、rs1803202CT,可能具有一定临床意义,尚需进一步分析验证。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735
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7 李e，
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