CD206、CD68基因单核苷酸多态性及其在巨噬细胞中的表达与肝细胞肝癌发病及预后的关联研究

发布时间：2018-05-11 16:23

本文选题：肝细胞肝癌 + 遗传易感性　；参考：《安徽医科大学》2017年博士论文

【摘要】：背景肝细胞肝癌(hepatocellular carcinoma,HCC)是世界上第五大致命的恶性肿瘤,恶性程度高、进展快、预后差,社会负担沉重。亚洲是HCC重灾区,而我国HCC的发病率和死亡率也呈上升趋势。尽管目前HCC基础和临床研究取得诸多成果,外科手术、介入治疗、射频消融等治疗方法使很多患者受益,但是HCC起病隐匿、早期症状不明显,晚期HCC十分常见且呈增加趋势,这些患者多伴远处转移,预后极差。同时,手术后复发也是HCC治疗的巨大挑战。因此,寻找更具潜力的生物标志物和治疗靶标将有助于HCC早期诊断和有效治疗。HCC是炎症相关性癌症的典型代表之一,肝脏中慢性炎症状态(以乙型肝炎病毒(Hepatitis virus B,HBV)感染常见)通过持续表达细胞因子和募集免疫细胞到肝脏,从而在肿瘤发生与发展中发挥作用。众所周知,遗传因素在HCC发生与发展中发挥重要作用,其中单核苷酸多态性(single nucleotide polymorphism,SNP)是基因多态性的主要形式之一,在人群中频率大于1%。SNP可以很好反映不同个体间的遗传差异,还可能调控基因表达,进而影响不同个体对某种疾病的易感性。SNP已被广泛应用于人类各种疾病的研究中,其中肿瘤相关SNP在个体对癌症易感性中扮演重要角色。巨噬细胞是肿瘤中浸润最多的免疫细胞,肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)是实体肿瘤中免疫浸润物的主要成分。TAMs在肿瘤发生、发展及预后中发挥的作用已被广泛研究。巨噬细胞一般被分为经典活化型(M1型巨噬细胞)和替代活化型(M2型巨噬细胞),一般认为TAMs属于M2型巨噬细胞,具有类似M2的标志物及促癌功能;但也有研究表明TAMs可以从M2样巨噬细胞向M1样细胞逆转,具有可逆性。TAMs这种复杂的生物学特点给临床研究和实践带来巨大挑战。然而,目前TAMs在肝癌发生发展中的角色仍未被完全阐明。CD68是重要的巨噬细胞特异性膜蛋白,也是泛巨噬细胞可靠的标志物。除了多种组织中定居的巨噬细胞,CD68还表达于肿瘤浸润巨噬细胞。CD206,又称巨噬细胞甘露糖受体(macrophage mannose receptor,MMR)或甘露糖受体C1型(MRC1)是一种具有噬菌和内吞作用的模式识别受体,主要表达于大部分巨噬细胞和树突细胞亚群,通常被用来鉴定M2型巨噬细胞。目前,CD68和CD206在人类疾病中的结构与功能已被广泛关注,但是有关CD68和CD206的基因多态性与人类HCC的易感性及预后是否存在关联、两者在HCC患者血清中表达水平尚未见文献报道。本研究从DNA、组织学、血清学三个不同层面比较系统研究了CD68和CD206在HCC中的表达及意义。目的本课题分为三个部分进行研究。第一部分:探究CD68和CD206三个位点(CD68 rs9901675、CD68rs9901673、CD206 rs941)多态性及其与HCC发病风险及患者预后的关联性,以期为HCC发病机制的研究提供新的切入点。第二部分:探讨CD68阳性巨噬细胞和CD206阳性巨噬细胞的数量和分布在HCC癌组织和癌旁组织中有无差异,评估其预后价值。第三部分:初步探讨HCC患者和健康对照者血清中可溶性CD68(a soluble form of s CD68,s CD68)和可溶性CD206(a soluble form of s CD206,s CD206)表达水平及其对HCC的诊断及预后意义,为HCC个体化临床诊治和长期随访管理提供有效的理论依据。方法第一部分:采用病例对照研究,连续收集符合条件的原发性HCC患者311例和健康体检者415名,采集研究对象的一般情况、临床及预后生存资料,同时收集全血标本、提取基因组脱氧核糖核酸(deoxyribonucleic acid,DNA),检测CD68和CD206基因三个位点的多态性,分析其与HCC发病风险、临床表型以及预后的关系。第二部分:选择268例行肝切除手术HCC患者进行回顾性研究。使用免疫组化方法检测HCC癌组织和癌旁组织中CD68阳性巨噬细胞和CD206阳性巨噬细胞,分析不同部位表达差异;采用受试者工作特征曲线(receiver operating characteristics curve ROC)评价CD68阳性和CD206阳性两种巨噬细胞对HCC的诊断价值;评估两者与HCC患者总体生存期和无瘤生存期的关系以及两者联合检测对HCC预后价值。第三部分:从第一部分研究样本中选取HCC术前患者123例和HCC术后患者60例、健康体检中心健康体检者60名,采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测血清中s CD68和s CD206水平,分析两者变化规律及其与HCC临床病理特征之间的关系,初步探讨两者对HCC的诊断价值及预后意义。结果第一部分:311例HCC患者和415名健康对照的SNP分析结果显示,CD68rs9901675多态性与HCC遗传易感性有关(OR=0.409,95%可信区间(confidence interval,CI)为0.206~0.812,P=0.009),该位点三种基因型GG、GA和AA的频率在HCC病例组为96.46%、3.54%和0%,对照组为92.53%、6.51%和0.96%,经调整性别年龄后差异具有统计学意义(P=0.019);但尚未发现CD68 rs9901673、CD206 rs941两个SNP位点与HCC易感性存在关联(P均0.05)。生存分析结果显示,CD68 rs9901675是HCC术后复发的独立预测因子[HR(95%CI)=8.114(1.103~59.675),P=0.040],但是该位点多态性与HCC主要临床特征之间无显著相关性(P0.05)。第二部分:(1)免疫组化检测结果显示:(1)CD68阳性和CD206阳性两种巨噬细胞在HCC癌旁组织中的表达量均显著高于癌组织(P均0.05);(2)不同年龄、不同血清甲胎蛋白(alpha fetoprotein,AFP)水平、是否伴有门脉高压的HCC患者癌组织中CD68阳性巨噬细胞表达量不同(P均0.05);而是否伴门脉高压或HBV阳性、不同AFP和肿瘤直径的HCC患者癌旁组织的CD68阳性巨噬细胞的浸润密度亦不同(P均0.05);(3)CD206阳性巨噬细胞在不同性别、Child-Pugh分级患者的癌组织中表达水平不同(P均0.05),而是否伴有肝硬化、不同血清总胆红素和TNM(tumor-nodes-metastasis)分期的患者癌旁组织中表达强度亦不同(P均0.05)。(2)ROC曲线结果显示,癌内CD68诊断临界值(cut-off value)设定为71个/每高倍视野(high-power fields,HPF),曲线下面积(area under curve,AUC)为0.614,95%CI为0.547~0.678,敏感性52.2%,特异性70.7%,对HCC诊断有意义(P=0.003);癌旁CD68最佳临界值为87个/每高倍视野(high-power fields,HPF),AUC为0.599,95%CI为0.534~0.661,敏感性49.5%%,特异性70.5%,对HCC诊断有意义(P=0.008)。(3)Kaplan-Meier法比较生存曲线显示,癌内组织高表达CD206的HCC患者预后差,总体生存期(P=0.014)和无瘤生存期(P=0.003)均短于低表达组患者;而癌旁组织高表达CD68的HCC患者总体生存时间(P=0.020)和无瘤生存时间(P=0.018)则更长。Cox回归分析结果显示,癌旁高表达的CD68阳性巨噬细胞是HCC患者总体预后[HR(95%CI)=0.510(0.311~0.837),P=0.008]和术后复发[HR(95%CI)=0.529(0.299~0.935),P=0.028]的保护性独立预后因子;而癌内高表达的CD206阳性巨噬细胞则是HCC患者预后的独立危险因素:总体生存期[HR(95%CI)=1.792,(1.116~2.879),P=0.016]和无瘤生存期[HR(95%CI)=2.222(1.280~3.856),P=0.005]。(4)联合分析癌旁CD68阳性肿瘤巨噬细胞和HBV的结果显示,癌旁CD68阳性巨噬细胞或癌内CD206阳性巨噬细胞与HBV联合分析,HCC患者的无瘤生存期差异均具有统计学意义(P均0.05),但是联合分析CD68阳性巨噬细胞与HBV对总生存期无明显影响(P=0.102)。第三部分:(1)HCC患者手术前和手术后血清s CD68和s CD206水平均明显高于正常对照组(P均0.05),但是术前与术后血清s CD68和s CD206水平变化不明显,差异无统计学意义(P均0.05)。同时,术前与术后s CD68/s CD206、术前s CD68与s CD206、术后s CD68和s CD206均存在相关性(P均0.05)。(2)进一步分析发现,两者与HCC重要临床特征(HBV、肝硬化、门脉高压和血清AFP水平)存在密切联系(P均0.05)。(3)CD68 rs9901675、CD68 rs9901673、CD206 rs941三个位点的不同基因型的HCC患者血清中s CD68/s CD206水平均无明显差异(P均0.05)。(4)ROC曲线分析结果表明,当术前血清s CD206为20.52ng/ml时,可获得最大的曲线下面积,术前血清s CD206具有诊断HCC价值(P=0.032),而术前血清s CD68则无统计学意义(P=0.174)。(5)生存分析结果显示,术后血清高表达s CD68、伴有门静脉癌栓、术前白蛋白降低、碱性磷酸酶升高、总胆红素升高、伴有淋巴结转移或远处转移的HCC患者总体生存时间较短,差异均具有统计学意义(P均0.05),进一步Cox回归分析结果显示,术后血清高表达s CD68是独立预后危险因素[HR(95%CI)=8.059(1.007~64.487),P=0.049]。而术前s CD68和s CD206以及术后s CD206对HCC患者预后未见统计学意义(P均0.05)。结论1.CD68 rs9901675多态性与HCC患病遗传易感性有关,该位点G等位基因和GG基因型可能是HCC发病风险因素,也是HCC术后复发的独立危险因子。2.定量分析HCC组织中CD68阳性和CD206阳性巨噬细胞可以为HCC患者术后分层管理提供线索,为再驯化HCC微环境中巨噬细胞提供新思路新方法。癌旁高表达的CD68是HCC患者预后独立保护因子,而癌内高表达的CD206是HCC患者预后独立危险因素。联合分析CD68或CD206阳性肿瘤巨噬细胞与HBV感染可能进一步提高HCC术后复发的预测能力。3.检测HCC患者血清s CD68和s CD206水平有助于观察免疫状态,术前血清s CD206水平可作为HCC患者早期诊断潜在标志物,而术后血清s CD68可为HCC术后患者总体生存期提供预测,对HCC患者的临床诊断和预后评估具有重要临床价值。
[Abstract]:Hepatocellular carcinoma (HCC) is the fifth most fatal malignant tumor in the world, with high malignancy, rapid progress, poor prognosis and heavy social burden. Asia is the hit area of HCC, and the incidence and mortality of HCC in China are also on the rise. Although many achievements have been made in the basic and clinical Research of HCC, surgical operations, Treatment, radiofrequency ablation and other treatment methods benefit many patients, but HCC is insidious and early symptoms are not obvious. Late HCC is very common and tends to increase. These patients are often accompanied by distant metastasis, and the prognosis is extremely poor. At the same time, recurrence is also a huge challenge for HCC treatment. Therefore, to find more potential biomarkers and target targets for treatment. It will help HCC early diagnosis and effective treatment of.HCC is one of the typical cases of inflammation related cancer. Chronic inflammatory state in the liver (hepatitis B virus (Hepatitis virus B, HBV) infection is common) through continuous expression of cytokines and recruitment of immune cells to the liver. It is known to play a role in the development and development of cancer. Genetic factors play an important role in the development and development of HCC, in which single nucleotide polymorphisms (single nucleotide polymorphism, SNP) are one of the main forms of genetic polymorphism. The frequency of more than 1%.SNP in the population can reflect the genetic differences among different individuals, and can also regulate gene expression, and then affect a certain number of individuals. The susceptibility to disease.SNP has been widely used in the study of various human diseases, in which tumor related SNP plays an important role in the susceptibility of cancer. Macrophages are the most infiltrating immune cells in tumor, and tumor related macrophages (tumor-associated macrophages, TAMs) are the main immune infiltrates in solid tumors. The role of component.TAMs in the occurrence, development and prognosis of tumor has been widely studied. Macrophages are generally divided into classical activated type (M1 type macrophages) and replacement activation type (M2 type macrophages). Generally, TAMs is considered as a M2 type macrophage, which has a marker of M2 like and a cancer promoting function; but there are also studies that TAMs can be derived from M2 like The reversal of macrophages to M1 like cells, which has the complex biological characteristics of reversible.TAMs, brings great challenges to clinical research and practice. However, the role of TAMs in the development of liver cancer is still not fully elucidated as an important macrophage specific membrane protein and a reliable marker of Pan macrophage. The colonization of macrophages, CD68 also expressed in tumor infiltrating macrophage.CD206, also known as macrophage mannose receptor (macrophage mannose receptor, MMR), or mannose receptor C1 type (MRC1), is a pattern recognition receptor with phagocytic and endocytosis, mainly expressed in most macrophages and dendritic cell subgroups, usually in the form of a subpopulation of macrophages and dendritic cells. It is used to identify M2 type macrophages. Currently, the structure and function of CD68 and CD206 in human diseases have been widely concerned, but there is a correlation between the genetic polymorphisms of CD68 and CD206 and the susceptibility and prognosis of human HCC. The level of expression in the serum of HCC patients has not yet been reported. This study from DNA, histology, serology three The expression and significance of CD68 and CD206 in HCC were studied at different levels. The purpose of this topic was divided into three parts. The first part was to explore the polymorphism of CD68 and CD206 (CD68 rs9901675, CD68rs9901673, CD206 rs941) and its association with HCC risk and prognosis in order to study the pathogenesis of HCC. The second part is to explore the number and distribution of CD68 positive macrophages and CD206 positive macrophages in the HCC and para cancerous tissues, and to evaluate their prognostic value. The third part: the preliminary study of the soluble CD68 in the serum of HCC and healthy controls (a soluble form of s CD68, s) and soluble solids 206 (a soluble form of s CD206, s CD206) expression level and its significance for the diagnosis and prognosis of HCC, providing an effective theoretical basis for the individualized clinical diagnosis and treatment of HCC and long-term follow-up management. Method first part: a case control study was used to collect 311 cases of primary HCC patients and 415 healthy physical examination persons. The general situation, the clinical and prognosis survival data, collection of whole blood samples, deoxyribonucleic acid, DNA, detection of the polymorphism of three loci of CD68 and CD206 gene, analysis of the relationship with the risk of HCC, the clinical phenotype and the prognosis. The second part: select 268 cases of hepatectomy for HCC patients. An immunohistochemical method was used to detect CD68 positive macrophages and CD206 positive macrophages in HCC carcinoma tissue and para cancerous tissue, and the difference of expression in different parts was analyzed. The diagnosis of CD68 positive and CD206 positive two macrophages on HCC was evaluated by the working characteristic curve of receiver operating characteristics curve ROC. The relationship between the total survival time and the tumor free life period of HCC patients and the value of the combined detection of HCC were evaluated. The third part: from the first part of the study, 123 cases of HCC patients and 60 patients after HCC, 60 healthy physical examination centers in the health check-up center were selected, and the enzyme linked immunosorbent assay (enzyme linked IM) was used. Munosorbent assay, ELISA) detected the level of s CD68 and s CD206 in serum, analyzed the relationship between the two changes and the clinicopathological features of HCC, and preliminarily discussed the diagnostic value and prognostic significance of both of them. Results the first part: the SNP analysis of 311 cases of HCC and 415 healthy controls showed that CD68rs9901675 polymorphism was associated with the remains. The OR=0.409,95% confidence interval (confidence interval, CI) was 0.206~0.812, P=0.009). The frequency of three genotypes of GG, GA and AA in the loci were 96.46%, 3.54% and 0% in HCC cases, and 92.53%, 6.51% and 0.96% in the control group. The difference was statistically significant after the adjustment of sex age (P=0.019). The two SNP loci of rs941 were associated with HCC susceptibility (P 0.05). Survival analysis showed that CD68 rs9901675 was an independent predictor of recurrence of HCC, [HR (95%CI) =8.114 (1.103~59.675), P=0.040], but there was no significant correlation between the polymorphism of the loci and the main clinical features. (1) (1) immunohistochemical detection results. (1) the expression of CD68 positive and CD206 positive macrophages in HCC para cancer tissues was significantly higher than that of cancer tissue (P 0.05); (2) the levels of alpha fetoprotein (AFP) in different ages, different serum alpha fetoprotein (alpha fetoprotein, AFP), and the expression of CD68 positive macrophages in the cancerous tissues of HCC patients with portal hypertension (P 0.05) (P 0.05); The infiltration density of CD68 positive macrophages in the paracancerous tissues of HCC patients with different AFP and tumor diameter was also different (P 0.05). (3) the expression level of CD206 positive macrophages in different sex and Child-Pugh grading patients was different (P 0.05), and whether there were cirrhosis, different serum total bilirubin and TNM (TU) (TU 0.05). Mor-nodes-metastasis) the expression intensity of the para cancerous tissue in the staging patients was also different (P 0.05). (2) the ROC curve showed that the critical value of CD68 (cut-off value) was set to 71 / each high field (high-power fields, HPF), and the area under the curve (area under curve) was 52.2%, and the sensitivity was 70.7%, The diagnosis of HCC was significant (P=0.003); the best critical value for CD68 was 87 / per high fold field of vision (high-power fields, HPF), AUC was 0.534~0.661, sensitivity 49.5%%, specificity 70.5%, significance for HCC diagnosis (P=0.008). (3) the prognosis of patients with high expression in cancer tissues was poor, and the overall survival rate was poor. The survival time (P=0.014) and the tumor free survival (P=0.003) were shorter than those in the low expression group, while the overall survival time (P=0.020) and the tumor free survival time (P=0.018) of the HCC patients with high expression of CD68 in the para cancer tissues were longer.Cox regression analysis, and the results showed that the high expression of CD68 positive macrophages beside the cancer was the overall prognosis of HCC patients [HR (95%CI) =0.510. P=0.008] and postoperative recurrence of [HR (95%CI) =0.529 (0.299~0.935) and P=0.028] were independent prognostic factors, while the high expression of CD206 positive macrophages in cancer were independent risk factors for the prognosis of HCC patients: the overall survival period [HR (95%CI) =1.792, (1.116~2.879), and tumor free survival period (4) union Analysis of the results of macrophages and HBV of the paracancerous CD68 positive tumor showed that the combination of CD68 positive macrophages or CD206 positive macrophages adjacent to the cancer was analyzed with HBV, and the difference of the tumor free survival time of HCC patients was statistically significant (P 0.05), but the combined analysis of CD68 positive macrophages and HBV had no significant effect on the total survival time (P=0.102). The three part: (1) the levels of serum s CD68 and s CD206 in patients with HCC before and after operation were significantly higher than those in normal control group (P 0.05), but there was no significant difference between preoperative and postoperative serum s CD68 and s CD206 levels, and the difference was not statistically significant (P 0.05). There was a correlation (P 0.05). (2) further analysis found that the two were closely related to the important clinical features of HCC (HBV, cirrhosis, portal hypertension and serum AFP level) (P 0.05). (3) there was no significant difference in the levels of CD68 rs9901675, CD68 rs9901673 and CD206 rs941 three loci. (4) the results of ROC curve analysis showed that the maximum subcurvilinear area was obtained when the serum s CD206 was 20.52ng/ml before operation. The preoperative serum s CD206 had the diagnostic value of HCC (P=0.032), while the preoperative serum s CD68 was not statistically significant (P=0.174). (5) the survival analysis showed that the postoperative serum high expression s, with the portal vein tumor thrombus, and the pre operation white eggs The total bilirubin increased, the total bilirubin increased, the total survival time of HCC patients with lymph node metastasis or distant metastasis was shorter, and the difference was statistically significant (P 0.05). Further Cox regression analysis showed that high expression of s CD68 after operation was [HR (95%CI) =8.059 (1.007~64.487), P=0.049]., the risk factor for independent prognosis. The prognosis of preoperative s CD68, s CD206 and postoperative s CD206 was not statistically significant (P 0.05). Conclusion 1.CD68 rs9901675 polymorphism is related to the genetic susceptibility to HCC disease, and the allele and genotype of this loci may be the risk factors for the onset of recurrence. Sex and CD206 positive macrophages can provide clues for postoperatively stratified management of HCC patients and provide a new way to train macrophages in HCC microenvironment. High expressed CD68 is an independent prognostic factor for HCC patients, and the high expression of CD206 in cancer is an independent risk factor for prognosis in HCC patients. Combined analysis of CD68 or CD206 positive swelling is combined. The tumor macrophage and HBV infection may further improve the predictive ability of recurrence after HCC.3. detection of the serum s CD68 and s CD206 levels in HCC patients can help to observe the immune state. The serum s CD206 level can be used as a potential marker for the early diagnosis of HCC patients. The clinical diagnosis and prognosis evaluation of patients has important clinical value.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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