【作者】 吴彬；

【导师】 樊嘉； 周俭； 徐泱； 代智；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【摘要】 肝癌是国内常见的恶性肿瘤之一,其侵袭转移与复发是患者死亡的主要原因之一。因此,探索肝癌侵袭转移与复发的发生机制,寻求有效的抗肝癌转移与复发治疗措施,对改善肝癌患者的预后具有重要意义。研究依据：CD88又称补体5片段受体(complement component 5a receptor, C5aR),是一种在细胞膜表面的G蛋白偶联受体(GPCR),其在体内主要分布在髓来源细胞系,参与一系列炎症反应过程,如趋化作用,促进多种细胞的释放反应(如前列腺素、5-HT、组胺、IL-1、IL-6等),增加血管通透性等。近来研究发现CD88在肝脏再生实质细胞中表达,并且在肝损伤后的肝细胞再生修复过程中发挥重要作用。CD88还参与了细胞抗失巢凋亡、新生血管生成等。CD88与其配体C5a结合后可激活下游的G蛋白、WASP、β-arresting等信号分子,引起PI3K/AKT、Ras/Raf/MEK/ERK和c-Src/JAK等信号通路反应,可激活CREB、NF-κB、STAT3等核转录因子。近来研究发现CD88在乳腺癌、肺癌、脑胶质细胞瘤组织中表达较正常组织明显升高。Markiewski等报道在小鼠子宫颈癌模型中,通过补体5敲除或CD88拮抗剂应用后小鼠子宫颈癌肿瘤的增殖明显受到抑制,其机制可能与微环境中髓来源抑制细胞(myeloid-derived suppressor cells, MDSC)增加并抑制肿瘤微环境中CD8+T细胞的数量和功能。在我所前期实验中发现Micro-RNA26表达较低的肝癌患者预后较高表达的患者预后差,通过对两者基因芯片扫描结果显示低表达Micro-RNA26的癌组织较高表达Micro-RNA26的癌组织中CD88表达明显升高,这提示低表达Micro-RNA26可能通过上调CD88表达的同时上调IL-6、NF-κB促进肿瘤发展。但迄今为止,它对肝癌的影响尚未有明确报道。本实验首先在肝癌、相应癌旁组织以及不同转移潜能肝癌细胞系中检测CD88表达差异；研究其与肝癌发生、发展以及预后的相关性；通过转染与干扰调节CD88表达后,探讨CD88与肝癌细胞侵袭与转移的关系。目的：研究CD88在肝癌细胞株及肝癌、相应癌旁组织中的表达差异,初步分析其与肝癌发生的关系。方法：应用荧光定量PCR、免疫组织化学及Western Blot方法检测CD88在56例肝癌、相应癌旁组织中的表达。应用荧光定量PCR、Western Blot及免疫荧光检测不同转移潜能肝癌细胞的CD88表达情况。结果：荧光定量PCR显示CD88在肝癌、相应癌旁中的相对表达量分别为0.056±0.014vs0.003±0.0004,肝癌与癌旁两者间差异明显(p<0.001)。Western Blot结果同样显示肝癌组织CD88表达高于癌旁组织(3.71±0.92vs.0.91±0.34,p<0.01)；在肝癌组织中早期复发组CD88表达要明显高于非早期复发组(4.43±0.48vs.1.64±0.28,p<0.05)。免疫组织化学显示两者都有CD88表达,但肝癌组织中的表达明显高于后者(p<0.05)。在肝癌细胞株中高转移潜能细胞HCCLM3和MHCC-97H的CD88mRNA和蛋白表达明显高于低转移潜能细胞MHCC-97L和HepG2。结论：CD88在肝癌发生发展中可能有重要作用。目的：研究CD88在原发性肝癌中的表达情况,探讨CD88在肝癌组织中表达的相关性、与原发性肝癌的关系及临床生物学意义。方法：组织芯片检测403例肝癌组织中CD88的表达,统计分析其与肝癌临床病理特征及预后的关系。结果：CD88蛋白的过表达与肝癌患者的血浆高AFP水平、肿瘤直径大于5cm、有肉眼癌栓、高BCLA临床分期和高CLIP分期等临床病理因素有明显关联(p<0.05)。CD88表达阳性组的7年总体生存率及无病生存率明显低于CD88阴性组(p<0.05),分层Kaplan-Meier分析显示在高AFP组、单个肿瘤组、高肿瘤分化组、无微血管侵犯组中肝癌组织中CD88过表达患者OS和DFS均较低表达组差。多因素分析表明血清GGT>54U/L、血清AFP>20ng/ml、有肝硬化、肿瘤直径>5cm、多个肿瘤、有微血管癌栓及CD88表达阳性是影响OS的独立预后因素；血清GGT>54U/L、血清AFP>20ng/ml、有肝硬化、多个肿瘤、有微血管癌栓及CD88表达阳性等为DFS的独立预后因素。结论：CD88与原发性肝癌的侵袭、转移及不良预后相关。目的：研究CD88与肝癌细胞侵袭与转移的关系。方法：转染pReceiver-M55-CD88 cDNA和GPHI/GFP/Neo-CD88-shRNA,筛选出稳定细胞株,经western blot, qRT-PCR验证CD88表达改变后,CSa刺激试验研究基质金属蛋白酶(MMP2、9)及血管内皮生长因子(VEGF) mRNA变化；MTT检测CD88表达改变后,细胞增殖能力变化；Transwell研究细胞迁移侵袭能力变化。细胞接种于裸鼠肝原位,研究CD88改变后,细胞成瘤与肺转移能力的改变;免疫组化检测移植瘤的CD88、MMP9和VEGF表达。结果：转染pReceiver-M55-CD88 cDNA和GPHI/GFP/Neo-CD88-shRNA于低转移能力的HepG2与高转移的HCCLM3,G418筛选出稳定细胞株,Western blot和qRT-PCR验证CD88表达明显上调或下调；Transwell实验结果显示上调或下调CD88表达后的肝癌细胞的迁移和侵袭能力明显增加或减少；rC5a刺激后细胞mRNA研究显示CD88的表达与MMP2,MMP9,VEGF的分泌呈正相关。CD88表达水平对肝癌细胞增殖有一定影响。高表达CD88的肝癌细胞体内成瘤能力较低表达肝癌细胞明显上升,高CD88表达组细胞的肺转移灶明显增加,且以Ⅲ、Ⅳ期为主；下调HCCLM3的CD88后较对照组MMP9和VEGF明显表达减少。结论：肝癌细胞CD88表达上调或下调能促进或降低肝癌的侵袭与转移能力。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common cancers in China in terms of number of cases, and the invasion and metastasis are the leading cause for the HCC death, so it is of great clinical importance to investigate the mechanism of invasion and metastasis, and the effectual therapeutic way of anti-metastasis and relapse of HCC.Previous study has showed that CD88, also known as complement component 5a receptor, is a kind of cell surface G protein-coupled receptor (GPCR). CD88 is mainly expressed in the surface of marrow-derived cells, are mainly distributed in line to participate in a series of inflammatory reactions, such as chemotaxis, promote multi-kinds of cells, the release reaction (such as prostaglandins,5-HT, histamine, IL-1, IL-6, etc.), increased vascular permeability and so on. Recently, research showed that CD88 is also expressed in the liver parenchymal cells and play an important role in the course of liver regeneration after partial liver resection. Recent it was found that CD88 mRNA expression in the tissue of primary glioblastomas was significantly higher than in normal brain tissue. Markiewski et al. reported that in the mouse cervical cancer model, knock-out the expression of CD88 in the mouse can reduce the tumor proliferation by increase the MDS cells infiltration and inhibit the number and function of intra-tumor CD8+ T cell. It has not yet been clearly reported the relationship of expression of CD88with hepatocellular carcinoma.In the present study, we investigated the expression of CD88 in HCC cell lines with different metastatic potential, and HCC and their adjacent nontumorous tissues, and then explored the changes of invasive and metastatic ability in non-metastatic cell line HepG2 through CD88 cDNA transfection and high metastatic cell line HCCLM3 through silencing CD88 in vivo and vitro. We assayed the clinical significance of CD88 in HCC samples using tissue microarrays (TMAs) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Objective: To investigate the relationship between the expression of CD88 and the carcinogenesis of HCC.Methods:The expression of CD88 was evaluated in HCC and their adjacent nontumorous by qRT-PCR, western blot and immunohistochemistry. qRT-PCR and western blot was used to detect the expression of CD88 in different HCC cell lines with different metastatic potential.Result: CD88 mRNA can be detected in HCC and their adjacent nontumorous tissue, the CD88 mRNA level was significantly higher in HCC than those in their adjacent nontumorous tissues (0.056±0.014 vs.0.003±0.0004,p＜0.001),. CD88 protein level detected by western blot was significantly higher in HCC than those in their adjacent nontumorous tissues (3.71±0.92 vs.0.91±0.34, P<0.01). The CD88 protein was located mainly in the cellular membrane of HCC cell, and partly in the cytoplasm. The level of CD88 protein and protein in HCCLM3 and MHCC-97H with high metastasis potential was higher than in MHCC-97L and HepG2 with low metastasis potential.Conclusion:The overexpression of CD88 may be related to the carcinogenesis of HCC.Objective: To investigate the correlation and clinical significance of CD88 with hepatocellular carcinoma.Methods:The TMA was used to detect the expression of CD88 in 403 HCC patients, then the clinical significance of overexpression of CD88 was analyzed by SPSS 15.Result: Overexpression CD88 accounted of 41.7%(168/403) of total patients. Overexpression CD88 was found to correlate significantly with high serum AFP level, tumor diameter greater than 5cm, portal vein tumor thrombus(PVTT), high BCLA clinical stage and high-CLIP staging (p<0.05).7-year overall survival and disease-free survival rate in the group of CD88-positive was significantly lower than that in the group of CD88-negative group (p<0.05). Multivariate analysis showed that serum GGT>54U/L, serum AFP>20ng/ml, cirrhosis, tumor diameter>5cm, multiple tumors, there is micro-vascular thrombosis and CD88 positive expression were independent prognostic factors for OS; serum GGT>54U/L, serum AFP>20ng/ml, cirrhosis, multiple tumors, there is micro-vascular thrombosis and positive expression of CD88 were independent prognostic factors for DFS.Conclusion:The overexpression of CD88 can be a new marker in predicting the prognosis of HCC.Objective: To investigate the relationship between the expression of CD88 and the invasion and metastasis of HCC cells.Method:We transfected the pReceiver-M55-CD88 cDNA and GPHI/GFP/Neo-CD88-shRNA plasmid into the non-metastatic cell line HepG2 and the highly metastatic cell line HCCLM3, the expression of CD88 was determined by qRT-PCR, western blot in the stable transfection HCC cell lines. MMP2,9 and VEGF expression change were detected by qRT-PCR after stimulated by recombination C5a in transfected HCC cell lines previously, and the ability of proliferation and invasion was tested by MTT and transwell in the the stable transfection HCC cell and their parental cell lines, respectively. At the last, HCC cell lines were implanted in the liver of nude mice. Metastasis assays in vivo was performed, and CD88, MMP9 and VEGF expression in the tumor were detected by immunohistochemistry.Result: Stable cell line HepG2 and HCCLM3 cells transfected by pReceiver-M55-CD88 cDNA and GPHI/GFP/Neo-CD88-shRNA plasmid respectively, were selected by G418. HCC cell lines with high CD88 expression increase higher mRNA expression of MMP2, MMP9, VEGF in the cell lines rather than the cell lines with low CD88 expression stimulated by recombination human C5a. Cell proliferation was lightly influenced by CD88 expression levels in HCC cell lines; high CD88 expression enhanced capability of cell movement and invasion in vitro, and remarkably increased lung metastases in vivo, especially in stageⅢandⅣ.Conclusion:The CD88 expression in HCC cell lines was positive correlation with the ability of HCC cell invasion and metastasis.更多还原