【作者】 周莹；

【导师】 张博恒；

【作者基本信息】 复旦大学 ， 肿瘤学， 2012， 硕士

【摘要】 背景与目的：肝细胞肝癌是世界上最常见的恶性肿瘤之一,在我国居恶性肿瘤死亡率第二位。转移一直是影响肝癌预后的重要因素。肝癌细胞获得侵袭转移能力,与其所在的肿瘤微环境是密切相关的。缺氧是实体肿瘤发展过程中的常见现象之一。研究认为缺氧微环境可以诱导肿瘤细胞发生上皮间质转化、生成新生血管和促进肿瘤侵袭和转移。趋化因子是一族分子量为8-14kDa大小的趋化作用的细胞因子,通过与其相应受体结合,发挥调节白细胞趋化到炎症部位的作用。近来不断有研究证实趋化因子及其受体在肿瘤的生长,侵袭,血管生成和转移中起到重要的作用。已有研究报道CCL2和CCR2与肿瘤转移有关,但是其他趋化因子在肿瘤发展中的作用仍知之甚少。本研究着重于探索CCL28在缺氧诱导的人肝癌转移过程中作用。方法：应用实时定量PCR检测50例肝细胞癌组织中CCL28和HIF-1α mRNA表达情况并分析两者表达的相关性,运用卡方检验,Kaplan-Meier分析HIF-1α及CCL28表达与年龄、性别、乙肝病毒标记物、肝硬化、分化、包膜、甲胎蛋白等临床病例特征及患者总体生存时间的关系；同时在体外细胞实验中,采用实时定量PCR,Western blot与酶联免疫吸附实验(ELISA)检测缺氧对肝癌细胞株HepG2、HCCLM3HIF-la和CCL28表达的影响；运用RNA干扰(siRNA)和Lipofectamine2000细胞转染技术下调HIF-1α基因表达对肝细胞癌HCCLM3细胞CCL28蛋白表达的影响。通过抑制CCL28表达,结合Transwell和BDMatrigel观察CCL28在缺氧诱导的肝癌细胞侵袭迁移中的作用；采用明胶酶谱法测定CCL28-siRNA干扰后细胞上清的金属蛋白酶含量的差异。结果：1)实时定量PCR结果发现癌组织中HIF-1αmRNA含量0.0654±0.0988,29/50(58%)例呈高表达。CCL28mRNA水平在肝癌组织为0.0254±0.0755,23/50(46%)例呈高表达。Spearman双变量相关性分析显示肝细胞癌组织中CCL28与HIF-1αmRNA水平呈高度相关(r=0.595,p<0.001)；二者表达水平增高皆与术后复发相关。2)体外细胞实验显示肝癌HepG2和HCCLM3细胞HIF-1α和CCL28mRNA水平在缺氧(1%O2)培养6h后开始增加,二者的蛋白表达水平在缺氧培养12h后开始增高,并呈现出时间依赖性。利用HIF-1a特异性siRNA阻断HIF-1α的表达后,CCL28蛋白的表达水平则随着HIF-1α表达抑制而减少；3)利用siRNA抑制HCCLM3细胞内CCL28表达,经实时定量PCR和ELISA证实mRNA和蛋白水平明显降低。研究发现干扰HCCLM3细胞内CCL28表达后,缺氧条件下迁移和穿膜细胞数目较空白组减少,细胞MMP-2的分泌也减少。结论：1)HIF-1α和CCL28在肝癌组织中mRNA表达增高,且表达水平呈高度相关性。二者表达水平增高皆与术后复发相关。2)缺氧可通过HIF-1α上调肝癌细胞CCL28表达,且呈时间依赖性。3)下调CCL28导致缺氧诱导的肝癌细胞侵袭力减弱。4)CCL28在缺氧诱导的肝癌细胞侵袭转移中发挥作用,可能与MMP-2的分泌有关。更多还原

【Abstract】 Background and Aims:Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the second most common cause of death from cancer in China. Metastasis is the major cause for dismal prognosis of HCC. For HCC cells, the ability to form metastasis phenotype is associated with the tumor microenvironment. Hypoxia is a feature of most solid tumors, and it is a negative prognostic factor owing to its contributions to epithelial-mesenchymal transition, vasculogenesis, invasiveness and metastasis. Chemokine are a superfamily of chemotactic cytokines of molecular weight8-14kDa. They bind to their receptors and regulate leukocyte trafficking to inflammation sites. Recently a growing body of evidence indicates that chemokines and their receptors play a key role in tumor growth, invasion, angiogenesis and metastasis. Although CCL2and CCR2have been reported to be associated with cancer metastasis, the role of other chemokines in cancer is poorly understood. We aimed to explore the status of CCL28in hypoxia-induced cell migration.Methods:The expression of HIF-la and CCL28were detected in50cases of hepatocellular carcinoma samples using real time PCR. Kaplan-Meier method, log-rank test and chi-square test were used to analyse the relationship of clinical characteristics with prognosis of HCC. In vitro, we used real time PCR, Western blot, and ELISAto detect the levels of HIF-1α and CCL28in HepG2and HCCLM3under hypoxia (1%O2); using siRNA and lipofectamine2000to seal up HIF-1α gene expression and detected the level of CCL28expression in HepG2/HCCLM3under hypoxia; using Trans-well and BD Matrigel to observe cellular mobility and invasion ability, using gelatin zymography to detect MMPs activity after CCL28siRNA transfection.Results:1) Real-time PCR showed that mRNA level of HIF-1α in hepatocellular carcinoma was0.0654±0.0988,29of50(58%) showed a high level of HIF-1αa; CCL28mRNA level in hepatocellular carcinoma was0.0254±0.0755, and23of50(46%) showed a high expression level. Spearman analysis indicated that CCL28expression strongly correlated with HIF-1α in hepatocellular carcinoma(r=0.595, p<0.001); high expression of HIF-1α and CCL28were both related with recurrence after surgery.2) In vitro, when cells HepG2and HCCLM3were subjected to hypoxia (1%O2), the mRNA levels of HIF-la and CCL28started to increase after6h hypoxia while the protein levels had a significant increase after12h hypoxia in a time-dependent manner; When HIF-1α siRNA was used to down-regulate HIF-1α expression, the protein level of CCL28under hypoxia was significant decreased (p<0.001);3) We used siRNA to inhibit CCL28expression in HCCLM3cells, and the mRNA and protein levels of CCL28were decreased. The number of invasion cells under hypoxia was significantly decreased as compared with control, and the activity of MMP-2was significantly decreased.Conclusions:1) Both of HIF-la and CCL28were highly expressed in hepatocellular carcinoma tissue, and their expression had a strong relationship. High level of HIF-la and CCL28were both related with recurrence after surgery.2) Hypoxia induced high expression of CCL28by up-regulated HIF-1α in a time-dependent manner.3) CCL28expression knockdown using siRNA inhibited the invasiveness of hypoxia-induced HCC cells invasion.4) CCL28played an important role in hypoxia-induced liver cancer cell invasion, which might be mediated by MMP-2.更多还原