肝星状细胞在肝癌细胞恶性行为中的作用及机制研究

发布时间：2018-02-28 01:26

本文关键词： 肝细胞性肝癌肝星状细胞 Sorafenib 上皮间质转化　出处：《南京医科大学》2015年博士论文　论文类型：学位论文

【摘要】：背景:肝细胞性肝癌(Hepatocellular Carcinoma,HCC)的发生通常伴有慢性炎症与纤维化,肝星状细胞(Hepatic stellate cells,HSCs)在炎症与纤维化过程中起到重要作用并构成HCC的肿瘤微环境。目的:共培养HSCs与HCC细胞,评价共培养体系中HCC细胞对靶向药物Sorafenib的敏感性,并检测共培养情况下HCC细胞表型是否存在变化。进一步深入研究HSCs引起HCC细胞对Sorafenib敏感性变化以及迁移侵袭能力变化的机制。方法:应用MTT法检测共培养条件下HCC细胞对Sorafenib敏感性的变化,进一步通过ELISA、Western Blotting、q PCR等方法检测与HSCs共培养后HCC细胞内可能导致Sorafenib敏感性变化的信号通路改变,并使用相应靶点的抑制剂干扰共培养的作用进行验证。另一方面,Western Blotting、q PCR方法检测HCC细胞内EMT相关蛋白、基因变化,划痕愈合实验、Transwell检测HCC细胞迁移、侵袭能力,使用si RNA干扰可能引起EMT的靶点后检测HCC细胞的EMT相关蛋白与迁移、侵袭能力。联合HSCs建立裸鼠原位肿瘤模型,检测肺转移能力的变化。结果:首先,我们发现Sorafenib通过凋亡途径抑制HCC细胞增殖,使用HSCs与HCC细胞共培养后,HCC细胞对Sorafenib的敏感性明显降低并且凋亡相关蛋白Caspase3、9、PARP有所减少,说明共培养引起HCC细胞对Sorafenib的耐受。HSCs条件培养基中的HGF水平明显升高,而且共培养后HCC细胞存活相关通路中AKT、STAT3被激活,外源性HGF作用于HCC细胞能够引起Sorafenib耐受,AKT、c-Met抑制剂能够抵消共培养引起的耐药,而STAT3激活不受上述抑制剂影响。然而共培养体系中STAT3明显激活,说明其激活可能由其他可溶性因子介导。抑制STAT3上游的JAK能够抑制STAT3激活并同样改善共培养引起的Sorafenib耐药,STAT3抑制剂S3I-201同样可以增加Sorafenib敏感性。其次,HSCs与HCC细胞共培养引起HCC细胞形态变化,这种变化与EMT诱导因子TGF-β引起的形态改变类似,与HSC共培养后HCC细胞的迁移与侵袭能力有所增强,上皮表型相关蛋白E-Cadherin减少、间质表型标记物Vimentin增加,免疫荧光染色显示共培养后HCC细胞Vimentin增加,而且m RNA变化与之一致,证实HSCs诱导HCC细胞发生了EMT。可能引起EMT的信号通路关键蛋白p-AKT、p-STAT3均明显增加,使用si RNA干扰这些靶点并检测E-Cadherin发现干扰AKT、ERK均不能影响共培养导致的EMT现象,而只有干扰STAT3才能逆转这一过程,并且干扰STAT3后HCC细胞的迁移侵袭能力减弱。HSCs条件培养基与HCC单独培养的细胞培养基中的TGF-β水平无差别。IL-6刺激HCC细胞不能引起EMT发生,而鞘氨醇1磷酸(Sphingosine-1-phosphate,S1P)刺激能够引起HCC细胞内STAT3持续激活并导致EMT。干扰S1P受体S1PR1能够降低HCC细胞的迁移与侵袭能力,并且通过STAT3/Slug进一步抑制共培养引起的EMT。同时注射HSCs能够增加裸鼠原位HCC肿瘤的转移能力。结论:HSCs能够通过分泌HGF激活HCC细胞内的c-Met/AKT通路从而引起Sorafenib耐药,另外HSCs还可通过激活JAK/STAT3引起Sorafenib耐药,分别靶向这两条通路均可以增加Sorafenib的敏感性;HSCs与HCC细胞共培养后引起EMT并促进HCC细胞迁移侵袭,S1P可能是HSCs与HCC细胞间交互作用的重要分子,HSCs可能通过S1PR1/STAT3/Slug引起HCC细胞发生EMT。
[Abstract]:Background: hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) the occurrence is usually accompanied by chronic inflammation and fibrosis, hepatic stellate cells (Hepatic stellate cells, HSCs) in the process of inflammation and fibrosis plays an important role in the tumor microenvironment and HCC. Objective: HSCs co cultured with HCC cells, the sensitivity to the target drug Sorafenib HCC cells in the co culture system of evaluation, and the detection of co cultured HCC cell phenotype under the existence of changes. Further research on the mechanism of HSCs HCC cell sensitivity to Sorafenib change and migration changes caused by invasion. Methods: MTT method was used to detect the changes of HCC cells co cultured under the condition of the sensitivity to Sorafenib, further by ELISA Western, Blotting, HSCs and Q PCR detection method of co culture may lead to Sorafenib signal pathway sensitivity changes in HCC cells, and the corresponding target The effect of co culture of inhibitors interference is verified. On the other hand, Western Blotting, HCC gene change detection of intracellular EMT related protein, Q PCR, wound healing assay, HCC Transwell detection of cell migration, invasion and migration of EMT related protein, HCC cells were detected by using Si RNA interference may cause EMT targets the invasion ability. Combined with HSCs to establish orthotopic tumor model, to detect the changes of pulmonary metastasis ability. Results: first, we found that Sorafenib inhibited the proliferation of HCC cells by apoptosis, using HSCs co cultured with HCC cells, the sensitivity of HCC cells to Sorafenib decreased significantly and apoptosis related protein Caspase3,9, PARP decreased, that caused by Sorafenib the tolerance of.HSCs HCC cell conditioned medium HGF was significantly increased in co cultured and co cultured HCC cell survival pathways in AKT, STAT3 is activated, exogenous Role of HGF in HCC cells can induce Sorafenib tolerance, AKT, c-Met inhibitors can cause the resistance of co culture offset, the activation of STAT3 is not affected by the inhibitor effect. However, in the co culture system of STAT3 was activated, the activation may be mediated by other soluble factors. Inhibition of STAT3 upstream of JAK can inhibit the activation of STAT3 and the same to improve the resistance of co cultured Sorafenib, STAT3 inhibitor S3I-201 can increase the sensitivity of Sorafenib. Secondly, HSCs HCC cells were co cultured with HCC cells induced by morphological changes, the changes induced by EMT and factor TGF- beta induced morphological changes similar to HSC were co cultured with the migration and invasion of HCC cells enhanced epithelial phenotype correlation protein E-Cadherin decreased, mesenchymal marker Vimentin, immunofluorescence staining showed that Vimentin co cultured HCC cells increased, and the change of RNA and one of M To confirm a EMT. may cause the EMT signaling pathway in HCC cells induced by HSCs protein p-AKT, p-STAT3 was significantly increased, the use of Si RNA interfere with the target detection of E-Cadherin and AKT EMT found that interference, caused by co culture of ERK were not affected, but only interfere with the process of STAT3 can be reversed, and the interference of STAT3 the migration and invasion of HCC cells reduced.HSCs conditioned medium and HCC cells cultured alone had no difference between the.IL-6 stimulation of HCC cells induced by EMT can occur in the medium of TGF- beta level training, and sphingosine 1 phosphate (Sphingosine-1-phosphate, S1P) stimulation on HCC cells and lead to sustained activation of STAT3 EMT. interference S1P receptor S1PR1 can reduce the migration and the invasion ability of HCC cells, and further inhibit the transfer ability by STAT3/Slug co culture induced EMT. and HSCs injection can increase HCC tumor in nude mice. Conclusion: HSCs can secrete HGF through activation of the c-Met/AKT pathway in HCC cells due to Sorafenib resistance, HSCs also can be caused by the activation of JAK/STAT3 Sorafenib resistance, respectively, targeting these two pathways can increase the sensitivity of Sorafenib; HSCs co cultured with HCC cells induced by EMT and HCC to promote cell migration and invasion, S1P may be an important molecular HSCs and HCC cell interaction, HSCs through S1PR1/STAT3/Slug of HCC cells induced by EMT.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

【相似文献】