【作者】 林海华；

【导师】 田德安；

【作者基本信息】 华中科技大学 ， 内科学， 2011， 硕士

【摘要】 背景和目的癌细胞的显著标志之一是细胞极性的改变,后者在很多生物学过程中发挥关键的作用。在肿瘤的转移过程中,癌细胞主要通过上皮间质转化(EMT)获得迁移、侵袭能力,细胞极性的丢失是EMT的启动阶段,但具体的机制仍不清楚。Netrin-1是层黏连蛋白相关的分泌蛋白,它在调节神经轴突生长、白细胞迁移的过程中发挥重要作用;同时,它还参与调控肿瘤细胞侵袭和转移能力。但其机制仍未完全明确。本项目旨在研究Netrin-1对肝癌细胞极性的作用,以及脂筏–依赖受体DCC在肝癌细胞极性中的调控机制。方法建立肝癌细胞物理缺氧模型,倒置显微镜下观察细胞形态的改变,激光共聚焦检测细胞极性的变化,检测shRNA干扰Netrin-1的表达对缺氧所致细胞极性改变的影响。转染Netrin-1质粒到肝癌细胞HepG2后,建立稳定表达Netrin-1的细胞株,激光共聚焦观察Netrin-1和其受体DCC在脂筏上的聚集及其对肝癌细胞极性的影响,用western-blot检测细胞极性改变后下游极性蛋白Cdc42、Rac-1表达的改变,同时免疫荧光观测细胞骨架的重排。用甲基-β-环糊精(MCD)破坏脂筏的完整性,观测其对缺氧及Netrin-1对肝癌细胞极性的影响及上皮标志蛋白E-钙粘素表达的变化。结果在缺氧培养条件下,肝癌细胞失去相互间的紧密连接,细胞分散,由上皮样转变为狭长的间质样,细胞极性丧失。在缺氧条件下shRNA干扰Netrin-1的表达后可以抑制细胞极性的改变。转染Netrin-1质粒后的HepG2细胞极性丧失,下游极性蛋白Cdc42、Rac-1表达增加,细胞骨架重排。激光共聚焦检测观察到Netrin-1的受体DCC和脂筏共定位于细胞膜上。用甲基-β-环糊精(MCD)破坏脂筏的完整性后,可以抑制Netrin-1对细胞极性的影响,同时上皮标志蛋白E-钙粘素表达增加。结论缺氧条件和Netrin-1可以促进肝癌细胞极性的丧失,抑制Netrin-1的表达后可以抑制肝癌细胞极性发生改变,Netrin-1作用的信号可能是通过脂筏-依赖性受体DCC平台传递。这为进一步探索细胞极性的丢失改变在肝癌早期转移的分子机制提供了重要的理论依据。更多还原

【Abstract】 Backgrounds and aimsCell polarity plays a key role in the biological process of cells and the loss of polarity is a hallmark of cancer. Previous research suggests that cancer cells obtain the ability of invasion principally through the EMT (epithelial-mesenchymal transition). The disruption of cell polarity is the initial stage of EMT, and the exact mechanism of which remains unclear. Netrin-1 is a diffusible laminin-like chemotactic protein which has been shown to act as guidance cues for developing axons and migrating neuroblasts and it also plays an important part in the regulation of the apoptosis and invasion of tumor cells. The aim of the project study is to investigate the role of Netrin-1 and the lipid rafts-dependence receptor DCC signaling in the regulation of cell polarity in hepatocellular carcinoma cells.MethodsWe established the physical hypoxia model of hepatocellular carcinoma cells. Cell images were captured by inverted microscope. Immunofluorescence staining and confocal laser microscopy were employed to detect the change of cell polarity and the influence of shRNA-Netrin-1 on the cell polarity under hypoxic conditions. The Netrin-1 plasid was stably transfected into heptocellular carcinoma cell HepG2. Immunofluorescence staining and confocal laser microscopy were employed to detect the the colocalization of Netrin-1’s receptor DCC and lipid rafts at the cell membranes, the disruption of cell polarity and actin cytoskeleton rearrangement of the cells. Western blot was used to measure the change of expression of specific polarity proteins such as Cdc42 and Rac-1. The influence of Netrin-1 on cell polarity and the change of E-cadherin were detected after the transfected cells were treated with methyl-β-cyclodextrin (MCD).ResultsAfter being cultured under hypoxia conditions, hepatocellular carcinoma cells, which transformed from epithelia to mesenchymal and lost cell contact. Hypoxic promoted the disruption of cell polarity. The shRNA of Netrin-1 could inhibit the disruption of cell polarity. The HepG2 cells transfected with Netrin-1 showed that the loss of cell polarity, the rearrangement of actin cytoskeleton and the increased expression of cell polarity proteins such as Cdc42 and Rac-1. We also found that the Netrin-1 receptor DCC and lipid rafts colocalized at the cell membranes. The disruption of cell polarity could be inhibited after the cells transfected with Netrin-1 being treated with methyl-β-cyclodextrin (MCD) and the sign of epithelial E- cadherin underwent substantial changes.ConclusionsHypoxic conditions and Netrin-1 can promote the disruption of cell polarity of hepatocellular cells, which occurred because of the Netrin-1 and the lipid rafts-dependence receptor DCC signaling. It reveals the role of cell polarity in the early stage of metastasis in hepatocellular carcinoma, which is important for us to continue to investigate the intervention targets in the metastasis of heptocellular carcinoma.更多还原