血管内皮细胞蛋白激酶C与锚蛋白、CD44分子生物学特性之间关系的研究

发布时间：2019-05-27 23:54

【摘要】：前言 CD44分子是广泛分布于血管内皮细胞、血细胞、淋巴细胞、成纤维细胞等细胞的重要粘附分子,与配体透明质酸、胶原蛋白、纤维连接素等结合后介导了淋巴细胞“归巢”和转化、肿瘤转移、信号传导等生理或病理过程。其分子胞浆侧与细胞骨架蛋白锚蛋白相连接,并存在一个高度特异性结合区域,此区域内含有PKC磷酸化位点。研究证实PKC对此位点的磷酸化能显著增强二者结合力。研究还发现,PKC活化对CD44变构体及ICAM-1、VCAM-1等粘附分子的表达有调节作用。因此,我们利用PMA活化HU-VECs PKC来观察PKC活性对血管内皮细胞CD44的表达及粘附性有无影响。锚蛋白是连接CD44跨膜糖蛋白与细胞内骨架网络的桥梁。在淋巴细胞上,研究发现PKC活化可引起细胞内锚蛋白的重新分布。那么,在血管内皮细胞上会不会也出现这种情况呢?并由此改变细胞骨架网络的立体结构而造成细胞的形态及细胞之间连接关系的变化,从而影响内皮细胞通透性呢?由于锚蛋白与CD44相连,如果有锚蛋白的移位,会不会同时牵动CD44的移位?我们利用免疫荧光标记法观察PKC活化后HUVECs CD44、锚蛋白的分布情况。PKC是一种信使传递酶,CD44分子与配体结合后胞内Ca~(2+)升高,而PKC是第二信使Ca~(2+)及DAG的靶蛋白,Ca~(2+)升高必然会引发PKC活化,通过其磷酸化作用将刺激信号传递下去。那么,信号传导的实际途径如何呢?Raf-1激酶是细胞内许多生物学信号传导第一步,可以接受酪氨酸激酶家族或PKC的活化,并进而引起酶链反应:Raf-1→MEK→ERK。我们利用PKC激活剂及抑制剂来观察HUVECs CD44基因的表达及CD44分子磷酸化情况,对Raf-1激酶活性进行分析,并比较了Raf-1激酶、MEK、ERK抑制剂对HUVECs CD44基因表达的影响,观察PKC能否直接活化上述途径,据此探讨PKC对CD44分子表达的信号调控
[Abstract]:CD44 molecule is an important adhesion molecule widely distributed in vascular endothelial cells, blood cells, lymphocytes, fibroblasts and other cells, and ligands hyaluronic acid, collagen. The binding of fibronectin mediates the physiological or pathological processes such as homing and transformation of lymphocytes, tumor metastasis, signal transduction and so on. The cytoplasm side of the molecule is connected with the cytoskeleton protein anchor protein, and there is a highly specific binding region, which contains the phosphorylation site of PKC. It has been confirmed that the phosphorylation of PKC to this site can significantly enhance the binding capacity of the two sites. It was also found that PKC activation regulated the expression of CD44 mutants and ICAM-1,VCAM-1 and other adhesion molecules. Therefore, we used PMA activated HU-VECs PKC to observe the effect of PKC activity on the expression and adhesion of CD44 in vascular endothelial cells. Anchor protein is a bridge between CD44 transmembrane glycoprotein and intracellular skeleton network. On lymphocytes, it was found that PKC activation could cause the redistribution of intracellular anchor protein. So, does this happen in vascular endothelial cells, too? Thus, the morphology of cells and the connection relationship between cells are changed by changing the three-dimensional structure of cytoskeleton network, which affects the permeability of endothelial cells. Because the anchor protein is connected to CD44, if there is the shift of anchor protein, will the shift of CD44 be affected at the same time? We observed the distribution of HUVECs CD44, anchor protein after PKC activation by immunofluorescence labeling. PKC is a messenger transfer enzyme. CD44 molecule increases intracellular Ca~ (2) after binding to ligands, while PKC is the target protein of second messenger Ca~ (2) and DAG. The increase of Ca~ (2) will inevitably lead to the activation of PKC, and the stimulation signal will be transmitted through its phosphorylation. So, what is the actual pathway of signal transduction? Raf- 1 kinase is the first step in many biological signal transduction in cells, which can accept the activation of tyrosine kinase family or PKC, and then lead to chain reaction: Raf-1 鈮，

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