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血管內(nèi)皮細(xì)胞蛋白激酶C與錨蛋白、CD44分子生物學(xué)特性之間關(guān)系的研究

發(fā)布時(shí)間:2019-05-27 23:54
【摘要】:前言 CD44分子是廣泛分布于血管內(nèi)皮細(xì)胞、血細(xì)胞、淋巴細(xì)胞、成纖維細(xì)胞等細(xì)胞的重要粘附分子,與配體透明質(zhì)酸、膠原蛋白、纖維連接素等結(jié)合后介導(dǎo)了淋巴細(xì)胞“歸巢”和轉(zhuǎn)化、腫瘤轉(zhuǎn)移、信號(hào)傳導(dǎo)等生理或病理過程。其分子胞漿側(cè)與細(xì)胞骨架蛋白錨蛋白相連接,并存在一個(gè)高度特異性結(jié)合區(qū)域,此區(qū)域內(nèi)含有PKC磷酸化位點(diǎn)。研究證實(shí)PKC對(duì)此位點(diǎn)的磷酸化能顯著增強(qiáng)二者結(jié)合力。研究還發(fā)現(xiàn),PKC活化對(duì)CD44變構(gòu)體及ICAM-1、VCAM-1等粘附分子的表達(dá)有調(diào)節(jié)作用。因此,我們利用PMA活化HU-VECs PKC來觀察PKC活性對(duì)血管內(nèi)皮細(xì)胞CD44的表達(dá)及粘附性有無影響。錨蛋白是連接CD44跨膜糖蛋白與細(xì)胞內(nèi)骨架網(wǎng)絡(luò)的橋梁。在淋巴細(xì)胞上,研究發(fā)現(xiàn)PKC活化可引起細(xì)胞內(nèi)錨蛋白的重新分布。那么,在血管內(nèi)皮細(xì)胞上會(huì)不會(huì)也出現(xiàn)這種情況呢?并由此改變細(xì)胞骨架網(wǎng)絡(luò)的立體結(jié)構(gòu)而造成細(xì)胞的形態(tài)及細(xì)胞之間連接關(guān)系的變化,從而影響內(nèi)皮細(xì)胞通透性呢?由于錨蛋白與CD44相連,如果有錨蛋白的移位,會(huì)不會(huì)同時(shí)牽動(dòng)CD44的移位?我們利用免疫熒光標(biāo)記法觀察PKC活化后HUVECs CD44、錨蛋白的分布情況。PKC是一種信使傳遞酶,CD44分子與配體結(jié)合后胞內(nèi)Ca~(2+)升高,而PKC是第二信使Ca~(2+)及DAG的靶蛋白,Ca~(2+)升高必然會(huì)引發(fā)PKC活化,通過其磷酸化作用將刺激信號(hào)傳遞下去。那么,信號(hào)傳導(dǎo)的實(shí)際途徑如何呢?Raf-1激酶是細(xì)胞內(nèi)許多生物學(xué)信號(hào)傳導(dǎo)第一步,可以接受酪氨酸激酶家族或PKC的活化,并進(jìn)而引起酶鏈反應(yīng):Raf-1→MEK→ERK。我們利用PKC激活劑及抑制劑來觀察HUVECs CD44基因的表達(dá)及CD44分子磷酸化情況,對(duì)Raf-1激酶活性進(jìn)行分析,并比較了Raf-1激酶、MEK、ERK抑制劑對(duì)HUVECs CD44基因表達(dá)的影響,觀察PKC能否直接活化上述途徑,據(jù)此探討PKC對(duì)CD44分子表達(dá)的信號(hào)調(diào)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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