發(fā)布時(shí)間：2018-03-19 15:29

  本文選題：褪黑素　切入點(diǎn)：褪黑素受體　出處：《中國協(xié)和醫(yī)科大學(xué)》2007年博士論文　論文類型：學(xué)位論文

【摘要】： 褪黑素對體外培養(yǎng)的臍靜脈內(nèi)皮細(xì)胞增殖和凋亡的影響 目的：第一部分：為了探討褪黑素(MEL)在血管新生方面的作用和可能的抗腫瘤血管新生機(jī)制，以體外培養(yǎng)的臍靜脈內(nèi)皮細(xì)胞(HUVECs)為模型，觀察MEL對HUVECs增殖和凋亡的影響。第二部分：在第一部分基礎(chǔ)上，探討高濃度MEL抑制HUVECs增殖和促其凋亡的可能細(xì)胞信號傳導(dǎo)通路。 方法：第一部分：原代培養(yǎng)HUVECs，免疫磁珠篩選后，并用免疫組化和電鏡鑒定內(nèi)皮細(xì)胞；MTT法測定不同濃度的MEL對HUVECs增殖的影響，并用流式細(xì)胞儀測定HUVECs細(xì)胞周期和細(xì)胞凋亡情況；免疫熒光、Western Blot和RT-PCR檢測MEL對HUVECs內(nèi)凋亡相關(guān)蛋白P53、Bcl-2和Bax及其基因表達(dá)情況。第二部分：在第一部分基礎(chǔ)上，RT-PCR檢測HUVECs褪黑素膜受體和核受體(MELR和RZR／ROR)的表達(dá)情況，以及MEL對其表達(dá)影響；檢測高濃度MEL對影響HUVECs增殖和凋亡的重要的細(xì)胞內(nèi)信號通路RTK／ERK／P13K／AKT／PKC／NF-κB蛋白表達(dá)影響；應(yīng)用MELR受體阻滯劑Luzindole、ERK1／2激活抑制劑U0126、PI3K／AKT抑制劑LY294002以及PKC激活劑PMA和抑制劑GF 109203X，觀察其對高濃度MEL抑制HUVECs增殖和相關(guān)細(xì)胞信號傳遞通路的影響。 結(jié)果：第一部分：(1)成功的分離培養(yǎng)原代HUVECs，免疫磁珠篩選后能繼續(xù)貼壁生長并傳代，倒置顯微鏡下HUVECs呈鵝卵石狀，Ⅷ因子內(nèi)皮細(xì)胞細(xì)胞免疫化學(xué)著棕色，電鏡下可見內(nèi)皮細(xì)胞特征性的Weibel-Palade小體。(2)極低濃度MEL(lnmol／L)對HUVECs增殖無明顯影響，但高濃度MEL(10nmol／L-1mmol／L)明顯抑制HUVECs增殖，并有濃度依賴傾向，1mmol／L抑制作用最明顯。(3)流式細(xì)胞儀顯示高濃度MEL能明顯阻滯HUVECs細(xì)胞周期，能阻止細(xì)胞進(jìn)入S期，引起“S期停滯”；凋亡分析顯示高濃度的MEL能明顯促使HUVECs凋亡，1mmol／L時(shí)最明顯。(4)高濃度MEL(100nmol／L和lmmol／L)明顯調(diào)節(jié)凋亡相關(guān)基因P53、Bax和Bcl-2的表達(dá)。(5)Western Blot和免疫熒光檢測高濃度MEL促進(jìn)HUVECs凋亡與細(xì)胞內(nèi)P53和Bax蛋白表達(dá)上調(diào)，Bcl-2蛋白下調(diào)有關(guān)。 第二部分：(1)正常HUVECs中褪黑素膜受體MELlaR和MELlbR都有明顯表達(dá)，而核受體家族中有RORa和RORb表達(dá)，以RORb表達(dá)更為突出，RORC幾乎無表達(dá)。(2) MEL受體參與MEL抑制HUVECs增殖機(jī)制，膜受體阻滯劑Luzindole只能部分阻斷高濃度MEL的增殖抑制效應(yīng)。(3)外源性MEL可使HUVECs上MEL受體表達(dá)下調(diào)，此過程可能與PKCα表達(dá)上調(diào)有關(guān)。(4) ERK1／2、PI3K／AKT和PKC信號通路與HUVECs增殖密切相關(guān)，其特異的通路抑制劑能明顯抑制HUVECs增殖。(5)高濃度MEL通過抑制ERK1／2、PI3K／AKT和PKC信號通路抑制了HUVECs增殖和促其凋亡，相應(yīng)的通路抑制劑或激活劑能分別部分促進(jìn)或阻斷MEL抑制HUVECs增殖效應(yīng)。(6) MEL抑制HUVECs中NF-κB的表達(dá)并抑制其和DNA結(jié)合，從而抑制HUVECs增殖和促其凋亡。(7)特異的ERK1／2、PI3K／AKT和PKC信號通路抑制劑能不同程度的阻斷HUVECs中NF-κB的活性，和MEL作用類似，說明MEL可通過抑制ERK1／2、PI3K／AKT和PKC信號通路抑制NF-κB的活性，，從而影響細(xì)胞增殖和凋亡。 結(jié)論：(1)高濃度的MEL能明顯抑制HUVECs增殖和促其凋亡；(2)高濃度的MEL抑制HUVECs的增殖和促其凋亡與P53和Bax／Bc1-2基因和蛋白的表達(dá)改變密切相關(guān)；(3)高濃度MEL抑制HUVECs的增殖和促其凋亡可能與MEL受體、PI3K／AKT／ERK／PKC／NF-κB等多條信號途徑密切相關(guān)。
[Abstract]:Effects of melatonin on proliferation and apoptosis of umbilical vein endothelial cells cultured in vitro
Objective: the first part: To investigate the effect of melatonin (MEL) on angiogenesis and the possible mechanism of anti angiogenesis, in vitro cultured human umbilical vein endothelial cells (HUVECs) as a model, the effect of MEL on proliferation and apoptosis of HUVECs. The second part: in the first part based on the possible signal transduction to explore the pathway of high concentration of MEL inhibited HUVECs proliferation and induced apoptosis.
Methods: the first part: the primary cultured HUVECs, immunomagnetic screening, and immunohistochemical and ultrastructural characterization of endothelial cells; Determination of proliferation of HUVECs cells with different concentrations of MEL and HUVECs MTT method, cell cycle and apoptosis were analyzed by flow cytometry; immunofluorescence detection of MEL, Western and RT-PCR on Blot HUVECs apoptosis related proteins P53, Bcl-2 and Bax and its gene expression. In the second part, on the basis of the first part, RT-PCR detection of HUVECs melatonin membrane and nuclear receptors (MELR and RZR / ROR) expression, and the effect of MEL on the surface of high concentration; influence the detection effect of MEL expression on proliferation and apoptosis of HUVECs the important intracellular signal pathway of RTK / ERK / P13K / AKT / PKC / NF- kappa B protein; application of MELR receptor antagonist Luzindole, ERK1 / 2 activation inhibitor U0126, PI3K inhibitor LY294002 and the activation of PKC / AKT The effect of PMA and inhibitor GF 109203X on the inhibition of HUVECs proliferation and related cell signaling pathway by high concentration MEL was observed.
Results: the first part: (1) to develop a successful separation of primary HUVECs, immunomagnetic screening to adherent growth and subculture, under the inverted microscope HUVECs showed a cobblestone like endothelial cell immune factor VIII, chemical brown, Weibel-Palade body visible characteristics of endothelial cells under electron microscope. (2) the extremely low concentrations of MEL (lnmol / L) had no obvious effect on the proliferation of HUVECs, but high concentration of MEL (10nmol / L-1mmol / L) significantly inhibited the proliferation of HUVECs, and the concentration dependence of 1mmol / L, the most obvious inhibition. (3) flow cytometry showed that high concentration of MEL could significantly block the cell cycle of HUVECs, can prevent the cell into S, cause S arrest; apoptosis analysis showed that high concentration of MEL can significantly promote the apoptosis of HUVECs, 1mmol / L is most obvious. (4) the high concentration of MEL (100nmol / L and lmmol / L) significantly regulate the apoptosis related gene P53, expression of Bax and Bcl-2 (5) Western. Blot and immunofluorescent detection of high concentration of MEL promote the apoptosis of HUVECs and the up regulation of P53 and Bax protein expression in the cells, and the downregulation of Bcl-2 protein.
The second part: (1) normal HUVECs melatonin receptors MELlaR and MELlbR have obvious expression, and nuclear receptor family in the expression of RORa and RORb, RORb expression was more prominent, almost no expression. RORC (2) MEL MEL receptor is involved in inhibition of HUVECs proliferation mechanism, membrane receptor blocker Luzindole only partially blocked the inhibitory effect the high concentration of MEL (3). The proliferation of exogenous MEL can make HUVECs on MEL receptor expression, this process may be related to PKC alpha expression upregulation. (4) ERK1 / 2, PI3K / AKT and PKC signaling pathway is closely related to the proliferation of HUVECs, via its specific inhibitor can significantly inhibit the proliferation of HUVECs (. 5) the high concentration of MEL through inhibition of ERK1 / 2, PI3K / AKT and PKC signaling pathway inhibits HUVECs proliferation and apoptosis, pathway inhibitors or activators can promote or inhibit MEL respectively inhibited HUVECs proliferation effect. (6) the expression of MEL HUVECs in NF- kappa B inhibition And its combination with DNA inhibition, inhibit HUVECs proliferation and apoptosis. (7) specific ERK1 / 2, PI3K / AKT and PKC signaling pathway inhibitors can different degree of blockade of HUVECs NF- K B activity, similar and MEL, indicating that MEL can inhibit ERK1 / 2, PI3K / AKT and PKC signaling inhibits NF- kappa B activity, thus affecting cell proliferation and apoptosis.
Conclusion: (1) the high concentration of MEL can inhibit HUVECs proliferation and apoptosis; (2) the expression of high concentration of MEL inhibited and apoptosis with P53 and Bax / Bc1-2 gene and protein HUVECs proliferation are closely related to the change; (3) the high concentration of MEL and inhibition of apoptosis may be related to MEL receptor the proliferation of HUVECs, PI3K / AKT / ERK / PKC / NF- kappa B multiple signaling pathways are closely related.

【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R363

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