褪黑素对体外培养的脐静脉内皮细胞增殖和凋亡的影响

发布时间：2018-03-19 15:29

  本文选题：褪黑素　切入点：褪黑素受体　出处：《中国协和医科大学》2007年博士论文　论文类型：学位论文

【摘要】： 褪黑素对体外培养的脐静脉内皮细胞增殖和凋亡的影响 目的：第一部分：为了探讨褪黑素(MEL)在血管新生方面的作用和可能的抗肿瘤血管新生机制，以体外培养的脐静脉内皮细胞(HUVECs)为模型，观察MEL对HUVECs增殖和凋亡的影响。第二部分：在第一部分基础上，探讨高浓度MEL抑制HUVECs增殖和促其凋亡的可能细胞信号传导通路。 方法：第一部分：原代培养HUVECs，免疫磁珠筛选后，并用免疫组化和电镜鉴定内皮细胞；MTT法测定不同浓度的MEL对HUVECs增殖的影响，并用流式细胞仪测定HUVECs细胞周期和细胞凋亡情况；免疫荧光、Western Blot和RT-PCR检测MEL对HUVECs内凋亡相关蛋白P53、Bcl-2和Bax及其基因表达情况。第二部分：在第一部分基础上，RT-PCR检测HUVECs褪黑素膜受体和核受体(MELR和RZR／ROR)的表达情况，以及MEL对其表达影响；检测高浓度MEL对影响HUVECs增殖和凋亡的重要的细胞内信号通路RTK／ERK／P13K／AKT／PKC／NF-κB蛋白表达影响；应用MELR受体阻滞剂Luzindole、ERK1／2激活抑制剂U0126、PI3K／AKT抑制剂LY294002以及PKC激活剂PMA和抑制剂GF 109203X，观察其对高浓度MEL抑制HUVECs增殖和相关细胞信号传递通路的影响。 结果：第一部分：(1)成功的分离培养原代HUVECs，免疫磁珠筛选后能继续贴壁生长并传代，倒置显微镜下HUVECs呈鹅卵石状，Ⅷ因子内皮细胞细胞免疫化学着棕色，电镜下可见内皮细胞特征性的Weibel-Palade小体。(2)极低浓度MEL(lnmol／L)对HUVECs增殖无明显影响，但高浓度MEL(10nmol／L-1mmol／L)明显抑制HUVECs增殖，并有浓度依赖倾向，1mmol／L抑制作用最明显。(3)流式细胞仪显示高浓度MEL能明显阻滞HUVECs细胞周期，能阻止细胞进入S期，引起“S期停滞”；凋亡分析显示高浓度的MEL能明显促使HUVECs凋亡，1mmol／L时最明显。(4)高浓度MEL(100nmol／L和lmmol／L)明显调节凋亡相关基因P53、Bax和Bcl-2的表达。(5)Western Blot和免疫荧光检测高浓度MEL促进HUVECs凋亡与细胞内P53和Bax蛋白表达上调，Bcl-2蛋白下调有关。 第二部分：(1)正常HUVECs中褪黑素膜受体MELlaR和MELlbR都有明显表达，而核受体家族中有RORa和RORb表达，以RORb表达更为突出，RORC几乎无表达。(2) MEL受体参与MEL抑制HUVECs增殖机制，膜受体阻滞剂Luzindole只能部分阻断高浓度MEL的增殖抑制效应。(3)外源性MEL可使HUVECs上MEL受体表达下调，此过程可能与PKCα表达上调有关。(4) ERK1／2、PI3K／AKT和PKC信号通路与HUVECs增殖密切相关，其特异的通路抑制剂能明显抑制HUVECs增殖。(5)高浓度MEL通过抑制ERK1／2、PI3K／AKT和PKC信号通路抑制了HUVECs增殖和促其凋亡，相应的通路抑制剂或激活剂能分别部分促进或阻断MEL抑制HUVECs增殖效应。(6) MEL抑制HUVECs中NF-κB的表达并抑制其和DNA结合，从而抑制HUVECs增殖和促其凋亡。(7)特异的ERK1／2、PI3K／AKT和PKC信号通路抑制剂能不同程度的阻断HUVECs中NF-κB的活性，和MEL作用类似，说明MEL可通过抑制ERK1／2、PI3K／AKT和PKC信号通路抑制NF-κB的活性，，从而影响细胞增殖和凋亡。 结论：(1)高浓度的MEL能明显抑制HUVECs增殖和促其凋亡；(2)高浓度的MEL抑制HUVECs的增殖和促其凋亡与P53和Bax／Bc1-2基因和蛋白的表达改变密切相关；(3)高浓度MEL抑制HUVECs的增殖和促其凋亡可能与MEL受体、PI3K／AKT／ERK／PKC／NF-κB等多条信号途径密切相关。
[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.

【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363

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