褪黑素抑制基质金属蛋白酶-9对人脐静脉内皮细胞屏障保护机制的研究

发布时间：2018-01-12 03:04

本文关键词：褪黑素抑制基质金属蛋白酶-9对人脐静脉内皮细胞屏障保护机制的研究　出处：《北京协和医学院》2012年博士论文　论文类型：学位论文

【摘要】：目的：第一部分：探讨大鼠脑微血管内皮细胞和周细胞的分离、培养方法和鉴定。第二部分：探讨褪黑素(Melatonin, Mel)通过抑制基质金属蛋白酶(matrix metalloproteinase, MMP)-9的表达和活性,来保护血管内皮细胞屏障功能的作用机制。方法：第一部分：取3周龄Wistar大鼠,无菌分离脑组织,采用两次酶消化和一次密度梯度离心法分离大鼠脑微血管片段后,通过不同的处理方法,接种于35mm培养皿上进行原代培养；使用vWF和GFAP抗体来鉴定脑微血管内皮细胞,免疫荧光染色观察紧密连接蛋白claudin-5在细胞间的分布；使用α-SMA、NG2、vWF和GFAP抗体来鉴定周细胞,利用MTT法测定周细胞的增殖。第二部分：原代分离培养人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs),并使用包被小鼠抗人CD31单克隆抗体的免疫磁珠对原代分离的HUVECs进一步纯化。以小鼠抗人vWF单克隆抗体及FITC标记的山羊抗小鼠荧光二抗鉴定纯化后的HUVECs。RT-PCR和Western blot检测不同干预条件下MMP-9、MMP-2、TIMP-1和TIMP-2mRNA和蛋白表达；明胶酶谱分析不同干预条件下MMP-9和MMP-2的活性；利用Transwell系统,检测不同干预条件下透过单层内皮的Na-F来反映MMP-9对内皮通透性的影响；用免疫荧光显微镜观察不同干预条件下内皮细胞黏着连接蛋白VE-钙粘素(VE-cadherin)和紧密连接蛋白occludin、 claudin-5和ZO-1的变化以及核转录因子p65核转位；使用Western blot检测VE-cadherin、occludin和p65的表达变化。结果：第二部分：成功分离培养原代脑微血管内皮细胞和周细胞,倒置显微镜下内皮细胞呈梭形,汇合后形成典型的“鹅卵石”样外观,并通过免疫荧光染色,GFAP表达阴性,vWF表达阳性,证实为微血管内皮细胞；同时也可以看到claudine-5连续完整表达在细胞边缘；倒置显微镜下周细胞胞体大,且表现出很多的突触,并通过免疫荧光染色证实为微血管周细胞。原代周细胞初始生长速度较缓慢,传代细胞36-60h进入对数生长期,72-108h进入平台期。第二部分：(1)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,vWF免疫荧光染色证实细胞确为HUVECs。细胞呈梭形、圆形或多角形。(2)与对照组相比,IL-1β (10ng/mL)处理组显著增加了MMP-9mRNA和蛋白的表达(P0.001),同时TIMP-1mRNA和蛋白的表达显著减少(P0.001),而MMP-2和TIMP-2mRNA和蛋白的表达没有明显变化(P0.05)；与IL-1p处理组相比,Mel (10μmol/L)预处理组显著抑制了MMP-9mRNA和蛋白的表达(P0.001),而TIMP-1mRNA和蛋白的表达显著增加(P0.001),MMP-2和TIMP-2mRNA和蛋白的表达没有明显变化(P0.05)；(3)明胶酶谱分析,与对照组相比,IL-1p处理组的MMP-9酶活性显著增加(P0.001)；与IL-1β处理组相比,Mel预处理显著抑制了MMP-9酶活性(P0.01)。然而,与对照组相比,IL-1β处理组和Mel预处理组的MMP-2酶活性无显著差异(P0.05)。(4)Transwell分析内皮细胞屏障对Na-F的通透性的变化,与对照组相比,IL-1p处理组在不同的时间点显著增加了内皮屏障对Na-F的通透性(P0.001)。而与IL-1p处理组相比,褪黑素预处理组显著降低了Na-F的通透性(P0.001)。(5)荧光显微镜观察：正常汇合后的内皮细胞表达VE-cadherin、occludin、claudin-5和ZO-1呈现出连续不间断的状态；IL-1β处理组细胞之间黏附连接蛋白VE-cadherin和紧密连接蛋白occludin、 claudin-5和ZO-1在细胞膜上的表达下调,并出现断裂。Mel(10μmol/L)预处理后,VE-cadherin、occludin、claudin-5和ZO-1的表达状态得到了改善。Western Blot结果表明：与对照组相比,IL-1β处理组HUVECs的VE-cadherin和occludin表达量下调。Mel可有效抑制MMP-9导致的VE-cadherin和occludin下调,但仍低于对照组(P0.01)。(6) Western blot和免疫荧光分析,IL-1β处理组可显著增加NF-KB/p65核转位。Mel(10μmol/L)预处理组可以有效抑制这一效应。结论：第一部分：成功分离出纯度较高的大鼠脑微血管内皮细胞和周细胞；第二部分：(1)MMP-9破坏了HUVECs黏附连接蛋白VE-cadherin和紧密连接蛋白occludin、claudin-5和ZO-1,使其表达降低,细胞间出现缝隙,通透性升高；(2)Mel通过抑制IL-1β诱导的NF-κB/p65信号转导,抑制了MMP-9活性和表达的增加,改善了内皮细胞黏着连接和紧密连接的状态,保护了内皮细胞屏障功能。
[Abstract]:Objective: the first part: To explore the isolation, culture and identification of rat brain microvascular endothelial cells and pericytes.
The second part is to explore the mechanism of Melatonin (Mel) protecting vascular endothelial barrier function by inhibiting the expression and activity of matrix metalloproteinase (MMP) -9.
Methods: the first part: 3 week old Wistar rats, sterile isolated brain tissue, using two enzyme digestion and a density gradient centrifugation of rat brain microvessels, through different treatment method, inoculated in 35mm culture dishes and cultured; using vWF and GFAP antibodies to identify brain microvascular endothelial cells, observe the distribution of tight junction protein claudin-5 in cell immunofluorescence staining; the use of alpha -SMA, NG2, vWF and GFAP antibodies to identify pericytes, determination of peripheral cell proliferation by MTT method.
The second part: the primary cultured human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVECs), and use of the package is further purified mouse anti human CD31 monoclonal antibody immunomagnetic beads to isolated HUVECs. Anti MMP-2 two identification of purified HUVECs.RT-PCR and Western blot to detect different intervention conditions with mouse anti human vWF monoclonal antibody and FITC labeled Goat anti mouse MMP-9, TIMP-1 fluorescence, and TIMP-2mRNA and protein expression; gelatinase spectrum analysis of different interference conditions MMP-9 and MMP-2 activity; using Transwell system to test different interference conditions through the endothelial monolayer Na-F to reflect the effects of MMP-9 on endothelial permeability of endothelial cell adhesion; observe different intervention under the condition of connexin VE- cadherin by immunofluorescence microscopy (VE-cadherin) and tight junction protein occludin and changes of claudin-5 and ZO-1 And nuclear transcription factor p65 nuclear transposition; Western blot was used to detect the changes in the expression of VE-cadherin, occludin and p65.
Results: the second part: the primary cultured brain microvascular endothelial cells and pericytes isolated, inverted microscope, endothelial cells were spindle shaped, formed after the confluence of the typical cobblestone like appearance, and by immunofluorescence staining, GFAP negative, vWF positive expression was confirmed by microvascular endothelial cells; at the same time also can see claudine-5 continuous and complete expression in the cell edge; inverted microscope cells next week, and show a lot of synapses, and confirmed pericytes by immunofluorescence staining. The primary cells initial growth rate is slow, 36-60h cells in logarithmic growth phase, 72-108h into the platform.
The second part: (1) the cultured HUVECs, immunomagnetic screening to adherent growth and subculture, under the inverted microscope HUVECs showed a cobblestone, vWF immunofluorescence staining showed that the cells of HUVECs. cells were spindle shaped, round or polygonal. (2) compared with the control group, IL-1 beta (10ng/mL) treatment group significantly increased the expression of MMP-9mRNA and protein (P0.001), while the expression of TIMP-1mRNA and protein was significantly reduced (P0.001), while no significant changes in the expression of MMP-2 and TIMP-2mRNA and protein (P0.05); compared with IL-1p group, Mel (10 mol/L) preconditioning group significantly inhibited the expression of MMP-9mRNA and protein (P0.001), while the expression of TIMP-1mRNA and protein were significantly increased (P0.001), no significant changes in the expression of MMP-2 and TIMP-2mRNA and protein (P0.05); (3) zymography analysis, compared with the control group, the activity of MMP-9 in IL-1p group increased significantly (P0.001); Compared with the IL-1 beta treatment group, Mel pretreatment significantly inhibited the activity of MMP-9 (P0.01). However, compared with the control group, IL-1 group and Mel pretreatment group. The activity of MMP-2 beta treatment had no significant difference (P0.05). Transwell (4) analysis of the changes of permeability of endothelial cell barrier to Na-F, compared with the control group, IL-1p treatment group at different time points significantly increased the permeability of endothelial barrier of Na-F (P0.001) and IL-1p treatment group. Compared to melatonin pretreatment group significantly decreased the permeability of Na-F (P0.001). (5) fluorescence microscopy: normal confluent endothelial cells express VE-cadherin, occludin, claudin-5 and ZO-1 showed a continuous state; group IL-1 beta cells between adhesion protein VE-cadherin and tight junction protein occludin, expression of claudin-5 and ZO-1 in the cell membrane, and the fracture of.Mel (10 mol/L) after the pretreatment, V E-cadherin, occludin, claudin-5 and ZO-1 expression to improve the.Western Blot results showed that: compared with the control group, IL-1 treatment group HUVECs beta VE-cadherin and down-regulation of occludin expression of.Mel can effectively inhibit MMP-9 induced down-regulation of occludin and VE-cadherin, but still lower than the control group (P0.01). (6) analysis of Western and blot immunofluorescence, IL-1 beta treatment group can significantly increase the nuclear translocation of NF-KB/p65.Mel (10 mol/L) preconditioning group can effectively inhibit this effect.
Conclusion: the first part: the high purity of the isolated rat brain microvascular endothelial cells and pericytes; the second part: (1) MMP-9 destroyed HUVECs adhesion protein VE-cadherin and tight junction protein occludin, claudin-5 and ZO-1, the expression decreased, the intercellular gap, the permeability increased; (2) Mel through the NF- B/p65 signal transduction inhibition induced by IL-1, inhibited the increase in MMP-9 activity and expression of endothelial cell adhesion, improve the connection and close connection, protect the barrier function of endothelial cells.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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