本文關(guān)鍵詞：褪黑素抑制基質(zhì)金屬蛋白酶-9對人臍靜脈內(nèi)皮細胞屏障保護機制的研究　出處：《北京協(xié)和醫(yī)學院》2012年博士論文　論文類型：學位論文

  更多相關(guān)文章： 內(nèi)皮細胞 周細胞 褪黑素 基質(zhì)金屬蛋白酶 金屬蛋白酶組織抑制劑 內(nèi)皮細胞連接蛋白 NF-κB

【摘要】：目的：第一部分：探討大鼠腦微血管內(nèi)皮細胞和周細胞的分離、培養(yǎng)方法和鑒定。 第二部分：探討褪黑素(Melatonin, Mel)通過抑制基質(zhì)金屬蛋白酶(matrix metalloproteinase, MMP)-9的表達和活性,來保護血管內(nèi)皮細胞屏障功能的作用機制。 方法：第一部分：取3周齡Wistar大鼠,無菌分離腦組織,采用兩次酶消化和一次密度梯度離心法分離大鼠腦微血管片段后,通過不同的處理方法,接種于35mm培養(yǎng)皿上進行原代培養(yǎng)；使用vWF和GFAP抗體來鑒定腦微血管內(nèi)皮細胞,免疫熒光染色觀察緊密連接蛋白claudin-5在細胞間的分布；使用α-SMA、NG2、vWF和GFAP抗體來鑒定周細胞,利用MTT法測定周細胞的增殖。 第二部分：原代分離培養(yǎng)人臍靜脈內(nèi)皮細胞(human umbilical vein endothelial cells, HUVECs),并使用包被小鼠抗人CD31單克隆抗體的免疫磁珠對原代分離的HUVECs進一步純化。以小鼠抗人vWF單克隆抗體及FITC標記的山羊抗小鼠熒光二抗鑒定純化后的HUVECs。RT-PCR和Western blot檢測不同干預條件下MMP-9、MMP-2、TIMP-1和TIMP-2mRNA和蛋白表達；明膠酶譜分析不同干預條件下MMP-9和MMP-2的活性；利用Transwell系統(tǒng),檢測不同干預條件下透過單層內(nèi)皮的Na-F來反映MMP-9對內(nèi)皮通透性的影響；用免疫熒光顯微鏡觀察不同干預條件下內(nèi)皮細胞黏著連接蛋白VE-鈣粘素(VE-cadherin)和緊密連接蛋白occludin、 claudin-5和ZO-1的變化以及核轉(zhuǎn)錄因子p65核轉(zhuǎn)位；使用Western blot檢測VE-cadherin、occludin和p65的表達變化。 結(jié)果：第二部分：成功分離培養(yǎng)原代腦微血管內(nèi)皮細胞和周細胞,倒置顯微鏡下內(nèi)皮細胞呈梭形,匯合后形成典型的“鵝卵石”樣外觀,并通過免疫熒光染色,GFAP表達陰性,vWF表達陽性,證實為微血管內(nèi)皮細胞；同時也可以看到claudine-5連續(xù)完整表達在細胞邊緣；倒置顯微鏡下周細胞胞體大,且表現(xiàn)出很多的突觸,并通過免疫熒光染色證實為微血管周細胞。原代周細胞初始生長速度較緩慢,傳代細胞36-60h進入對數(shù)生長期,72-108h進入平臺期。 第二部分：(1)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,vWF免疫熒光染色證實細胞確為HUVECs。細胞呈梭形、圓形或多角形。(2)與對照組相比,IL-1β (10ng/mL)處理組顯著增加了MMP-9mRNA和蛋白的表達(P0.001),同時TIMP-1mRNA和蛋白的表達顯著減少(P0.001),而MMP-2和TIMP-2mRNA和蛋白的表達沒有明顯變化(P0.05)；與IL-1p處理組相比,Mel (10μmol/L)預處理組顯著抑制了MMP-9mRNA和蛋白的表達(P0.001),而TIMP-1mRNA和蛋白的表達顯著增加(P0.001),MMP-2和TIMP-2mRNA和蛋白的表達沒有明顯變化(P0.05)；(3)明膠酶譜分析,與對照組相比,IL-1p處理組的MMP-9酶活性顯著增加(P0.001)；與IL-1β處理組相比,Mel預處理顯著抑制了MMP-9酶活性(P0.01)。然而,與對照組相比,IL-1β處理組和Mel預處理組的MMP-2酶活性無顯著差異(P0.05)。(4)Transwell分析內(nèi)皮細胞屏障對Na-F的通透性的變化,與對照組相比,IL-1p處理組在不同的時間點顯著增加了內(nèi)皮屏障對Na-F的通透性(P0.001)。而與IL-1p處理組相比,褪黑素預處理組顯著降低了Na-F的通透性(P0.001)。(5)熒光顯微鏡觀察：正常匯合后的內(nèi)皮細胞表達VE-cadherin、occludin、claudin-5和ZO-1呈現(xiàn)出連續(xù)不間斷的狀態(tài)；IL-1β處理組細胞之間黏附連接蛋白VE-cadherin和緊密連接蛋白occludin、 claudin-5和ZO-1在細胞膜上的表達下調(diào),并出現(xiàn)斷裂。Mel(10μmol/L)預處理后,VE-cadherin、occludin、claudin-5和ZO-1的表達狀態(tài)得到了改善。Western Blot結(jié)果表明：與對照組相比,IL-1β處理組HUVECs的VE-cadherin和occludin表達量下調(diào)。Mel可有效抑制MMP-9導致的VE-cadherin和occludin下調(diào),但仍低于對照組(P0.01)。(6) Western blot和免疫熒光分析,IL-1β處理組可顯著增加NF-KB/p65核轉(zhuǎn)位。Mel(10μmol/L)預處理組可以有效抑制這一效應。 結(jié)論：第一部分：成功分離出純度較高的大鼠腦微血管內(nèi)皮細胞和周細胞；第二部分：(1)MMP-9破壞了HUVECs黏附連接蛋白VE-cadherin和緊密連接蛋白occludin、claudin-5和ZO-1,使其表達降低,細胞間出現(xiàn)縫隙,通透性升高；(2)Mel通過抑制IL-1β誘導的NF-κB/p65信號轉(zhuǎn)導,抑制了MMP-9活性和表達的增加,改善了內(nèi)皮細胞黏著連接和緊密連接的狀態(tài),保護了內(nèi)皮細胞屏障功能。
[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.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R329

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