本文關(guān)鍵詞： 中性粒細(xì)胞 Percoll密度梯度離心法 Ficoll-Hypaque密度梯度離心法 裂解紅細(xì)胞法 Dextran紅細(xì)胞沉降法 Antiflammin-1(AF-1) 粘附 中性粒細(xì)胞 內(nèi)皮細(xì)胞　出處：《中南大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 第一章人外周血中性粒細(xì)胞不同分離方法的比較 目的:從血液中分離中性粒細(xì)胞(neutrophil)是在細(xì)胞水平研究其生物學(xué)特性及功能的基礎(chǔ)。根據(jù)目前國內(nèi)外文獻(xiàn)資料顯示的常用中性粒細(xì)胞分離方法,選取四種,即Percoll非連續(xù)密度梯度離心法、Ficoll-Hypaque密度梯度離心法、裂解紅細(xì)胞法、Dextran作用下紅細(xì)胞自然沉降法進(jìn)行細(xì)胞純度、回收率、存活率比較,旨在尋找一種簡單、高效的中性粒細(xì)胞分離方法。 方法:采取健康人外周靜脈血,分別采用以上四種方法進(jìn)行中性粒細(xì)胞分離,對分離純化的中性粒細(xì)胞純度、回收率、存活率進(jìn)行比較。 結(jié)果:Percoll非連續(xù)密度梯度離心法與Ficoll-Hypaque密度梯度離心法分離得到的細(xì)胞純度均大于90%,兩者間比較無顯著差異(P＞0.05);裂解紅細(xì)胞法和Dextran作用下紅細(xì)胞自然沉降法分離得到的細(xì)胞純度略低于Percoll非連續(xù)密度梯度離心法和Ficoll-Hypaque密度梯度離心法(P＜0.05或P＜0.01);Percoll非連續(xù)密度梯度離心法、Ficoll-Hypaque密度梯度離心法和裂解紅細(xì)胞法的回收率均高于Dextran作用下紅細(xì)胞自然沉降法(P＜0.01或P＜0.05);Percoll非連續(xù)密度梯度離心法回收的中性粒細(xì)胞存活率明顯高于其它三組(P＜0.01或P＜0.05)。 結(jié)論:Percoll非連續(xù)密度梯度離心法分離中性粒細(xì)胞,純化程度好,回收率和細(xì)胞存活率高,是一種簡單、高效的中性粒細(xì)胞分離方法,適于臨床和科研中廣泛應(yīng)用。 第二章AF-1對于內(nèi)毒素誘導(dǎo)的中性粒細(xì)胞與內(nèi)皮細(xì)胞粘附的影響及機(jī)制研究 目的:中性粒細(xì)胞(neutrophil)在肺內(nèi)聚集是急性肺損傷(acutelung injury,ALI)發(fā)病的最初環(huán)節(jié),中性粒細(xì)胞與肺血管內(nèi)皮細(xì)胞粘附是炎癥反應(yīng)的最初現(xiàn)象,是其進(jìn)一步移行入肺組織的基礎(chǔ)。子宮珠蛋白(uteroglobin,UG)是一種多功能非類固醇類激素蛋白,具有抗炎、抗氧化、免疫調(diào)節(jié)、抑制腫瘤發(fā)生等作用。來源于子宮珠蛋白C末端第三個(gè)α螺旋的九肽Antiflammin-1(AF-1),也被證實(shí)具有強(qiáng)烈的抗炎作用。關(guān)于AF-1抗炎作用的機(jī)制至今尚未完全明了,因此本研究旨在通過觀察AF-1對于內(nèi)毒素誘導(dǎo)的中性粒細(xì)胞與內(nèi)皮細(xì)胞粘附及粘附分子表達(dá)的影響,檢測中性粒細(xì)胞和內(nèi)皮細(xì)胞子宮珠蛋白結(jié)合蛋白(Uteroglobin-binding protein,UGBP)的表達(dá)情況,通過應(yīng)用制備的抗-UGBP抗體觀察抗-UGBP抗體對AF-1粘附抑制及粘附分子表達(dá)的影響,還通過檢測AF-1、抗-UGBP抗體對于P38/MAPK信號(hào)轉(zhuǎn)導(dǎo)通路的影響,初步探討AF-1產(chǎn)生抗炎作用的機(jī)制,為ALI的治療提供新的靶點(diǎn)。 方法:采取健康人外周靜脈血分離純化中性粒細(xì)胞,培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞(HUVEC-12),分別應(yīng)用不同濃度、不同作用時(shí)間的AF-1預(yù)處理中性粒細(xì)胞和內(nèi)皮細(xì)胞,觀察中性粒細(xì)胞與內(nèi)皮細(xì)胞粘附率的變化及應(yīng)用流式細(xì)胞儀檢測粘附分子表達(dá)的變化;通過應(yīng)用免疫熒光、流式細(xì)胞術(shù)、RT-PCR一系列實(shí)驗(yàn)技術(shù)檢測中性粒細(xì)胞和內(nèi)皮細(xì)胞UGBP的表達(dá)情況;應(yīng)用制備的抗-UGBP抗體預(yù)處理觀察抗-UGBP多抗對AF-1粘附抑制及粘附分子表達(dá)的影響;進(jìn)一步應(yīng)用Western-Blot技術(shù)檢測AF-1、抗-UGBP抗體對于P38/MAPK信號(hào)轉(zhuǎn)導(dǎo)通路的影響。 結(jié)果: 1.應(yīng)用AF-1(100μmol/L)分別預(yù)處理內(nèi)皮細(xì)胞、中性粒細(xì)胞15min～2h時(shí)可以明顯抑制內(nèi)毒素誘導(dǎo)的中性粒細(xì)胞與內(nèi)皮細(xì)胞的粘附,與LPS組比較均有統(tǒng)計(jì)學(xué)意義(P＜0.01),其中以30min預(yù)處理抑制作用最明顯,隨著預(yù)處理時(shí)間延長,其粘附抑制效應(yīng)均降低,預(yù)處理時(shí)間為4h時(shí),其粘附抑制作用與LPS組比較沒有統(tǒng)計(jì)學(xué)差異;不同濃度AF-1分別預(yù)處理內(nèi)皮細(xì)胞、中性粒細(xì)胞30min,研究發(fā)現(xiàn)AF-1 1μmol/L對中性粒細(xì)胞與內(nèi)皮細(xì)胞的粘附即產(chǎn)生抑制作用(P＜0.05),隨著AF-1濃度增高其粘附抑制作用逐漸增強(qiáng),呈劑量依賴性。流式細(xì)胞儀檢測結(jié)果發(fā)現(xiàn)AF-1可以抑制內(nèi)毒素誘導(dǎo)的中性粒細(xì)胞粘附分子CD11b和內(nèi)皮細(xì)胞粘附分子CD54的表達(dá),而AF-1本身對于靜息狀態(tài)下的中性粒細(xì)胞的CD11b和內(nèi)皮細(xì)胞的CD54表達(dá)沒有影響。 2.免疫熒光、RT-PCR、流式細(xì)胞實(shí)驗(yàn)均證實(shí)在內(nèi)皮細(xì)胞和中性粒細(xì)胞上均存在UGBP的表達(dá),并且中性粒細(xì)胞的表達(dá)強(qiáng)于內(nèi)皮細(xì)胞。 3.粘附率測定及粘附分子表達(dá)測定結(jié)果顯示抗-UGBP抗體預(yù)處理可以抑制AF-1的粘附抑制效應(yīng)。 4.進(jìn)一步應(yīng)用Western-Blot檢測AF-1、抗-UGBP抗體對于P38/MAPK磷酸化水平的影響。結(jié)果顯示,AF-1可以明顯下調(diào)內(nèi)毒素誘導(dǎo)的內(nèi)皮細(xì)胞P38/MAPK的磷酸化水平,抗-UGBP抗體可以抑制AF-1作用引起的下調(diào)效應(yīng)。 結(jié)論: 1.AF-1可以抑制內(nèi)毒素誘導(dǎo)的中性粒細(xì)胞與內(nèi)皮細(xì)胞的粘附及粘附分子表達(dá),說明AF-1抗炎作用的產(chǎn)生與調(diào)節(jié)粘附分子的表達(dá)有關(guān)系。 2.中性粒細(xì)胞和內(nèi)皮細(xì)胞均有UGBP表達(dá),以細(xì)胞膜表達(dá)最為強(qiáng)烈,細(xì)胞漿內(nèi)也有少量表達(dá),并且中性粒細(xì)胞UGBP的表達(dá)水平高于內(nèi)皮細(xì)胞。 3.抗-UGBP抗體預(yù)處理可以抑制AF-1對于粘附的抑制作用及粘附分子表達(dá)變化,說明AF-1的粘附抑制作用同UGBP密切相關(guān)。 4.AF-1可以通過與UGBP結(jié)合調(diào)節(jié)細(xì)胞內(nèi)P38/MAPK磷酸化水平而影響細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)。 因此我們認(rèn)為AF-1通過結(jié)合細(xì)胞表面UGBP進(jìn)而調(diào)節(jié)細(xì)胞內(nèi)信號(hào)通路P38/MAPK的磷酸化水平而抑制中性粒細(xì)胞與內(nèi)皮細(xì)胞的粘附及粘附分子的表達(dá),產(chǎn)生抗炎作用。
[Abstract]:A comparison of different methods of separation of neutrophils in human peripheral blood
Objective: to isolate neutrophils from the blood (neutrophil) is the base of the research on the biological characteristics and function at the cellular level. According to the separation method commonly used at home and abroad literature shows that the neutral granulocyte, select four kinds, namely Percoll discontinuous density gradient centrifugation and Ficoll-Hypaque density gradient centrifugation, red blood cell lysis method. The effect of Dextran natural erythrocyte sedimentation method cell purity, recovery rate, survival rate, in order to find a simple and efficient method for the separation of neutrophils.
Methods: peripheral blood was taken from healthy people. Neutrophils were separated by the above four methods. The purity, recovery rate and survival rate of purified neutrophils were compared.
Results: the Percoll non isolated cells purity continuous density gradient centrifugation and Ficoll-Hypaque density gradient centrifugation were higher than 90%, there is no significant difference between them (P > 0.05); erythrocyte lysis method and Dextran effect of natural erythrocyte sedimentation method isolated cell purity was slightly lower than Percoll discontinuous density gradient centrifugation and Ficoll-Hypaque density gradient centrifugation (P < 0.05 or P < 0.01); Percoll discontinuous density gradient centrifugation. The recovery of Ficoll-Hypaque density gradient centrifugation and erythrocyte lysis method were higher than the natural erythrocyte sedimentation under the action of Dextran (P < 0.01 or P < 0.05); neutrophil Percoll discontinuous density gradient centrifugal recovery survival rate was significantly higher than the other three groups (P < 0.01 or P < 0.05).
Conclusion: Percoll discontinuous density gradient centrifugation is a simple and effective method for isolation of neutrophils. It is suitable for clinical and scientific research.
The effect and mechanism of second chapter AF-1 on endotoxin induced neutrophils adhesion to endothelial cells
Objective: neutrophil (neutrophil) in the lung of acute lung injury is the aggregation (acutelung injury ALI) the first link of the incidence of neutropenia and pulmonary vascular endothelial cell adhesion is the initial phenomenon of inflammatory reaction, is the basis of further migration into the lung tissue. Uteroglobin (Uteroglobin, UG) is a kind of multifunctional non steroid hormone protein, anti-inflammatory, antioxidant, immune regulation, tumor suppression effect. Nine peptide Antiflammin-1 derived from the Uteroglobin C terminal third alpha helix (AF-1), has also been shown to have anti-inflammatory effects strongly. On the mechanism of the anti-inflammatory effect of AF-1 is not yet fully understood, so this study in order to effect by observing the AF-1 for endotoxin induced neutrophil adhesion to endothelial cells and the expression of adhesion molecules, detection of neutrophils and endothelial cells (Uteroglobin binding protein Uteroglobin-b Inding protein, UGBP) expression, through the application of the preparation of anti -UGBP antibody to observe the effect of anti -UGBP antibody on the expression of AF-1 inhibited the adhesion and adhesion molecules, but also through the detection of AF-1, effects of anti -UGBP antibody for P38/MAPK signal transduction pathway, to explore the mechanism of AF-1 anti inflammatory effect, provide a new target for the treatment of ALI.
Methods: healthy human peripheral venous blood purification of neutrophils, cultured human umbilical vein endothelial cells (HUVEC-12), respectively with different concentrations, different time of AF-1 pretreatment of neutrophils and endothelial cells, to observe the changes of neutrophil adhesion and changes of endothelial cells and the rate of application of flow cytometry to detect the expression of adhesion molecules; by using immunofluorescence and flow cytometry, the expression of RT-PCR in a series of experimental techniques for the detection of neutrophils and endothelial cells UGBP; application of the preparation of anti -UGBP antibody pretreatment effects of anti -UGBP anti adhesion inhibition on the expression of AF-1 and adhesion molecules; the further application of AF-1 Western-Blot detection technology, influence anti -UGBP antibodies to P38/MAPK signal transduction pathway.
Result:
Application of 1. AF-1 (100 mol/L) were treated with endothelial cells, adhesion of neutrophil 15min ~ 2H could inhibit LPS induced neutrophil and endothelial cells, compared with LPS group were statistically significant (P < 0.01), of which 30min pretreatment inhibited the most obvious, with the increasing of pretreatment time the inhibitory effect was decreased, adhesion, pretreatment time was 4h, the inhibition compared with the LPS group had no significant difference; different concentrations of AF-1 were treated with endothelial cells, neutrophils, 30min, the study found that the adhesion of AF-1 1 mol/L on neutrophils and endothelial cells is inhibited (P < 0.05), with inhibition of AF-1 concentration increased the adhesion increased gradually in a dose-dependent manner. Flow cytometry results showed that neutrophil adhesion molecule CD11b and AF-1 can inhibit the adhesion of endothelial cells induced by lipopolysaccharide The expression of molecular CD54, while AF-1 itself has no effect on the CD54 expression of CD11b and endothelial cells in resting state neutrophils.
2. immunofluorescence, RT-PCR and flow cytometry demonstrated that UGBP expression was present on endothelial cells and neutrophils, and the expression of neutrophils was stronger than that of endothelial cells.
3. determination of adhesion rate and expression of adhesion molecules showed that anti -UGBP antibody preconditioning could inhibit the adhesion inhibition effect of AF-1.
4., we further applied Western-Blot to detect AF-1 and the effect of anti -UGBP antibody on P38/MAPK phosphorylation level. The results showed that AF-1 could significantly reduce the level of P38/MAPK induced by endotoxin, and -UGBP antibody could inhibit the down-regulation effect induced by AF-1.
Conclusion:
1.AF-1 can inhibit the adhesion of neutrophils to endothelial cells and expression of adhesion molecules induced by endotoxin, indicating that the production of AF-1 is related to regulating the expression of adhesion molecules.
2. neutrophils and endothelial cells had UGBP expression, the most strongly expressed in cell membrane, and a few in cytoplasm, and the expression level of UGBP in neutrophils was higher than that in endothelial cells.
3. anti -UGBP antibody pretreatment could inhibit the inhibitory effect of AF-1 on adhesion and the changes in the expression of adhesion molecules, indicating that the adhesion inhibition of AF-1 is closely related to UGBP.
4.AF-1 can affect intracellular signal transduction by modulating the level of P38/MAPK phosphorylation in cells with UGBP.
Therefore, we think that AF-1 can regulate the level of phosphorylation of intracellular signaling pathway P38/MAPK and inhibit the expression of adhesion molecules and neutrophils, and produce anti-inflammatory effect by binding the cell surface UGBP to further regulate the level of phosphorylation of intracellular signaling pathway.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 張?zhí)旖?AF-1對LPS誘導(dǎo)的巨噬細(xì)胞IL-10表達(dá)和分泌的影響[D];中南大學(xué);2009年

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