本文選題：巴曲酶　切入點(diǎn)：內(nèi)皮祖細(xì)胞　出處：《安徽醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的內(nèi)皮祖細(xì)胞(endothelial progenitor cells,EPCs)又稱為內(nèi)皮前體細(xì)胞。它們不僅參與胚胎時(shí)期的血管發(fā)生,也存在于成年體內(nèi)的骨髓和外周血中,參與血管的修復(fù)和新生。目前,抗動(dòng)脈硬化以及促血管新生的研究是臨床研究的熱點(diǎn)問(wèn)題,已經(jīng)證明自體EPCs的動(dòng)員和移植是治療性血管再生的一個(gè)有效方法。巴曲酶(batroxobin,DF-521)是從南美具竅腹蛇的蛇毒中分離出、提純出的一種具有抗凝溶纖作用的絲氨酸蛋白酶�，F(xiàn)廣泛應(yīng)用于包括腦梗塞和慢性下肢動(dòng)脈硬化性閉塞癥在內(nèi)的多種缺血性心腦血管疾病的治療�？紤]DF-521可能通過(guò)影響EPCs來(lái)改善血管內(nèi)皮結(jié)構(gòu)與功能,促進(jìn)血管新生,從而發(fā)揮臨床作用。本研究旨在通過(guò)觀察DF-521在體外環(huán)境下對(duì)EPCs的增殖、分化、成血管能力以及NO分泌能力的影響,來(lái)探討DF-521的作用機(jī)制,并為EPCs的體外增殖、分化尋找可能的新誘導(dǎo)劑。方法取經(jīng)過(guò)rhG-CSF動(dòng)員的健康成人的外周血,密度梯度離心法提取單個(gè)核細(xì)胞,接種在hFN包被的培養(yǎng)板上,DMEM條件培養(yǎng)液(含F(xiàn)BS和VEGF)培養(yǎng)。培養(yǎng)至第6d,對(duì)貼壁細(xì)胞進(jìn)行EPCs的鑒定:(1)倒置顯微鏡下觀察貼壁細(xì)胞形態(tài);(2)激光共聚焦顯微鏡觀察經(jīng)DiI-acLDL和FITC-UEA-I雙染色的貼壁細(xì)胞;(3)流式細(xì)胞術(shù)檢測(cè)貼壁細(xì)胞CD34、CD31、vWF、VEGFR的表達(dá)情況。EPCs隨機(jī)為對(duì)照組(基本培養(yǎng)基)、實(shí)驗(yàn)組A(0.05U/ml DF-521),實(shí)驗(yàn)組B(0.1U/ml DF-521),實(shí)驗(yàn)組C(含0.2U/ml DF-521)。培養(yǎng)24h,NO試劑盒檢測(cè)上清液NO濃度。繼續(xù)48h,對(duì)EPCs進(jìn)行計(jì)數(shù)。3d換液一次,培養(yǎng)至第14d和21d用流式細(xì)胞術(shù)對(duì)各組EPCs做CD31和vWF的表型檢測(cè)。收集培養(yǎng)至21d的細(xì)胞,制備電子顯微鏡標(biāo)本,電子透射電子顯微鏡觀察細(xì)胞超微結(jié)構(gòu)。鑒定完EPCs后,將EPCs按等濃度接種至基質(zhì)膠上,按上述4組培養(yǎng)72h,觀察EPCs生長(zhǎng)情況,并用DiI-acLDL和FITC-UEA-I雙染色,激光共聚焦顯微鏡再觀察。結(jié)果EPCs的鑒定:EPCs呈梭形或圓形;DiI-acLDL和FITC-UEA-I雙染色陽(yáng)性,發(fā)黃色熒光;流式細(xì)胞術(shù)顯示EPCs的表型為CD34+(26.13±8.51)%,CD31+(5.15±2.15)%,VEGFR-2+(15.16±1.31)%,vWF+(5.12±1.01)%。各實(shí)驗(yàn)組的NO濃度以及EPCs數(shù)量均明顯大于對(duì)照組(P0.05),并且這個(gè)效應(yīng)隨DF-521濃度升高而增加(P0.05)。在第14d、21d,實(shí)驗(yàn)組的CD31和vWF表達(dá)強(qiáng)于對(duì)照組。在第21d,在實(shí)驗(yàn)組B,C的細(xì)胞呈鋪路石樣排列。電子透射顯微鏡觀察到ECs特異性結(jié)構(gòu),即懷布爾-帕拉德小體。高劑量干預(yù)組的EPCs形成了條梭狀結(jié)構(gòu)和管腔樣結(jié)構(gòu)。結(jié)論在體外培養(yǎng)條件下,DF-521促進(jìn)EPCs的增殖和成血管功能,并誘導(dǎo)其向ECs分化,改善其分泌NO的能力; DF-521對(duì)EPCs增殖和分泌NO能力的影響呈現(xiàn)劑量依賴關(guān)系;DF-521的這些作用可能與內(nèi)皮型一氧化氮合酶有關(guān)。
[Abstract]:Objective Endothelial progenitor cells (EPCs) are also called endothelial progenitor cells (EPCs).They are not only involved in angiogenesis at embryonic stage, but also in bone marrow and peripheral blood in adults, and participate in the repair and regeneration of blood vessels.At present, the research of anti-arteriosclerosis and angiogenesis is a hot issue in clinical research. It has been proved that the mobilization and transplantation of autogenous EPCs is an effective method for the treatment of vascular regeneration.Batroxobin DF-521) is a serine protease with anticoagulant and fibrinolytic activity isolated from the venom of the South American snake.It is widely used in the treatment of ischemic cardiovascular and cerebrovascular diseases, including cerebral infarction and chronic arteriosclerotic obliteration of lower extremities.It is considered that DF-521 may improve the structure and function of vascular endothelium and promote angiogenesis by affecting EPCs.The aim of this study was to investigate the effect of DF-521 on the proliferation, differentiation, vascularization and no secretion of EPCs in vitro, to explore the mechanism of DF-521, and to find a new inducer for the proliferation and differentiation of EPCs in vitro.Methods Mononuclear cells were extracted from the peripheral blood of healthy adults mobilized by rhG-CSF and cultured in the conditioned medium (including FBS and VEGF) on the hFN coated culture plate by density gradient centrifugation.Identification of adherent cells by EPCs on the 6th day of culture: 1) observation of adherent cell morphology under inverted microscope) observation of adherent cells by confocal laser microscopy (DiI-acLDL and FITC-UEA-I double staining) flow cytometry to detect the expression of CD34- CD31vWFEGFR in adherent cellsEPCs were randomly divided into two groups: control group (basic medium, A(0.05U/ml DF-521), experimental group (B(0.1U/ml DF-521), and experimental group C (including 0.2U/ml DF-521).The concentration of no in supernatant was detected by 24 h culture.EPCs was counted for 48h. The phenotypes of CD31 and vWF were detected by flow cytometry on the 14th and 21st day after culture. The phenotypes of CD31 and vWF were detected by flow cytometry.The cells cultured for 21 days were collected and the ultrastructure of the cells was observed by electron transmission electron microscopy (TEM).After EPCs was identified, EPCs was inoculated into the matrix gel at the same concentration and cultured for 72 hours according to the above mentioned four groups. The growth of EPCs was observed by double staining of DiI-acLDL and FITC-UEA-I, and then observed by laser confocal microscope.Results EPCs were double stained with double staining of DiI-acLDL and FITC-UEA-I, and the phenotype of EPCs was CD34 26.13 鹵8.51. The phenotype of EPCs was CD34 26.13 鹵8.51. The phenotype of EPCs was 5.15 鹵2.15 鹵5.16 鹵1.31. The phenotype of EPCs was 5.12 鹵1.01.The concentration of no and the amount of EPCs in each experimental group were significantly higher than those in the control group, and the effect increased with the increase of DF-521 concentration.The expression of CD31 and vWF in the experimental group was stronger than that in the control group at day 14 and day 21.On the 21 th day, the cells of BMC in the experimental group were arranged like paving stones.The specific structure of ECs was observed by electron transmission microscope (TEM).In the high dose intervention group, EPCs formed a spindle-like structure and a lumen like structure.Conclusion DF-521 promotes the proliferation and angiogenesis of EPCs in vitro and induces its differentiation into ECs.The effects of DF-521 on the proliferation and secretion of no in EPCs were dose-dependent. These effects of DF-521 may be related to endothelial nitric oxide synthase.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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