本文關(guān)鍵詞： 微血管內(nèi)皮細(xì)胞 造血干細(xì)胞 增殖　出處：《西南醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究微血管內(nèi)皮細(xì)胞(MEC)對造血干細(xì)胞(HSC)增殖的影響,尋找促進(jìn)HSC增殖的方法。方法:1.MEC分離、培養(yǎng)及鑒定:將肺組織經(jīng)I型膠原酶消化獲得的細(xì)胞,在20%FBS、2ng/mlVEGF、肝素100u/ml、DMEM-F12中培養(yǎng),24h全換液,此后每2-3天全量換液,鏡下觀察細(xì)胞形態(tài)變化及生長情況,取第8天細(xì)胞進(jìn)行FITC-CD31免疫熒光鑒定,“鋪路石”細(xì)胞用于流式鑒定、傳代培養(yǎng)和共培養(yǎng)。連續(xù)8天觀察P1代MEC生長情況、形態(tài)變化,并逐日細(xì)胞計(jì)數(shù),繪制P1代MEC生長曲線。2.GFP小鼠HSC的分離、鑒定:密度梯度離心法獲得小鼠骨髓單個核細(xì)胞(MNC),MACS-CD117+磁珠分選HSC。應(yīng)用流式細(xì)胞計(jì)數(shù)儀(FACS)檢測陽性細(xì)胞的純度及HSC CD117CD34共表達(dá)率。3.微血管內(nèi)皮細(xì)胞對造血干細(xì)胞增殖的研究:設(shè)立對照組(HSC)、共培養(yǎng)組(MEC+HSC),體外培養(yǎng)7天,觀察HSC生長情況、細(xì)胞計(jì)數(shù)、繪制生長曲線、計(jì)算HSC擴(kuò)增倍數(shù)。收集共培養(yǎng)組HSC,FACS檢測共培養(yǎng)組HSC CD117CD34共表達(dá)率,與共培養(yǎng)前對比。結(jié)果:1.MEC分離、培養(yǎng)及鑒定:?原代細(xì)胞:6h貼壁,24h少數(shù)細(xì)胞形態(tài)不規(guī)則,多數(shù)細(xì)胞呈圓形。48h見多角形、短梭形細(xì)胞。第3d細(xì)胞聚集生長,形成形態(tài)大小較一致的細(xì)胞團(tuán)。第6d相鄰細(xì)胞偽足彼此相連,第10d細(xì)胞繼續(xù)增多,第14d細(xì)胞生長達(dá)80%融合,呈“鋪路石”外觀。?傳代細(xì)胞:4h貼壁,P1代MEC第3d見多角形、短梭形細(xì)胞。第4d細(xì)胞聚集生長,形成形態(tài)大小較一致的細(xì)胞團(tuán),第7d細(xì)胞密集生長,多數(shù)呈梭形、不規(guī)則形。第8d細(xì)胞生長近80%融合。p1代mec生長曲線:第4到7天為對數(shù)生長期,隨后進(jìn)入平臺期。cd31抗體免疫熒光檢測陽性率為54.5%。mecfacs鑒定vwf、cd31、cd34、cd45表達(dá)率分別為:81.39%、45.8%、57.48%、0.17%。2.gfp小鼠hsc的分離、鑒定:本實(shí)驗(yàn)中平均每只gfp小鼠骨髓經(jīng)密度梯度離心后可以獲得約1.1×108個mnc,活細(xì)胞率為98%。mnc經(jīng)macscd117磁珠分選后可以獲得約4.7×105個cd117+細(xì)胞,活率為97%。骨髓mnc中cd117+細(xì)胞含量約0.43%。磁珠分選后的cd117+hsc純度為99.51%,hsccd117cd34共表達(dá)率為75.28%。熒光顯微鏡下:hsc呈綠色,圓形,大小均一。3.微血管內(nèi)皮細(xì)胞對造血干細(xì)胞增殖的研究:培養(yǎng)時間增加,兩組hsc數(shù)均增加,共培養(yǎng)組較對照組更明顯。共培養(yǎng)組與對照組細(xì)胞數(shù)的比值:第1d,共培養(yǎng)組細(xì)胞數(shù)約為對照組的1.21倍(p0.01),第2d,共培養(yǎng)組約為對照組的1.35倍(p0.05),第3d,共培養(yǎng)組約為對照組的1.50倍(p0.01),第4d,共培養(yǎng)組約為對照組的1.72倍(p0.01),第5d,共培養(yǎng)組約為對照組的1.71倍(p0.01),第6d,共培養(yǎng)組約為對照組的1.75倍(p0.01),第7d,共培養(yǎng)組約為對照組的1.78倍(p0.01)。共培養(yǎng)組與對照組細(xì)胞數(shù)比值變化曲線示:比值逐漸增加,第4d變化最明顯。d1-d7較d0的hsc數(shù)比較:第7d與第0d相比,共培養(yǎng)組擴(kuò)增約12.31倍(p0.01),對照組擴(kuò)增約6.92倍(p0.01);第6d與第0d相比,共培養(yǎng)組擴(kuò)增約11.28倍(P0.01),對照組擴(kuò)增約6.45倍(P0.01);第5d與第0d相比,共培養(yǎng)組擴(kuò)增約9.72倍(P0.01),對照組擴(kuò)增約5.66倍(P0.01);第4d與第0d相比,共培養(yǎng)組擴(kuò)增約7.31倍(P0.01),對照組擴(kuò)增約4.25倍(P0.01);第3d與第0d相比,共培養(yǎng)組擴(kuò)增約3.45倍(P0.01),對照組擴(kuò)增約2.30倍(P0.01);第2d與第0d相比,共培養(yǎng)組擴(kuò)增約1.92倍(P0.01),對照組擴(kuò)增約1.42倍(P0.01);第1d與第0d相比,共培養(yǎng)組擴(kuò)增約1.20倍(P0.01),對照組擴(kuò)增約0.99倍(P0.05)。共培養(yǎng)7天后,共培養(yǎng)組HSC CD117CD34共表達(dá)率為92.06%。結(jié)論:1.微血管內(nèi)皮細(xì)胞對造血干細(xì)胞增殖有促進(jìn)作用,共培養(yǎng)第4天促進(jìn)作用最明顯。2..微血管內(nèi)皮細(xì)胞可以促進(jìn)造血干細(xì)胞CD34的表達(dá)。
[Abstract]:Objective: To study the microvascular endothelial cells (MEC) on hematopoietic stem cells (HSC) proliferation, looking for the method to promote the proliferation of HSC. Methods: 1.MEC isolation, culture and identification: the lung tissue by type I collagenase digestion cells obtained in 20%FBS, 2ng/mlVEGF, 100u/ml, heparin DMEM-F 12 24h, culture, change the liquid, then every 2-3 days for liquid, to observe the changes in cell morphology and growth under microscope were identified by immunofluorescence of FITC-CD31 cells from eighth days, paving stone cells for flow cytometry, and co culture were cultured for 8 consecutive days. Observe the P1 generation MEC growth, morphological changes, cell counting and daily P1, separation, drawing generation growth curve of MEC.2.GFP mice HSC identification: mouse bone marrow mononuclear cells were obtained by density gradient centrifugation (MNC), MACS-CD117+ multisort HSC. by flow cytometry (FACS) detection and HSC CD117CD34 positive cells purity table Study on hematopoietic stem cell proliferation rate of.3. microvascular endothelial cells: the establishment of the control group (HSC), co culture group (MEC+HSC), cultured for 7 days, to observe the growth condition, HSC cell count, draw the growth curve, calculation of HSC amplification ratio. Group HSC co culture collection, FACS detection of HSC co culture group CD117CD34 co expression rate, and co culture before comparison. Results: 1.MEC isolation, culture and identification: primary cells: 6h? 24h a few cells attached to the wall, irregular shape, most of the cells were round.48h polygonal and spindle shaped cells. The accumulation of 3D cells growth, forming the shape and size of cell clusters is consistent the 6D of adjacent cells. The 10d cells were connected to each other, continue to increase, the growth of 14d cells reached 80% confluence, a paving stone appearance.?: 4H cell wall, P1 generation MEC 3D see the polygonal and spindle shaped cells. The 4D cell aggregation growth form with the same size the cell group, seventh D cell density growth, most fusiform, irregular shape. The growth of 8D cells in 80% generation fusion.P1 MEC growth curve: fourth to 7 days for the logarithmic growth phase, then entered the positive rate of.Cd31 antibody was detected by immunofluorescence for identification of vWF platform 54.5%.mecfacs, CD31, CD34, CD45 expression rate respectively: 81.39% 45.8%, 57.48%, 0.17%.2.gfp, separation, identification of HSC mice: in this experiment, the average GFP of mouse bone marrow by density gradient centrifugation can be obtained after about 1.1 * 108 MNC, the cell survival rate of 98%.mnc by macscd117 magnetic beads can be obtained after about 4.7 x 105 cd117+ cells, the survival rate for cd117+ cells in 97%. bone marrow MNC 0.43%. multisort cd117+hsc after about 99.51% purity, hsccd117cd34 co expression rate of 75.28%. under the fluorescence microscope: HSC is green, round, uniform size of.3. microvascular endothelial cells on the proliferation of hematopoietic cells: culture time increased, the two groups hsc鏁板潎澧炲姞,鍏卞煿鍏葷粍杈冨鐓х粍鏇存槑鏄，

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