微血管内皮细胞对造血干细胞增殖的研究

发布时间：2018-03-01 04:35

  本文关键词： 微血管内皮细胞 造血干细胞 增殖　出处：《西南医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究微血管内皮细胞(MEC)对造血干细胞(HSC)增殖的影响,寻找促进HSC增殖的方法。方法:1.MEC分离、培养及鉴定:将肺组织经I型胶原酶消化获得的细胞,在20%FBS、2ng/mlVEGF、肝素100u/ml、DMEM-F12中培养,24h全换液,此后每2-3天全量换液,镜下观察细胞形态变化及生长情况,取第8天细胞进行FITC-CD31免疫荧光鉴定,“铺路石”细胞用于流式鉴定、传代培养和共培养。连续8天观察P1代MEC生长情况、形态变化,并逐日细胞计数,绘制P1代MEC生长曲线。2.GFP小鼠HSC的分离、鉴定:密度梯度离心法获得小鼠骨髓单个核细胞(MNC),MACS-CD117+磁珠分选HSC。应用流式细胞计数仪(FACS)检测阳性细胞的纯度及HSC CD117CD34共表达率。3.微血管内皮细胞对造血干细胞增殖的研究:设立对照组(HSC)、共培养组(MEC+HSC),体外培养7天,观察HSC生长情况、细胞计数、绘制生长曲线、计算HSC扩增倍数。收集共培养组HSC,FACS检测共培养组HSC CD117CD34共表达率,与共培养前对比。结果:1.MEC分离、培养及鉴定:?原代细胞:6h贴壁,24h少数细胞形态不规则,多数细胞呈圆形。48h见多角形、短梭形细胞。第3d细胞聚集生长,形成形态大小较一致的细胞团。第6d相邻细胞伪足彼此相连,第10d细胞继续增多,第14d细胞生长达80%融合,呈“铺路石”外观。?传代细胞:4h贴壁,P1代MEC第3d见多角形、短梭形细胞。第4d细胞聚集生长,形成形态大小较一致的细胞团,第7d细胞密集生长,多数呈梭形、不规则形。第8d细胞生长近80%融合。p1代mec生长曲线:第4到7天为对数生长期,随后进入平台期。cd31抗体免疫荧光检测阳性率为54.5%。mecfacs鉴定vwf、cd31、cd34、cd45表达率分别为:81.39%、45.8%、57.48%、0.17%。2.gfp小鼠hsc的分离、鉴定:本实验中平均每只gfp小鼠骨髓经密度梯度离心后可以获得约1.1×108个mnc,活细胞率为98%。mnc经macscd117磁珠分选后可以获得约4.7×105个cd117+细胞,活率为97%。骨髓mnc中cd117+细胞含量约0.43%。磁珠分选后的cd117+hsc纯度为99.51%,hsccd117cd34共表达率为75.28%。荧光显微镜下:hsc呈绿色,圆形,大小均一。3.微血管内皮细胞对造血干细胞增殖的研究:培养时间增加,两组hsc数均增加,共培养组较对照组更明显。共培养组与对照组细胞数的比值:第1d,共培养组细胞数约为对照组的1.21倍(p0.01),第2d,共培养组约为对照组的1.35倍(p0.05),第3d,共培养组约为对照组的1.50倍(p0.01),第4d,共培养组约为对照组的1.72倍(p0.01),第5d,共培养组约为对照组的1.71倍(p0.01),第6d,共培养组约为对照组的1.75倍(p0.01),第7d,共培养组约为对照组的1.78倍(p0.01)。共培养组与对照组细胞数比值变化曲线示:比值逐渐增加,第4d变化最明显。d1-d7较d0的hsc数比较:第7d与第0d相比,共培养组扩增约12.31倍(p0.01),对照组扩增约6.92倍(p0.01);第6d与第0d相比,共培养组扩增约11.28倍(P0.01),对照组扩增约6.45倍(P0.01);第5d与第0d相比,共培养组扩增约9.72倍(P0.01),对照组扩增约5.66倍(P0.01);第4d与第0d相比,共培养组扩增约7.31倍(P0.01),对照组扩增约4.25倍(P0.01);第3d与第0d相比,共培养组扩增约3.45倍(P0.01),对照组扩增约2.30倍(P0.01);第2d与第0d相比,共培养组扩增约1.92倍(P0.01),对照组扩增约1.42倍(P0.01);第1d与第0d相比,共培养组扩增约1.20倍(P0.01),对照组扩增约0.99倍(P0.05)。共培养7天后,共培养组HSC CD117CD34共表达率为92.06%。结论:1.微血管内皮细胞对造血干细胞增殖有促进作用,共培养第4天促进作用最明显。2..微血管内皮细胞可以促进造血干细胞CD34的表达。
[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,鍏卞煿鍏荤粍杈冨�圭収缁勬洿鏄庢槪�

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