大鼠骨髓间充质干细胞PKH26标记后生物学性状及其向血管内皮细胞分化研究

发布时间：2018-01-21 08:51

  本文关键词： PKH26 大鼠骨髓间充质干细胞 生物学性状 血管内皮细胞　出处：《山西医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 背景 细胞标记是体内外研究间充质干细胞(mesenchymal stem cells,MSCs)的关键问题,研究报道PKH26标记技术操作简单,易于检测,标记率高,体内移植60d仍可观察到荧光表达,国内类似报道较少。 系统性血管炎属于结缔组织病,易侵犯多系统、多脏器,发病率较高,不及时治疗,常危及生命。血管内皮细胞损伤、炎性细胞浸润是主要发病机制。研究发现MSCs在适宜条件下可分化为血管内皮细胞,具有免疫调节作用,参与组织重建和修复,这为调节血管炎患者机体免疫紊乱,修复受损的血管内皮细胞带来了新的希望。 目的 1.探讨大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cells,rBMSCs)PKH26标记后生物学性状,建立一种简单、实用、快速的rBMSCs标记方法。 2.体外诱导rBMSCs向血管内皮细胞分化,为系统性血管炎及其他血管炎患者的临床治疗提供新的思路。 方法 1. rBMSCs的分离培养与传代:密度梯度离心法结合贴壁法分离rBMSCs,用含10%胎牛血清的L-DMEM培养液培养,细胞至80%融合时进行传代,0.25%胰酶消化2-5min,按1:3进行传代扩增; 2. rBMSCs的鉴定:CD45、CD31分别是造血干细胞、骨髓内皮细胞特有的表面标志,骨髓来源的MSCs上述2种抗原呈阴性,MSCs无特异性表面标志,表达CD44、CD71。我们通过流式细胞仪检测rBMSCs表面抗原CD44、CD71、CD31及CD45的表达,通过排除法鉴定所培养细胞为rBMSCs; 3.检测rBMSCs在PKH26标记后生物学性状:PKH26标记第3代rBMSCs,荧光显微镜下观察细胞形态学;流式细胞仪检测细胞标记率;MTT法测定生长曲线,比较两组细胞群体倍增时间;流式细胞仪检测细胞周期,比较两组细胞增殖指数;流式细胞仪检测两组细胞的表面抗原CD44、CD71、CD31及CD45; 4.体外诱导rBMSCs向血管内皮细胞分化:含40ng/mlVEGF的L-DMEM诱导液诱导第3代rBMSCs向血管内皮细胞分化,于诱导后第14d流式细胞仪鉴定其表面抗原CD31表达水平,免疫组化鉴定其表面FⅧ的表达。 结果 1.经流式细胞仪检测表面抗原,结果显示CD44阳性,CD71弱阳性,CD31、CD45阴性,鉴定为间充质干细胞; 2.荧光显微镜下观察PKH26标记后的rBMSCs大部分呈长梭形,排列规则,呈平行、旋涡状生长,与未标记细胞比较无明显变化;标记后10d流式细胞仪检测细胞标记率达91.03%,标记后40d仍可在荧光显微镜下观察到荧光表达;t检验结果显示两组细胞群体倍增时间及增殖指数差异不具有统计学意义(P0.05);流式细胞仪检测结果显示两组细胞均为CD44阳性,CD71弱阳性,CD31、CD45阴性; 3.体外诱导rBMSCs向血管内皮细胞分化,14d后流式细胞仪检测表明CD31表达较未诱导前增加37.36%,免疫组化鉴定FⅧ表达阳性。 结论 1. PKH26标记方法操作简单,细胞标记率高,标记时间达6周左右,未影响rBMSCs的生物学性状,为需求大量标记后rBMSCs的科学研究提供了一种实用、简单、快速的标记方法; 2.体外诱导条件下rBMSCs可向血管内皮细胞分化,为系统性血管炎及其他血管炎患者的临床治疗提供新的思路和方法。
[Abstract]:background
Cell labeling is a key issue in the study of mesenchymal stem cells (MSCs) in vivo and in vitro. The PKH26 labeling technology is simple, easy to detect, and has high labeling rate. The expression of 60d in vivo can still be observed. Similar reports are rare in China.
Systemic vasculitis belong to connective tissue disease, easy invasion of multi system, multi organ, high incidence, not timely treatment, often life-threatening. The injury of vascular endothelial cells, inflammatory cell infiltration is the main pathogenesis. MSCs was found in suitable conditions can differentiate into vascular endothelial cells, has immunomodulatory effects, participate in tissue reconstruction and restoration, the regulation of immune disorders in patients with vasculitis, repair damaged endothelial cells has brought new hope.
objective
1., we explored the biological characteristics of rat bone marrow mesenchymal stem cells (rBMSCs) PKH26 markers, and set up a simple, practical and fast rBMSCs labeling method.
2. the differentiation of rBMSCs into vascular endothelial cells in vitro provides a new way of thinking for the clinical treatment of systemic vasculitis and other vasculitis.
Method
1. rBMSCs isolation culture and passage: rBMSCs was separated by density gradient centrifugation combined with adherent method, and cultured in L-DMEM containing 10% fetal bovine serum. The cells were passaged to 80% fusion and 2-5min was digested by 0.25% trypsin, and then amplified by 1:3.
Identification of 2. rBMSCs: CD45, CD31 respectively, hematopoietic stem cells, bone marrow endothelial cell specific surface markers of bone marrow derived MSCs 2 antigens were negative, MSCs has no specific surface markers, expression of CD44 CD71., we detected by rBMSCs surface antigen CD44, CD71 flow cytometry, the expression of CD31 and CD45. By excluding identification of cultured cells is rBMSCs;
Detection of rBMSCs in biological characters of 3. PKH26 markers: PKH26 marks the third generation of rBMSCs, cell morphology was observed under the fluorescence microscope detection rate of labeled cells; flow cytometry; Determination of growth curve of MTT was compared between the two groups, the doubling time of the cells; flow cytometry cell cycle and cell proliferation index were compared between the two groups; flow cytometry cell surface antigen was detected two groups of cells in the CD44, CD71, CD31 and CD45;
4. in vitro differentiation of rBMSCs induced vascular endothelial cells: 40ng/mlVEGF containing L-DMEM was induced by the third generation of rBMSCs induced differentiation into vascular endothelial cells, the expression level of CD31 antigen on the surface of the 14d flow cytometry after induction by immunohistochemistry to identify the surface expression of F VIII.
Result
1. the surface antigen was detected by flow cytometry. The results showed that CD44 was positive, CD71 was weak positive, CD31 and CD45 were negative, which were identified as mesenchymal stem cells.
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