BMSCs的软骨向诱导分化与调控及经淫羊藿苷干预的研究

发布时间：2018-04-02 08:12

  本文选题：骨髓间充质干细胞　切入点：软骨损伤　出处：《暨南大学》2009年硕士论文

【摘要】： 目的: 建立一种持续、稳定地向软骨细胞表型分化的BMSCs体外培养体系,观察淫羊藿苷对BMScs生物学行为的影响,并初步探讨对BMSCs的增殖及软骨向分化的作用及其可能机制,从而优化组织工程的两大因素:种子细胞及其分化调控,为改善修复软骨的质量提供参考。 方法: 1.运用密度梯度离心法和全骨髓贴壁法分离培养BMSCs。 2.采用相差显微镜观察其生长情况,扫描电镜观察细胞结构,流式细胞法观察细胞周期和细胞活力,MTT比色法描绘生长曲线。 3.用特定诱导液将BMSCs向成骨细胞和成脂肪细胞定向诱导分化,并进行ALP、钙结节和油红O等染色,对各种染色进行分析鉴定。 4.采用MTT比色法,观察不同浓度组淫羊藿苷对BMSCs增殖的影响。 5.将第3代BMSCs分为淫羊藿苷组、空白组、经典诱导组、淫羊藿苷与经典诱导液协同作用组,干预其软骨向分化,并定性分析——Ⅱ型胶原和甲苯胺蓝染色。 6.二甲基亚甲蓝显色法定量检测硫酸糖胺聚糖的分泌情况,并观察经典诱导液与淫羊藿苷的交互作用。 结果: 1.密度梯度离心法和贴壁生长法均能获得BMSCs,全骨髓贴壁法较优,分离后24h基本贴壁,12-15d达到融合。 2.经过成骨、成脂和成软骨诱导培养,BMSCs形态发生变化,分别表现出成骨细胞、脂肪细胞和软骨细胞表型,ALP、钙结节、油红O染色为阳性。 3.经MTT法检测发现1×10~(-5)mol/L、2×10~(-6)mol/L的淫羊藿苷浓度组OD值均高于对照组(P＜0.05),有促进BMSCs增殖的作用。 4.经以上淫羊藿苷浓度组诱导BMSCs软骨向分化,Ⅱ型胶原和甲苯胺蓝染色为阴性,且与所设浓度无相关性。 5.此淫羊藿苷浓度与经典诱导液协同作用组在软骨诱导培养条件下,Ⅱ型胶原和甲苯胺蓝染色为阳性,并促进硫酸糖胺聚糖分泌,且含量高于经典组(P＜0.01)。 结论: 通过体外对BMSCs的分离培养与鉴定,可以建立较完善的BMSCs培养体系;淫羊藿苷可明显促进BMSCs增殖,并且淫羊藿苷与经典诱导液存在交互协同作用,可促BMSCs软骨向分化;淫羊藿苷影响BMSCs分化软骨细胞生物学行为的可能机制是,通过促进体外培养的BMSCs增殖或者参与调控BMSCs向软骨分化,从而促进硫酸糖胺聚糖和Ⅱ型胶原的分泌使分化过程中的软骨组织不断成熟,改善修复软骨质量;这可以反映中医药补益先天等相关机理。
[Abstract]:Purpose :


To establish an in vitro culture system in vitro and stably for the phenotypic differentiation of chondrocytes , and observe the effect of Icariin on the biological behavior of BMScs , and to investigate the effect and possible mechanism of the proliferation and the differentiation of cartilage in vitro , so as to optimize the two factors of tissue engineering : seed cells and their differentiation and regulation , and provide a reference for improving the quality of repair cartilage .


Method :


1 . Bone marrow cells were isolated by density gradient centrifugation and full - marrow adherent method .


2 . The growth of cells was observed by phase contrast microscope . The cell structure was observed by scanning electron microscope . Cell cycle and cell viability were observed by flow cytometry , and the growth curve was depicted by MTT colorimetric method .


3 . The differentiation was induced into osteoblasts and adipogenic cells by specific induction liquid . ALP , calcium nodules and oil red O staining were performed to identify the various kinds of staining .


4 . MTT assay was used to observe the effect of Icariin on the proliferation of bone marrow cells .


5 . The third generation of bone marrow cells were divided into epimedin group , blank group , classical induction group , epimedin and classical induction liquid synergistic action group , which interfered with the differentiation of cartilage and qualitative analysis of type 鈪，

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