本文選題：骨髓間充質干細胞　切入點：軟骨損傷　出處：《暨南大學》2009年碩士論文

【摘要】： 目的: 建立一種持續(xù)、穩(wěn)定地向軟骨細胞表型分化的BMSCs體外培養(yǎng)體系,觀察淫羊藿苷對BMScs生物學行為的影響,并初步探討對BMSCs的增殖及軟骨向分化的作用及其可能機制,從而優(yōu)化組織工程的兩大因素:種子細胞及其分化調控,為改善修復軟骨的質量提供參考。 方法: 1.運用密度梯度離心法和全骨髓貼壁法分離培養(yǎng)BMSCs。 2.采用相差顯微鏡觀察其生長情況,掃描電鏡觀察細胞結構,流式細胞法觀察細胞周期和細胞活力,MTT比色法描繪生長曲線。 3.用特定誘導液將BMSCs向成骨細胞和成脂肪細胞定向誘導分化,并進行ALP、鈣結節(jié)和油紅O等染色,對各種染色進行分析鑒定。 4.采用MTT比色法,觀察不同濃度組淫羊藿苷對BMSCs增殖的影響。 5.將第3代BMSCs分為淫羊藿苷組、空白組、經(jīng)典誘導組、淫羊藿苷與經(jīng)典誘導液協(xié)同作用組,干預其軟骨向分化,并定性分析——Ⅱ型膠原和甲苯胺藍染色。 6.二甲基亞甲藍顯色法定量檢測硫酸糖胺聚糖的分泌情況,并觀察經(jīng)典誘導液與淫羊藿苷的交互作用。 結果: 1.密度梯度離心法和貼壁生長法均能獲得BMSCs,全骨髓貼壁法較優(yōu),分離后24h基本貼壁,12-15d達到融合。 2.經(jīng)過成骨、成脂和成軟骨誘導培養(yǎng),BMSCs形態(tài)發(fā)生變化,分別表現(xiàn)出成骨細胞、脂肪細胞和軟骨細胞表型,ALP、鈣結節(jié)、油紅O染色為陽性。 3.經(jīng)MTT法檢測發(fā)現(xiàn)1×10~(-5)mol/L、2×10~(-6)mol/L的淫羊藿苷濃度組OD值均高于對照組(P＜0.05),有促進BMSCs增殖的作用。 4.經(jīng)以上淫羊藿苷濃度組誘導BMSCs軟骨向分化,Ⅱ型膠原和甲苯胺藍染色為陰性,且與所設濃度無相關性。 5.此淫羊藿苷濃度與經(jīng)典誘導液協(xié)同作用組在軟骨誘導培養(yǎng)條件下,Ⅱ型膠原和甲苯胺藍染色為陽性,并促進硫酸糖胺聚糖分泌,且含量高于經(jīng)典組(P＜0.01)。 結論: 通過體外對BMSCs的分離培養(yǎng)與鑒定,可以建立較完善的BMSCs培養(yǎng)體系;淫羊藿苷可明顯促進BMSCs增殖,并且淫羊藿苷與經(jīng)典誘導液存在交互協(xié)同作用,可促BMSCs軟骨向分化;淫羊藿苷影響B(tài)MSCs分化軟骨細胞生物學行為的可能機制是,通過促進體外培養(yǎng)的BMSCs增殖或者參與調控BMSCs向軟骨分化,從而促進硫酸糖胺聚糖和Ⅱ型膠原的分泌使分化過程中的軟骨組織不斷成熟,改善修復軟骨質量;這可以反映中醫(yī)藥補益先天等相關機理。
[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，

本文編號：1699514