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全骨髓贴壁法获取组织工程髓核种子细胞的相关研究*
引用本文:康健,侯洋 周许辉 杨立利 陈华江 唐先业 袁文.全骨髓贴壁法获取组织工程髓核种子细胞的相关研究*[J].生物骨科材料与临床研究,2014,11(1):10-15.
作者姓名:康健  侯洋 周许辉 杨立利 陈华江 唐先业 袁文
作者单位:[1]第二军医大学附属长征医院骨科,上海200003 [2]山东省泰安市解放军88医院骨科中心,山东泰安271000
基金项目:国家自然科学基金资助项目(81071509)
摘    要:目的明确兔骨髓间充质干细胞的分化能力及分化条件,以TGF-β3和BMP-7诱导兔骨髓间充质干细胞向类髓核细胞分化,为组织工程髓核的构建提供良好的种子细胞。方法利用全骨髓贴壁法分离获取兔骨髓间充质干细胞,并对细胞进行培养和传代,以特定的环境及细胞诱导液进行诱导,促使其向特定的中胚层细胞分化。再将细胞分为四组,A:空白对照组,B:BMP-7诱导组,C:TGF-β3诱导组,D:TGF-β3与BMP-7共同诱导组,诱导21天后对蛋白聚糖、Ⅰ型胶原、Ⅱ型胶原、Ⅹ型胶原和SOX9基因进行realtime-PCR检测。结果全骨髓贴壁法分离获得的原代兔骨髓间充质干细胞生长良好,2周后显微镜下观察细胞间形态较为一致,成纺锤形。细胞传代后生长良好。细胞表面表达CD29、CD105、CD166表面标记物。在成骨诱导培养液、成软骨诱导培养液和脂肪诱导培养液的诱导下,培养21天后,通过特定染色发现兔骨髓间充质干细胞向预定方向分化生长。以TGF-β3和BMP-7生长因子诱导21天后,蛋白聚糖、Ⅱ型胶原和SOX9基因表达C组和D组明显高于A组和B组(P0.05),X型胶原表达A组和B组明显高于C组和D组(P0.05),Ⅰ型胶原表达四组之间无明显差异。结论全骨髓贴壁法可以成功分离获得状态良好的兔骨髓间充质干细胞,在成骨诱导培养液、成软骨诱导培养液和脂肪诱导培养液的诱导下,兔骨髓间充质干细胞可向预定的方向分化且生长良好。TGF-β3和BMP-7诱导兔骨髓间充质干细胞后可明显提高其类髓核细胞具有的下游基因表达水平。

关 键 词:兔骨髓间充质干细胞  全骨髓贴壁法  诱导分化  转化生长因子3  骨形态发生蛋白7

The study of obtaining the seed cells in engineering nucleus pulposus through all bone marrowadherence method
Kang Jian,Hou Yang,Zhou Xuhui,et al.The study of obtaining the seed cells in engineering nucleus pulposus through all bone marrowadherence method[J].Orthopaedic Biomechanics Materials and Clinical Study,2014,11(1):10-15.
Authors:Kang Jian  Hou Yang  Zhou Xuhui  
Institution:Kang Jian, Hou Yang, Zhou Xuhui,et al. (1 Orthopaedic Surgery, Changzheng Hospital,Second Military Medical University,Shanghai,200003;2 Orthopaedic Surgery,No.88 Hospital of People's Liberation Army,Tai'an Shandong,271000,China)
Abstract:Objective To identify the demanding environment of differentiation and differentiation ability of BMSCs,in order to choose a suitable seed cells for tissue engineering nucleus pulposus. Methods Obtain the rabbit BMSCs withthe method of direct way. Be sure of the BMSCs through the surface mark, and then induce it to differentiate into threedifferent mesoderm cells. Then devide BMSCs into four groups A blank control group, B singly induced by TGF- 3,C Singly induced by BMP-7, Dcombination induced by TGF- 3 and BMP-7. Measure the expression of ACAN、CollagenⅠ、CollagenⅡ、CollagenⅩand SOX9 in mRNAlevel through Realtime PCR after 21 days cultivation. ResultsThe cells got through the all bone marrow adherence method are able to express cell surface marker of CD29, CD105,CD166 according to flowcytometry detection. After 21 days induced by certain kinds of induction medium, the BMSCsdifferentiated into at least three kinds of mesoderm cells: osteoblast, chondroblast and adipocyte. The expression levelof ACAN, CollagenⅡand SOX9 in groupCandDwas obviously higher than groupAandB( 〈0.05). The expressionlevel of CollagenⅩin group Aand B was much higher than group C and D( 〈0.05). And there was no obviously differencebetween four groups with CollagenⅠ expression level. Conclusion The BMSCs could be obtained successfullythrough the all bone marrow adherence method, and the cells induced by induction medium are able to differentiate intosome kinds of mesodermcells. The BMSCs was able to show a high expression level of downstreamgene in NPCs.
Keywords:BMSCs  All bone marrow adherence method  Induced differentiation  TGF- 3  BMP-7
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