高纯度大鼠下颌骨来源间充质干细胞的分离、培养及生物学特性

Isolation,culture and biological characteristics of high-purity orofacial-bone-derived mesenchymal stem cells of the rats

背景:以募集颌骨来源间充质干细胞来修复颌骨骨缺损这种新观点逐渐被广泛认可,但目前对颌骨来源间充质干细胞的研究相对较少,主要由于颌骨来源间充质干细胞分离困难。目的:建立大鼠下颌骨来源间充质干细胞的体外分离、培养方法,并观察研究其相关生物学特性。方法:解剖分离大鼠下颌骨,剥离表面附着肌肉后剪碎,用Ⅱ型胶原酶消化疏松密质骨,利用间充质干细胞的迁徙和贴壁生长能力分离下颌骨间充质干细胞。采用倒置显微镜观察细胞形态;流式细胞仪检测细胞表面抗原;MTT法检测细胞增殖情况并绘制细胞生长曲线;克隆形成实验计算克隆形成率;成骨、成脂诱导实验研究细胞的多向分化潜能。结果与结论:胶原酶消化骨片培养法分离的细胞阳性表达CD29、CD44、Sca-1,阴性表达CD31、CD34和CD45;细胞增殖能力检测提示下颌骨间充质干细胞生长经历了潜伏期、对数生长期和平台期,具有较强的增殖能力;细胞克隆形成率达20%且处于DNA合成期的细胞占52.5%;成骨、成脂诱导后行茜素红和油红O染色均呈阳性,表明该细胞具有多向分化潜能。结果表明胶原酶消化骨片培养法可从大鼠下颌骨分离足量、高纯度的下颌骨间充质干细胞,且该细胞具有较强的增殖和成骨分化能力,为以种植体骨缺损修复为代表的颌骨骨组织工程提供充足的种子细胞来源。

BACKGROUND:It is widely accepted to recruit mesenchymal stem cells from the jaws to repair the defects of the jaws.However,there are relatively few researches on orofacial-bone-derived mesenchymal stem cells,mainly due to the difficulty in separating mesenchymal stem cells from the jaws.OBJECTIVE:To establish the methods of in vitro isolation and culture of rat orofacial-bone-derived mesenchymal stem cells and observe and study its related biological characteristics.METHODS:The mandibles of rats were dissected.The attached muscles were stripped and cut into pieces.Cortical bone was loosened by digesting with collagenase II.The migration and adherent growth ability of mesenchymal stem cells was used to isolate orofacial-bone-derived mesenchymal stem cells.Cell morphology was observed by inverted microscope.Surface markers of the cell were detected by flow cytometry.Cell proliferation was detected by MTT assay and cell growth curve was drawn.Fibroblast colony forming rate was calculated by colony formation.Osteogenic and lipogenic induction experiments were conducted to study the multi-directional differentiation potential of cells.RESULTS AND CONCLUSION:Cells isolated by collagenase digestion and bone slice culture were positive for CD29,CD44 and Sca-1,and negative for CD31,CD34 and CD45.Cell proliferation test showed that the growth curves of orofacial-bone-derived mesenchymal stem cells exhibited incubation period,logarithmic phase and platform period.In addition,the cells had a strong ability of proliferation,and the cell clone formation rate was 20%and the cells in DNA synthesis stage accounted for 52.5%.Alizarin red and oil red O staining showed positive reaction after osteogenic and lipogenic induction,indicating that the cells have the potential of multi-directional differentiation.It is concluded that the method of bone fragment culture after digestion with collagenase II could separate orofacial-bone-derived mesenchymal stem cells sufficiently and purely.Besides,the orofacial-bone-derived mesenchymal stem cells show strong proliferative and osteogenic differentiation capacities.Thus,it provides abundant source of seed cells for bone tissue engineering of maxillofacial represented by bone defects repairing of implants.