骨髓间充质干细胞在Matrigel凝胶支架上的生长与变化  

Bone marrow mesenchymal stem cells on Matrigel: growing and changing

作　　者：张斌斌 高全文[2] 李冰[3] 黄沙[4] 姚斌[4] Zhang Bin-bin;Gao Quan-wen;Li Bing;Huang Sha;Yao Bin(Department of Stomatology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China;Department of Burns and Plastic Surgery, First Hospital Affiliated to General Hospital of PLA, Beijing 100048, China;Hospital of Stomatology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China;Institute of Basic Medical Sciences, General Hospital of PLA, Beijing 100853, China)

机构地区：[1]山西医科大学口腔医学系,山西省太原市030001 [2]解放军总医院第一附属医院烧伤整形科,北京市100048 [3]山西医科大学口腔医院,山西省太原市030001 [4]解放军总医院基础医学研究所,北京市100853

出　　处：《中国组织工程研究》2018年第13期1993-1998,共6页Chinese Journal of Tissue Engineering Research

基　　金：北京市自然科学基金青年基金项目(7144249)~~

摘　　要：背景:研究表明,Matrigel作为一种细胞外基质复合物加入到细胞培养体系中,对细胞的增殖、分化、胶原分泌具有促进作用。目的:建立Matrigel复合骨髓间充质干细胞的三维培养模型,观察Matrigel三维培养条件下骨髓间充质干细胞的形态、增殖及存活情况。方法:全骨髓贴壁法分离培养骨髓间充质干细胞,进行成骨、成脂诱导鉴定;CCK-8测定第3代骨髓间充质干细胞的生长曲线;选取生长状态良好的第2代骨髓间充质干细胞,与Matrigel混合形成细胞凝胶复合物,采用相差显微镜和苏木精-伊红染色观察骨髓间充质干细胞的形态及增殖情况,Live/Dead染色评价细胞活性。结果与结论:(1)全骨髓贴壁法分离培养的比格犬骨髓间充质干细胞具备向成骨、成脂细胞分化的能力;(2)骨髓间充质干细胞的生长曲线呈S形,符合正常细胞的生长特性;(3)相差显微镜下可见骨髓间充质干细胞在Matrigel中逐渐伸长相互交联,呈三维网状生长,增殖状态良好,随着时间延长(7 d后),基质胶逐渐变软塌陷,14 d时仍可见少部分细胞网状交联生长;苏木精-伊红染色可见第4天时细胞胞质较大,第7天时细胞细长、相互交联;(4)三维培养第1,4,7天,骨髓间充质干细胞在Matrigel中活性百分比分别为92.57%,95.54%,97.37%,呈逐渐升高趋势;(5)结果表明,骨髓间充质干细胞在Matrigel中的增殖能力较强,活性较高。BACKGROUND： Matrigel as an extracellular matrix complex can facilitate cell proliferation, differentiation and collagen secretion in a cell culture system. OBJECTIVE： To establish a three-dimensional culture model of Matrigel combined with bone marrow mesenchymal stem cells （BMSCs） and to observe the morphology, proliferation and survival of BMSCs in the Matrigel three-dimensional culture model. METHODS： BMSCs were isolated and cultured by the whole bone marrow adherent method, followed by osteogenic and adipogenic induction and identification. The growth curve of passage 3 BMSCs was determined through the CCK-8 experiment. Passage 2 BMSCs were combined with Matrigel, and the morphology and proliferation of BMSCs were observed by hematoxylin-eosin staining under a phase contrast microscope. The viability of the cells was evaluated by the Live/Dead staining. RESULTS AND CONCLUSION： （1） BMSCs cultured by the whole bone marrow adherent method had the ability to differentiate into osteoblasts and adipocytes. （2） BMSCs exhibited an ＂S＂-shaped growth curve, which was consistent with the growth characteristics of normal cells. （3） Under the phase contrast microscope, BMSCs cultured by the Matrigel model were extended and interconnected in a three-dimensional network growth state with good proliferation. The Matrigel gradually became soft and collapsed over time （7days） and a few cells were still in a network growth after 14 days. Hematoxylin-eosin staining results showed that the cell cytoplasm was larger at 4 days and the cells became thinned and mutually cross-linked at 7 days. （4） The active percentage of BMSCs in the Matrigel model was 92.57%, 95.54% and 97.37% at 1, 4, 7 days of culture, respectively. To conclude, BMSCs cultured on the Matrigel has good proliferation and high viability.

关 键 词：干细胞 骨髓间充质干细胞 三维培养 MATRIGEL 细胞增殖 细胞活性 北京市自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]