摘要
目的:观察成年大鼠体外原代培养骨髓间充质干细胞向神经细胞的转化情况。方法:实验于2005-08/2006-02在青岛大学医学院附属医院脑血管病研究所细胞生物工程实验室进行,取2只4个月龄Wistar大鼠,获取胫骨和股骨骨髓,提取骨髓间充质干细胞,无血清和有血清培养基进行细胞克隆,应用碱性成纤维细胞生长因子和表皮生长因子进行细胞扩增及诱导分化。采用IX-70型荧光倒置显微镜进行形态追踪观察。诱导分化后的细胞分别用巢蛋白抗体、神经元特异性烯醇化酶抗体、胶质原性纤维酸性蛋白抗体进行标记。结果:2只实验大鼠均进入结果分析。①原代细胞的生长情况和形态变化:初接种的细胞呈悬浮状态,接种后1d内,骨髓间充质干细胞逐渐开始贴壁,2d后贴壁细胞开始增殖。3d后细胞呈长梭形及扁平状,出现聚集细胞团并有纤维发出,细胞间呈单层成纤维网状结构,纯化的骨髓间充质干细胞随培养时相的推进,细胞数呈幅度性扩增,细胞分化扩增呈散在性和聚集性细胞集落,出现干细胞克隆团。②诱导分化细胞的生长情况和形态变化:两周后分别加入碱性成纤维细胞生长因子和表皮生长因子诱导骨髓间充质干细胞向神经细胞定向性分化。第3周换液,细胞克隆球趋于衰老期呈分裂崩解过程,形成片状细胞,向神经元分化。4周后片状干细胞发出大量的轴突及树突丝状纤维向神经元及神经胶质细胞分裂增殖。③神经细胞特异性标志的表达:镜下观察(x200/视野)结果显示:经培养一两周后,分化细胞经巢蛋白抗体标记呈棕黄色深染为神经干样细胞,约占80%;3周后,分化细胞经神经元特异性烯醇化酶抗体标记呈棕黄色深染为神经元,约占70%;4周后,分化细胞经神经胶质纤维酸性蛋白抗体标记呈棕黄色深染为神经胶质细胞,约占90%。结论:骨髓间充质干细胞具有较强的自我更新能力和多分化潜能,在合适的环境条件下,可诱导分化出神经元和神经胶质细胞,是较为理想的种子细胞。
AIM： To observe the transformation of bone marrow messenchymal stem cells （BMSCs） of adult rats cultured in vitro into neural cells. METHODS： The experiment was conducted in the Cell Bioengineering Laboratory of Institute of Cerebrevascular Diseases, Affiliated Hospital of Qingdao University Medical College between August 2005 and February 2006. Two 4-month old Wistar rats were selected, and the marrow in tibia and femoral bone was taken to extract the BMSCs, which were cultured in medium containing or without serum for cell clone. The basic fibroblast growth factor （bFGF） and epidermal growth factor （EGF） were used for cell amplification and differentiation. The cell morphous was observed with IV-70 fluorescence inverted microscope. The differentiated cells were labeled by nidogen antibody, neurenspecific enolase antibody and glial fibrillary acidic protein （GFAP） antibody. RESULTS： The two rats were involved in the result analysis. （1）Growth condition and morphologic changes of the primary cells： The cells floated initially after seeding, and BMSCs began to adhere in 1 day and proliferate after 2 days. Three days later, the cells showed long fusiform shape and thin and fiat, accompanying with assembled cell groups and fiber;, there was monolayer fiber network structure betweencells, and the number of purified cells amplified extently with phase; the cells differentiated scatteredly, and aggregated cell colony was found, accompanying with stem cell cloning group. （2）Grewth condition and morphologic changes of differentiated cells： Two weeks later, the bFGF and EGF were added to induce the BMSCs to differentiate into neural cells. The solution was changed at the 3rd week; the cell cloned ball trended to ageing and began to cleavage. The lamellar cells were observed and began to differentiate into neurons. Four weeks later, the lamellar cells erupted a great amount of axon and cytodendrite-like fiber, which divided into neurons and glia cells. （3）Expression of neuronspecific marks： The results observed under microscope （x200/field） showed that after 1-2 weeks culture, the differentiated cells showed deep stained nerve cord-like cells by nidogen antibody labeling, accounted for 80%; three weeks later, the differentiated cells showed brown yellow neurons by neuronspecific enolase antibody labeling, accounted for 70%; four weeks later, the differentiated cells showed brown yellow glia cells by GFAP antibody labeling, accounted for 90%. CONCLUSION： BMSCs have strong potency of self-renewal and multi differentiation; they can be induced to differentiate into neurons and gUa cells under suitable environment, which can be regarded as seed cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第7期1243-1246,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
