摘要
目的:目前对脂肪基质细胞向神经元样细胞分化基本采用人工合成的化学诱导剂的方法进行诱导,其中部分组合试剂费用昂贵,不适合进行大规模基础和临床实验。因此以复合诱导液作替代,观察其能否在体外诱导人脂肪基质细胞向神经元样细胞方向分化。方法:实验于2004-10/2005-06在华北煤炭医学院中学实验室完成。①实验材料:腹部皮下的脂肪组织来源于30例自愿捐献者,年龄20～35岁,通过外科手术获得。复合诱导液的组成:alpha-MEM+BHA(200μmol/L)+KCl(5mmol/L)+丙戊酸钠(2μmol/L)+IBMX(0.5mmol/L)+氢化可的松(1μmol/L)+胰岛素(5mg/L)+乙醇(0.5%)+青霉素(100U/mL)+链霉素(100mg/L)。②实验方法:离体的脂肪组织消化、离心、过滤,进行人脂肪基质细胞的原代培养。按8×103/cm2接种,消化传代。取第3代人脂肪基质细胞,接种到孔中预先放置无菌盖玻片的24孔培养板,细胞生长达50%～60%融合时,去除培养液,换为复合诱导液进行诱导;空白对照组培养液为DMEM/F12培养基。③实验评估:自加入诱导剂后在倒置相差显微镜下观察细胞生长情况和形态变化,应用免疫细胞化学方法检测神经前体细胞的特异性标志神经巢蛋白、神经细胞的特异性标志神经元特异性烯醇化酶及微管联合蛋白2、神经胶质细胞的特异性标志胶质纤维酸性蛋白的表达。结果:①体外培养过程人脂肪基质细胞的形态学观察:刚分离接种的细胞呈圆形,悬浮状态。接种24h内细胞大多已贴壁,呈梭形、圆形或多角形,有粗大突起,核居中,1～2个核仁。48h后贴壁细胞开始分裂增殖,多呈梭形。1周后细胞融合成单层,排列出现方向性,但有少量圆形及卵圆形细胞混杂生长。②复合诱导液诱导脂肪基质细胞向神经元样细胞分化的形态学观察:人脂肪基质细胞胞体回缩,呈圆形或锥形,胞体周围具有较强的光晕,胞体有突起伸展,具有典型的神经细胞样
AIM： At present, the differentiation of human adipose-derived adult stromal cell （hADASc） into neuron-like cells （NLCs） is induced by chemical reagents, but some of which are so expensive that they are .not suitable for the basic and clinical trials on a large scale. This paper describes whether differentiation cocktail can induce hADASc differentiate into NLCs in vitro. METHODS： The experiment was carried out in the Central Laboratory of North China Coal Medical College from October 2004 to June 2005. ①Adipose tissues were obtained surgically from 30 volunteers, who aged 20-35 years. Differentiation cocktail included alpha-MEM＋BHA （200 μmol/L）＋KCI （5 mmol/L）＋sodium valproate （2μmol/L） ＋3-isobutyl-l-methylxanthine （0.5 mmol/L）＋hydrocortisone （1μmol/L）＋insulin （5 mg/L）＋alcohol （0.5%）＋penicillin （100 U/mL）＋streptomycin （100 mg/L）.②lsolated adipose tissues were digested, centrifuged and filtrated. The hADASc were pdmadly cultured at the density of 8×10^3cm^2 to the third passage, then were inoculated on 24-well plate of cover glass. When the calls reached 50%-60% confluence, differentiation cocktail was used instead of culture liquid to induce hADASc, while the blank control group adopted DMEM/F12 medium.③lnverted phase contrast microscope was used to observe the growth and morphology of differentiated hADASc. The technique of immunocytochemistry was used to identify the neural-specific markers： Nestin, neural specific enolase （NSE）, microtubule associated protein-2 （MAP2） and glial fibrillary acidic protein （GFAP） of hADASc. RESULTS： ①Morphology of hADASc cultured in vitrσ The isolated cells were round and suspended. Within 24 hours Of inoculation, the calls were shaped as fusiform, round or polygon, with the appearances of prominence, middle nucleus and 1-2 nucleoli. Most of the cells were adhesive. With 48 hours of inoculation, the proliferation of adhesive cells was found and the shape was fusiform. One week later, cel
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第28期5469-5472,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
