摘要
背景:研究发现脂肪间充质干细胞核型具有不稳定性,且干细胞移植后长期安全评价等问题尚未从根本上得到解决。目的:观察体外分离培养的人脂肪间充质干细胞传代培养的遗传稳定性。设计、时间及地点:细胞学体外观察,于2008-03/11在解放军兰州军区兰州总医院骨科研究中心完成。材料:成人腹部大网膜脂肪组织来源于单纯性阑尾炎患者,由解放军兰州军区兰州总医院普通外科提供。方法:胶原酶消化法体外分离培养人脂肪间充质干细胞,加入含体积分数为10%胎牛血清的HG-DMEM培养基吹打细胞沉淀,过滤后于37℃、体积分数为5%的CO2饱和湿度条件下培养,待细胞生长至70%～80%融合时消化传代。分别取第3,25,30代脂肪间充质干细胞进行相关指标检测。主要观察指标:脂肪间充质干细胞表面标志表达、细胞增殖、核型分析、超微结构电镜观察、恶性肿瘤特异性生长因子的表达。结果:第3代脂肪间充质干细胞CD13,CD44,CD59均呈阳性表达,而造血干细胞表面标志CD34及成纤维细胞表面标志HLA-DR均呈阴性表达;染色体的形态、结构和数目均未见异常,呈二倍体结构;镜下可见完整的高尔基复合体,数量较多的粗面内质网,部分糖元颗粒分布在胞浆中。第25代脂肪间充质干细胞分裂增殖作用强于第3代脂肪间充质干细胞;多条染色体出现断裂畸变,并且染色体数目出现异常,呈亚二倍体或超二倍体核型;核仁增大,异染色质及线粒体数量增多,内质网数量少,细胞较为幼稚。第3,25,30代脂肪间充质干细胞恶性肿瘤特异性生长因子的表达吸光度值无明显差异(P>0.05)。结论:分离获得的人脂肪间充质干细胞长期体外培养具有遗传不稳定性,有向肿瘤细胞突变的倾向。
BACKGROUND：Studies have shown that karyotype of adipose-derived mesenchymal stem cells（ADMSCs） is unstable. The long-term safety of stem cell transplantation has not been completely solved. OBJECTIVE：To explore the genetic stability of ADMSCs cultured in vitro. DESIGN,TIME AND SETTING：The cytological in vitro study was performed at the Research Center of Orthopaedics,Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from March to November 2008. MATERIALS：Adult abdomen greater omentum adipose tissue obtained from appendicitis patients was supplied by the Department of General Surgery,Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA. METHODS：ADMSCs were isolated from adult adipose tissue by collagenase digestion in vitro,and then treated with HG-DMEM medium containing 10% fetal bovine serum. Following filtration,ADMSCs were incubated at 37 ℃,5% CO2 saturated humidity. Till 70%-80% confluency,ADMSCs were digested and subcultured. At passages 3,25 and 30,ADMSCs were used for relative detection. MAIN OUTCOME MEASURES：The following parameters were measured：ADMSCs surface marker expression,cell proliferation,karyotype analysis,ultrastructure（electron microscope） and malignant tumor specific growth factor. RESULTS：ADMSCs at passage 3 were positive for CD13,CD59 and CD44,but negative for CD34 and HLA-DR. Chromosomal morphous,structure and number were normal,showing diplont structure. Under the microscope,there were complete Golgi complex,plenty of rough endoplasmic reticulum,and some glycogen granules in cytoplasm. ADMSCs at passage 25 had strong proliferative ability compared with the passage 3. At the same time,chromosomes of ADMSCs were broken and aberrated,with abnormal chromosome number,which manifested subdiploid karyotype or hyperdiploid karyotype. There were bigger nuclei,heterochromatin,an increased number of chondrosomes,a decreased number of endoplasmic reticulum,and immature cells. At passages 3,25 and 30,no significant difference in absorbance of ADMSCs malignant tumor specific growth factor was detected（P 0.05） . CONCLUSION：After cultured for a long time in vitro,ADMSCs have genetic instability and tend to mutate into tumor cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第40期7833-7837,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
