大鼠骨髓间质干细胞基本生物学特性的研究

Study on the biological characteristics of rat bone marrow mesenchymal stem cells

目的:研究成年大鼠骨髓间质干细胞(rBMMSCs)的基本生物学特性,并与成人骨髓间质干细胞(hB-MMSCs)作比较。方法:分别采用贴壁法和Ficoll-Paque淋巴细胞分离法分离获得rBMMSCs和hBMMSCs,进行体外传代、扩增、纯化,比较分析不同代数的rBMMSCs和hBMMSCs的增殖能力和克隆形成能力,观察传代次数对rBMM-SCs的神经分化能力的影响,流式细胞仪检测rBMMSCs表面抗原表达;采用中期分裂相秋水仙素阻抑法分析rBMM-SCs的核型。结果:原代rBMMSCs和hBMMSCs在体外扩增后可分别获得(7-8)×10~5和(5-6)×10~5个细胞,体外扩增5代后可分别获得1.7×10~8和1×10~8个细胞,体外扩增15代后,可分别获得(4-8)×10^(12)和(3-4)×10^(12)个细胞,随着传代次数的增加,细胞的增殖能力、克隆形成能力和神经分化能力有所下降,但各代rBMMSCs的增殖能力和克隆形成能力都比hBMMCs较强。流式细胞仪检测结果显示rBMMSCs的CDllb、CD45、CD61、CD71、CD80、CD86、MHCⅠ、MHCⅡ表达为阴性,而CD29和CD44表达阳性。rBMMSCs为正常大鼠二倍体核型。结论:rBMMSCs具有极强的自我更新能力和神经分化能力,是一种具有正常二倍体核型的间质干细胞,在组织工程中具有广阔的应用前景。

AIM: To investigate the basic biological characteristics of adult rat bone marrow mesenchymal stem cells(rBMMSCs), and compare to that of human BMMSCs (hBMMSCs). METHODS: rBMMSC and hBMMSCs were separated from bone marrow with the difference of adherence and Ficoll-Paque reagent, and expanded in culture medium in vitro, respectively. The proliferation and growth characteristics of the primary and different passage culture of rBMMSCs and hBMMSCs were analysed. The neural differentiation capacity of rBMMSCs with passages were observed. To detect the surface antigens of rBMMSCs, the labeled cells were analysed on a FACScan flow cytometer. The karyotype of rBMMSCs were detected by blocking cellular fission with colchicines. RESULTS: rBMMSCs and hBMMSCs have a strong self-renewal capacity. Approximately (4-8)×10^(12) and (3-4)×10^(12) cells were obtained after passage 15 in vitro, respectively. The ability of proliferation, CFU-Fs, and neural differentiation of rBMMSCs and hBMMSCs were decreased gradually with passages, but the ability of proliferation and CFU-Fs of rBMMSCs were higher than that of hBMMSCs at different passage. FACScan result showed rBMMSCs were uniformly positive for CD29 and CD44, and negative for CD11b, CD45, CD61, CD71, CD80, CD86,MHC Ⅰ and MHC Ⅱ. rBMMSCs had an normal karyotype, which had an average of 37.0±4.0 to 40.5±2.5 chromosomes. CONCLUSION: Adult rBMMSCs have strong self-renewal and neural differentiation capacity, and have an normal karyotype. So rBMMSCs can be used as the seed cells for tissue engineering.