SD大白鼠骨髓多能间质干细胞QY1细胞系的生物稳定性分析

Biological stability analysis of QY1 pluripotential mesenchymal stem cell line from Sprague Dawley rat bone marrow

目的：探索建立Sprague-Dawley大白鼠骨髓间质干细胞（mesenchymat stem cells，MSCs）系体外长期培养的适宜条件及其生物学特性，并分析其稳定性，为进一步研究MSCs的诱导分化和应用提供理想的细胞模型。方法：应用细胞生物学、干细胞组织工程学等方法培养并纯化骨髓MSCs，检测细胞生长的形态、换液时间、生长曲线、倍增时间、贴壁率、染色体、培养条件和反复冻存后复苏对其生物学特性的影响。结果：多次传代后的细胞呈均一的成纤维细胞样；骨髓MSCs原代培养10～12d时融合至80％～90％，不同的首次换液时间之间差异没有统计学意义（P〉0．05）；P1，P3，P10，P20代细胞生长曲线基本相同，细胞的倍增时间P1为34．2h；P3为33．9h；P10为31．8h；P20为30．6h。4代细胞任意两代之间的细胞数差异没有统计学意义（P〉0．05）；传代后细胞迅速贴壁，约89％的细胞贴壁主要发生在接种后10h内。4代细胞任意两代之间的贴壁率差异没有统计学意义（P〉0．05）；在浓度为20％的标准胎牛血清中，骨髓MSCs的增殖活性最高；在种植细胞密度为8×10^4／mL时，骨髓MSCs的增殖活性最高；吉姆萨染色显示P8代骨髓MSCs胞浆为淡紫红色，胞核为深蓝色，细胞内可见1～2个核仁；P8和P20代细胞染色体形态及数目均正常，染色体核型组成均为42，XY，符合大鼠正常二倍体细胞系特征；反复冻存后复苏骨髓MSCs，其活细胞数〉85％。至今已体外培养10个多月。结论：QY1细胞系是纯的、可长期传代的细胞系，可扩增并保持未分化状态，且其生物学性能稳定。

Objective To explore the optimal culture conditions in vitro and the biological characterizations of the QY1 pluripotential mesenchymal stem cell （ MSC ） line from Sprague-Dawley rat bone marrow and to analyze the biological stability of this MSC line so as to provide an ideal cell model for the further differentiation and actual application. Methods The methods and technologies of cell biology and stem cell tissue engineering were used to purify MSCs. We determined the effects of morphology of cell proliferation, the time of change medium, growth curves, doubling time, adhesive rates, chromosome, culture conditions, and repeated frost on the biological characterizations of MSCs. Results The cells had a fibroblastic-like morphology and were well spread out; 80% 90% cells became confluent between 10 and 12 days in primary culture. The growth curves of Passage 1,3,10, and 20 were quite similar（P〉0.05）. The doubling time of passage 1, 3, 10, and 20 were 34.2, 33.9, 31.8, and 30.6 hours, respectively. The adhesive rates of Passage 1 , 3, 10, and 20 were very much similar（P 〉 0.05 ）and about 89% subcuhured cells adhered to the wall in 10 hours. The most appropriate concentration of serum was 20% and the most appropriate concentration of cell number was 8 × 10^4/mL. The cells of Passage 8 showed light red cytoplasm and heavy blue nuclear stained by Giemsa staining. The chromosomes of Passage 8 and 20 were normal in appearance and their karyotypes were 42, XY. The cells were resuscitable after repeated frozen, and more than 85 % cells were alive. The cells had been continuously cultured for more than 10 months so far. Conclusion The QY1 MSCs line from Sprague-Dawley rat bone marrow is a purified cell line, which can maintain its undiffirentiation and the stable biological characterizations for the long term.