摘要
目的探讨短期冻存对兔骨髓间充质干细胞(BMSCs)的增殖、生物特征及成骨能力的影响。方法选取第5代兔BMSCs冻存,30d后复苏、体外培养至第10代作为实验组,设相同代数未冻存的BMSCs为对照组。MTT、细胞周期、细胞表面标志及成骨诱导分化后碱性磷酸酶(ALP)水平、Ⅰ型胶原及钙结节染色作为其增殖、生物特征及成骨能力的评价指标。结果冻存后BMSCs生长潜伏期延长,但生长状态无明显影响;增殖指数和G1期细胞所占比例分别为42.9%±3.4%、57.0%±3.4%,细胞表面标志CD44阳性率为93.62%±1.05%,不表达CD45;成骨诱导第7、14天ALP水平(U/gprot)分别为6.73±1.92、15.99±4.36,第14、21天,Ⅰ型胶原、钙结节茜素红染色阳性;以上结果与对照组比较差异均无统计学意义(P>0.05)。结论短期冻存对兔BMSCs增殖、生物特征及成骨分化潜能无明显影响,冻存后可继续进一步研究。
Objective To study the effects of short-term cryopreservation on the proliferation ,bio-characteristics and osteogenic capability of rabbit BMSCs and lay the foundation for the further study .Methods The 5th generation of rabbit BMSCs were cryo-preservated by liquid nitrogen for 30 days ,and then thawing the cells to culture it to the 10th generation in vitro ,put the non-cryo-preservated and the same generation BMSCs as the control group .The MTT ,cell cycle ,cell surface marker ,the content of ALP and the immunohistochemical staining for collagen typeⅠ and alizarin red staining for calcium after differentiation induced by osteogene-sis were used to evaluate the proliferation ,bio-characteristic and osteogenic differentiation capability of rabbit BMSCs .Results The growth incubation period of BMSCs after cryopreservated was extended ,but it gradually recover through serial passage .The prolif-eration index and the proportion of BMSCs in G1 phase were 42 .9% ± 3 .4% and 57 .0% ± 3 .4% respectively .The positive rate of cell surface marker of CD44 was 93 .62% ± 1 .05% without expression of CD45 .The contents of ALP（U/gprot） were 6 .73 ± 1 .92 and 15 .99 ± 4 .36 in BMSCs after 7th day and 14th day with osteogenic induction ,respectively .The collagen typeⅠ and alizarin red staining for calcium indicated positive at BMSCs after 14th day and 21th day with osteogenic induction ,respectively .These results showed no significant difference compared with the control group （P〉0 .05） .Conclusion Short-term cryopreservation has no obvi-ous impacts on the proliferation ,bio-characteristic and osteogenic capability of BMSCs and it could be used for further study .
出处
《重庆医学》
CAS
CSCD
北大核心
2014年第4期459-461,464,共4页
Chongqing medicine
基金
国家自然科学基金资助项目(31160199)
关键词
骨髓间充质干细胞
冻存
生物特性
bone marrow mesenchymal stem cells
cryopreservation
bio-characteristic
