摘要
目的 探讨组织工程肌腱保存过程中细胞存活率研究的方法。 方法 采用碘化丙啶 (PI)将第 4代人成纤维细胞的死细胞染色 ,双苯并咪唑染料 (Ho)染色活细胞后经适当波长紫外光激发 ,分别产生红色荧光和蓝色荧光 ,利用流式细胞仪区分活细胞和死亡细胞 ;将组织工程化肌腱种子细胞悬液 4 ml(6 .0× 10 5个 / ml)平分成两管 ,其中一管反复冻融 ,将细胞冻死 ;另一管不冻。再将两管细胞按不同比例混合 ,采用荧光染色流式细胞仪分析 ,研究其量效关系 ;并用荧光摄影 ,图像分析 ,即刻及 2小时荧光标记到流式细胞仪 ,检测对细胞存活率的影响。 结果 荧光染色流式细胞分析可反映加入冻融死细胞比例与存活细胞的量效关系 ,即细胞存活率与未冻融细胞和冻融细胞比例分别成正、负相关 ,相关系数 r=0 .970 (P<0 .0 5 ) ;荧光摄影后图像分析也显示了同样的量效关系 ,r=0 .982 (P<0 .0 5 ) ;荧光标记后 2小时检测比即刻检测细胞存活率降低 ,但差异无统计学意义 (P>0 .0 5 ) ;通过荧光显微镜可直接观察组织工程化肌腱上的细胞。 结论 PI和 Ho荧光染色后流式细胞仪分析及荧光摄影是组织工程肌腱保存过程中细胞存活率研究的较好方法。
Objective To study the research method of cell survival rate at the procedure of cryopreservation of tissue engineered tendons.Methods In the 4th generation of human fibroblasts, the dead cells were stained with propidium iodine (PI), while the living cells with Hoechst 33342(Ho). The living cells and dead cells emitted fluorescence of red and blue respectively after they were stimulated by suitable ultra-violet, then flow cytometry was applied to distinguish them. The seeding cells were collected to make them to be the cell suspension of suitable concentration(6.0×10 5 cell/ml) before they were divided into two parts. We cryopreserved and defrosted one part three times to kill the cells and did not cryopreserve the other part, then we made cell suspension at different ratios of cryopreserved cell to non-cryopreserved cells. The fluorescence staining and flow cytometry were used to study the correlation between cell ratios of cryopreserved cell to non-cryopreserved cell and the cell survival rates. We compared the cell survival rates between immediate flow cytometry and that 2 hours after fluorescence staining. Results The results of flow cytometry showed that correlation between the ratio of cryopreservation and the cell survival rate was significant (r=0.970,P<0.05), image analysis study also showed the correlation was significant (r=0.982,P<0.05).The cell survival rate decreased by use of flow cytometry two hours after fluorescence staining, but there was no significant difference when compared with that of immediate flow cytometry (P>0.05). We could also observe the cells on the tissue engineered tendons by fluorescence image directly. Conclusion Flow cytometry and fluorescence image after PI and Ho staining is a good way in study cell survival rate at the procedure of cryopreservation of tissue engineered tendons.
出处
《中国修复重建外科杂志》
CAS
CSCD
2004年第2期123-126,共4页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家高技术研究发展计划 (863)资助项目(2 0 0 1 AA2 1 60 1 1 )~~
关键词
组织工程肌腱
细胞存活率
低温保存
流式细胞仪
实验
保存过程
Tissue engineered tendons Cryopreservation Fluorescence staining Flow cytometry Cell survival rate
