几种成年小鼠胸腺上皮细胞分离和富集方法效果的比较

doi:10.13481/j.1671-587x.20160106

Abstract

Abstract:

Objective: To compare the effects between adult murine thymic epithelial cells (TECs)isolation and enrichment methods,and to find a high-efficiency TECs enrichment method with accurate invivo representation.Methods: Twenty-seven BABL/c mice (6-8 weeks)were divided into nine groups,and each group contained three mice. ① The isolation experiment contained five isolation groups: collagenase Ⅴ 1.0 g·L^-1 group and 0.5,0.7,1.0,2.0 U·mL^-1 Liberase^TM groups. Thymus digestion was performed according to relevant enzyme working concentrations. ② The enrichment experiment contained four enrichment groups: control,magnetic activated cell sorting (MACS),Percoll and Ficoll enrichment groups;in each group,thymus digestion was performed using the optimal working concentration (1.0 U·mL^-1)of Liberase ^TM and the digests were continued for TECs enrichment by MACS,Percoll,Ficoll respectively (except control group). For isolation experiment,in each group,the cell viability and the yield of thymic stromal cells (TSCs)were examined by flow cytometry. For the enrichment experiment,the cell viability,the percentage of TSCs and the expression of epithelial cell adhesion molecule (EpCAM)in TSCs in each group were examined by flow cytometry. Furthermore,in both Percoll enrichment group and control group,the percentages of cortical TECs (cTECs)and medullary TECs (mTECs)were examined as well.Results: For the isolation experiment,the yield of TSCs in 1.0 U·mL^-1 Liberase^TM group was higher than those in 0.5 and 0.7 U·mL^-1 Liberase^TM groups(P<0.01);the cell viability and digestion time in 1.0 U·mL^-1 Liberase^TM group were superior to 1.0 g·L^-1 collagenase Ⅴ group (P<0.01). The cell viabilities in three enrichment groups were higher,and there were no significant differences between them (P > 0.05).Compared with control group,the percentages of TECs in three enrichment groups were increased (P<0.05 or P<0.01). And among three enrichment groups,the percentage of TECs in Percoll enrichment group was higher than those in other two groups (P<0.01). There were no significant differences in the percentages of cTECs and mTECs as well as the ratios of cTEC to mTEC between control group and Percoll enrichment group (P > 0.05). Conclusion: Liberase ^TM is more suitable for adult murine TECs isolation than collagenase Ⅴ and its optimal digestion concentration is 1.0 U·mL^-1.All the three enrichment methods mentioned above can increase the percentage of TECs,and they all do little harm to the cells;Percoll is the most efficient method for adult murine TECs enrichment among three enrichment methods,and it doesn't affect the ratio of cTEC to mTEC,thereby making it easier for the following study.

Key words: thymic epithelial cells, cell enrichment, flow cytometry

CLC Number:

R-331

QI Huihui, CHEN Bing, CHEN Xiaona, WANG Yaoqi, TONG Qingyue, SUN Liguang, YANG Yongguang. Comparison of effects between adult murine TEC isolation and enrichment methods[J].Journal of Jilin University Medicine Edition, 2016, 42(01): 24-30.

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Comparison of effects between adult murine TEC isolation and enrichment methods

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