几种成年小鼠胸腺上皮细胞分离和富集方法效果的比较
发布时间：2019-08-15 12:39
【摘要】：目的:通过比较几种成年小鼠胸腺上皮细胞(TECs)分离和富集方法的效果,寻找一种具有准确体内代表性的高效TECs富集方法。方法:27只6~8周龄BABL/c小鼠分为9组,每组3只。1分离实验分为5个分离组,包括1.0g·L~(-1) collagenaseⅤ组和0.5、0.7、1.0及2.0U·mL~(-1) LiberaseTM组,按照相应的酶工作浓度对胸腺组织进行消化处理;2富集实验分为4个富集组,包括对照组和免疫磁珠(MACS)、Percoll及Ficoll富集组,各组用1.0U·mL~(-1) LiberaseTM消化胸腺组织后,将所得的单细胞悬液分别用MACS、Percoll和Ficoll法进行细胞富集(对照组除外)。分离组通过流式细胞术检测细胞活力及胸腺基质细胞(TSCs)的产量;富集组通过流式细胞术检测细胞活力、TSCs比例和TSCs中TECs重要标志上皮细胞黏附分子(EpCAM)的表达丰度,检测Percoll富集组和对照组TECs的2个亚群——皮质TECs(cTECs)和髓质TECs(mTECs)比例。结果:1.0U·mL~(-1) Liberase TM组中TSCs的产量明显高于0.5和0.7U·mL~(-1) LiberaseTM组(P0.01),且其细胞活力及消化时间均优于1.0g·L~(-1)collagenaseⅤ组(P0.01)。3个富集组的细胞活力均较高,但组间比较差异无统计学意义(P0.05);与对照组比较,3个富集组TECs比例明显增加(P0.05或P0.01);在3个富集组中,Percoll富集组富集的TECs比例高于其他2个富集组(P0.01);与对照组比较,Percoll富集组cTECs和mTECs的百分比及cTEC/mTEC比值无明显变化(P0.05)。结论:Liberase TM较collagenaseⅤ更适用于消化成年小鼠胸腺,其最适消化浓度为1.0U·mL~(-1)。本研究采用的3种富集方法均可以提高TECs比例,对细胞的破坏均较小;Percoll富集法最有效,在富集后不影响TECs亚群细胞比例,便于后续实验操作。
[Abstract]:Aim: to compare the effects of several methods for isolation and enrichment of (TECs) in thymic epithelial cells of adult mice, and to find an accurate and efficient TECs enrichment method in vivo. Methods: 27 BABL/c mice aged 6 脳 8 weeks were divided into 9 groups with 3 mice in each group. 1 the isolation experiment was divided into 5 isolation groups, including 1.0g 路L ~ (- 1) collagenase V group and 0.5g 路L ~ (- 1) mL ~ (- 1) LiberaseTM group. The thymic tissue was digested according to the corresponding enzyme concentration. 2 enrichment test was divided into four enrichment groups, including control group, immunomagnetic beads (MACS), Percoll and Ficoll enrichment group. After thymic tissue was digested with 1.0U 路mL ~ (- 1) LiberaseTM, the single cell suspension was enriched by MACS,Percoll and Ficoll respectively (except control group). The cell vitality and (TSCs) production of thymic stroma cells were detected by flow cytometry in the isolated group, the cell vitality, the proportion of TSCs and the expression abundance of (EpCAM), an important marker of TECs in TSCs, and the ratio of cortical TECs (cTECs) and medulla TECs (mTECs), two subsets of TECs in the Percoll enrichment group and the control group, were detected by flow cytometry. Results: the yield of TSCs in 1.0U 路mL ~ (- 1) LiberaseTM group was significantly higher than that in 0.5U 路mL ~ (- 1) LiberaseTM group (P01), and the cell activity and digestion time were better than 1.0g 路L ~ (- 1) collagenase group (P 0.01). The cell vitality of the three enrichment groups was higher, but there was no significant difference between the three groups (P 0.05). Compared with the control group, the proportion of TECs in the three enrichment groups was significantly increased (P 0.05 or P 0.01). In the three enrichment groups, the proportion of TECs enriched in the Percoll enrichment group was higher than that in the other two enrichment groups (P 0.01). Compared with the control group, the percentage of cTECs and mTECs and the ratio of cTEC/mTEC in the Percoll enrichment group had no significant change (P 0.05). Conclusion: Liberase TM is more suitable for digesting the thymic gland of adult mice than collagenase V, and the optimum digestion concentration is 1.0U 路mL ~ (- 1). The three enrichment methods used in this study can increase the proportion of TECs and destroy the cells little, and the Percoll enrichment method is the most effective, which does not affect the proportion of TECs subsets after enrichment, which is convenient for subsequent experimental operation.
【作者单位】： 吉林大学第一医院转化医学研究院免疫学研究所;吉林大学中日联谊医院麻醉科;吉林大学公共卫生学院营养与食品卫生学教研室;吉林大学第一医院甲状腺外科;
【基金】：国家自然科学基金资助课题(81202379)
【分类号】：R392-33
本文编号：2526998
[Abstract]:Aim: to compare the effects of several methods for isolation and enrichment of (TECs) in thymic epithelial cells of adult mice, and to find an accurate and efficient TECs enrichment method in vivo. Methods: 27 BABL/c mice aged 6 脳 8 weeks were divided into 9 groups with 3 mice in each group. 1 the isolation experiment was divided into 5 isolation groups, including 1.0g 路L ~ (- 1) collagenase V group and 0.5g 路L ~ (- 1) mL ~ (- 1) LiberaseTM group. The thymic tissue was digested according to the corresponding enzyme concentration. 2 enrichment test was divided into four enrichment groups, including control group, immunomagnetic beads (MACS), Percoll and Ficoll enrichment group. After thymic tissue was digested with 1.0U 路mL ~ (- 1) LiberaseTM, the single cell suspension was enriched by MACS,Percoll and Ficoll respectively (except control group). The cell vitality and (TSCs) production of thymic stroma cells were detected by flow cytometry in the isolated group, the cell vitality, the proportion of TSCs and the expression abundance of (EpCAM), an important marker of TECs in TSCs, and the ratio of cortical TECs (cTECs) and medulla TECs (mTECs), two subsets of TECs in the Percoll enrichment group and the control group, were detected by flow cytometry. Results: the yield of TSCs in 1.0U 路mL ~ (- 1) LiberaseTM group was significantly higher than that in 0.5U 路mL ~ (- 1) LiberaseTM group (P01), and the cell activity and digestion time were better than 1.0g 路L ~ (- 1) collagenase group (P 0.01). The cell vitality of the three enrichment groups was higher, but there was no significant difference between the three groups (P 0.05). Compared with the control group, the proportion of TECs in the three enrichment groups was significantly increased (P 0.05 or P 0.01). In the three enrichment groups, the proportion of TECs enriched in the Percoll enrichment group was higher than that in the other two enrichment groups (P 0.01). Compared with the control group, the percentage of cTECs and mTECs and the ratio of cTEC/mTEC in the Percoll enrichment group had no significant change (P 0.05). Conclusion: Liberase TM is more suitable for digesting the thymic gland of adult mice than collagenase V, and the optimum digestion concentration is 1.0U 路mL ~ (- 1). The three enrichment methods used in this study can increase the proportion of TECs and destroy the cells little, and the Percoll enrichment method is the most effective, which does not affect the proportion of TECs subsets after enrichment, which is convenient for subsequent experimental operation.
【作者单位】： 吉林大学第一医院转化医学研究院免疫学研究所;吉林大学中日联谊医院麻醉科;吉林大学公共卫生学院营养与食品卫生学教研室;吉林大学第一医院甲状腺外科;
【基金】：国家自然科学基金资助课题(81202379)
【分类号】：R392-33
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