幾種成年小鼠胸腺上皮細胞分離和富集方法效果的比較
發(fā)布時間：2019-08-15 12:39
【摘要】：目的:通過比較幾種成年小鼠胸腺上皮細胞(TECs)分離和富集方法的效果,尋找一種具有準確體內代表性的高效TECs富集方法。方法:27只6~8周齡BABL/c小鼠分為9組,每組3只。1分離實驗分為5個分離組,包括1.0g·L~(-1) collagenaseⅤ組和0.5、0.7、1.0及2.0U·mL~(-1) LiberaseTM組,按照相應的酶工作濃度對胸腺組織進行消化處理;2富集實驗分為4個富集組,包括對照組和免疫磁珠(MACS)、Percoll及Ficoll富集組,各組用1.0U·mL~(-1) LiberaseTM消化胸腺組織后,將所得的單細胞懸液分別用MACS、Percoll和Ficoll法進行細胞富集(對照組除外)。分離組通過流式細胞術檢測細胞活力及胸腺基質細胞(TSCs)的產量;富集組通過流式細胞術檢測細胞活力、TSCs比例和TSCs中TECs重要標志上皮細胞黏附分子(EpCAM)的表達豐度,檢測Percoll富集組和對照組TECs的2個亞群——皮質TECs(cTECs)和髓質TECs(mTECs)比例。結果:1.0U·mL~(-1) Liberase TM組中TSCs的產量明顯高于0.5和0.7U·mL~(-1) LiberaseTM組(P0.01),且其細胞活力及消化時間均優(yōu)于1.0g·L~(-1)collagenaseⅤ組(P0.01)。3個富集組的細胞活力均較高,但組間比較差異無統(tǒng)計學意義(P0.05);與對照組比較,3個富集組TECs比例明顯增加(P0.05或P0.01);在3個富集組中,Percoll富集組富集的TECs比例高于其他2個富集組(P0.01);與對照組比較,Percoll富集組cTECs和mTECs的百分比及cTEC/mTEC比值無明顯變化(P0.05)。結論:Liberase TM較collagenaseⅤ更適用于消化成年小鼠胸腺,其最適消化濃度為1.0U·mL~(-1)。本研究采用的3種富集方法均可以提高TECs比例,對細胞的破壞均較小;Percoll富集法最有效,在富集后不影響TECs亞群細胞比例,便于后續(xù)實驗操作。
[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.
【作者單位】： 吉林大學第一醫(yī)院轉化醫(yī)學研究院免疫學研究所;吉林大學中日聯(lián)誼醫(yī)院麻醉科;吉林大學公共衛(wèi)生學院營養(yǎng)與食品衛(wèi)生學教研室;吉林大學第一醫(yī)院甲狀腺外科;
【基金】：國家自然科學基金資助課題(81202379)
【分類號】：R392-33
本文編號：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.
【作者單位】： 吉林大學第一醫(yī)院轉化醫(yī)學研究院免疫學研究所;吉林大學中日聯(lián)誼醫(yī)院麻醉科;吉林大學公共衛(wèi)生學院營養(yǎng)與食品衛(wèi)生學教研室;吉林大學第一醫(yī)院甲狀腺外科;
【基金】：國家自然科學基金資助課題(81202379)
【分類號】：R392-33
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