【摘要】：目的:通過比較幾種成年小鼠胸腺上皮細(xì)胞(TECs)分離和富集方法的效果,尋找一種具有準(zhǔn)確體內(nèi)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(shí)驗(yàn)分為5個(gè)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(yīng)的酶工作濃度對胸腺組織進(jìn)行消化處理;2富集實(shí)驗(yàn)分為4個(gè)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(xì)胞懸液分別用MACS、Percoll和Ficoll法進(jìn)行細(xì)胞富集(對照組除外)。分離組通過流式細(xì)胞術(shù)檢測細(xì)胞活力及胸腺基質(zhì)細(xì)胞(TSCs)的產(chǎn)量;富集組通過流式細(xì)胞術(shù)檢測細(xì)胞活力、TSCs比例和TSCs中TECs重要標(biāo)志上皮細(xì)胞黏附分子(EpCAM)的表達(dá)豐度,檢測Percoll富集組和對照組TECs的2個(gè)亞群——皮質(zhì)TECs(cTECs)和髓質(zhì)TECs(mTECs)比例。結(jié)果:1.0U·mL~(-1) Liberase TM組中TSCs的產(chǎn)量明顯高于0.5和0.7U·mL~(-1) LiberaseTM組(P0.01),且其細(xì)胞活力及消化時(shí)間均優(yōu)于1.0g·L~(-1)collagenaseⅤ組(P0.01)。3個(gè)富集組的細(xì)胞活力均較高,但組間比較差異無統(tǒng)計(jì)學(xué)意義(P0.05);與對照組比較,3個(gè)富集組TECs比例明顯增加(P0.05或P0.01);在3個(gè)富集組中,Percoll富集組富集的TECs比例高于其他2個(gè)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(jié)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(xì)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(xì)胞比例,便于后續(xù)實(shí)驗(yàn)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(xué)第一醫(yī)院轉(zhuǎn)化醫(yī)學(xué)研究院免疫學(xué)研究所;吉林大學(xué)中日聯(lián)誼醫(yī)院麻醉科;吉林大學(xué)公共衛(wèi)生學(xué)院營養(yǎng)與食品衛(wèi)生學(xué)教研室;吉林大學(xué)第一醫(yī)院甲狀腺外科;
【基金】：國家自然科學(xué)基金資助課題(81202379)
【分類號】：R392-33

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