SPF级C57BL/6小鼠肠道固有层单细胞悬液制备的酶消化法比较研究
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中国中医科学院西苑医院

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国家自然科学基金项目(面上项目);中国中医科学院科技创新工程面上项目


A comparative study of enzymatic digestion methods for the preparation of single cell suspensions from the lamina propria of the mouse intestine
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Xiyuan Hospital of China Academy of Chinese Medical Sciences

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The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)

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    摘要:

    目的 探究小鼠小肠固有层单细胞悬液制备的最佳酶消化法。方法 采集10根长度一致的小鼠小肠,随机分为两组,每组分别采用以胶原酶A或胶原酶Ⅷ为主的酶消化法制备固有层单细胞悬液,比较这两种酶消化法对单细胞悬液细胞获得率、细胞活性和细胞表面抗原的影响,然后对较优酶消化法制备的单细胞悬液进行流式检测。结果 与以胶原酶Ⅷ为主的酶消化法相比,以胶原酶A为主的酶消化法可以获得更高的细胞产量(9.48?.10)?09 VS(4.18?.02)?09、活细胞比例(86.36?.32)% VS(61.62?0.93)%以及活性CD45 细胞比例(57.19?.11)% VS (26.01?1.44)%、活性CD3 细胞比例(8.73?.89)% VS (4.52?.49)%、活性CD4 细胞比例(6.19?.09)% VS(3.22?.91)%和活性B220 细胞比例(15.06?.27)% VS(5.07?.20)%,为后续流式检测提供了高质量细胞。结论 以胶原酶A为主的酶消化法更适合于小鼠小肠固有层单细胞悬液的制备。

    Abstract:

    Objective To investigate the best digestion method for the preparation of single cell suspensions from mouse small intestinal lamina propria. Methods Ten mouse small intestines of uniform length were collected and randomly divided into two groups. Each group prepared the lamina propria single cell suspensions by enzymatic digestion with collagenase A or collagenase VIII, and compared the effects of these two enzymatic digestion methods on the cell yield, cell activity and cell surface antigen of the single cell suspensions, and then the single cell suspensions prepared by the superior enzymatic digestion method were subjected to flow assay. Results Compared to collagenase VIII-based enzymatic digestion, collagenase A-based enzymatic digestion resulted in higher cell yields (9.48 ?1.10) ?109 vs. (4.18 ?1.02) ?109, live cell proportions (86.36 ?3.32) % vs. (61.62 ?10.93) % and active CD45 cells (57.19 ?5.11) ) % vs (26.01?1.44) %, active CD3 cells (8.73?.89) % vs (4.52?.49) %, active CD4 cells (6.19?.09) % vs (3.22?.91) % and active B220 cells (15.06?.27) % vs (5.07?.20) %, providing high quality cells for subsequent flow assays. Conclusion The collagenase A-based enzymatic digestion method is more suitable for the preparation of single cell suspensions from the lamina propria of the mouse small intestine.

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  • 收稿日期:2023-06-06
  • 最后修改日期:2023-12-05
  • 录用日期:2024-05-10
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