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肾病科,北京 100091

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R-33


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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Department of Nephrology, Xiyuan Hospital, Chinese Academy of Traditional Chinese Medicine, Beijing 100091, China

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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±1. 10)×109 vs(4. 18±1. 02)×109、活细胞比例(86. 36±3. 32)% vs(61. 62±10. 93)%以及活性 CD45+细胞比例(57. 19±5. 11)% vs(26. 01±11. 44)%、活性 CD3+细胞比例(8. 73±2. 89)% vs(4. 52±2. 49)%、活性 CD4+细胞比例(6. 19±2. 09)% vs(3. 22±1. 91)%和活性 B220+细胞比例(15. 06±4. 27)% vs(5. 07±2. 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 optimal 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 was used to prepare lamina propria single-cell suspensions by enzymatic digestion with collagenase A or collagenase VIII. We compared the effects of these two enzymatic digestion method on the cell yield, cell activity, and cell surface antigens of the single-cell suspensions. Single-cell suspensions prepared by the superior enzymatic digestion method were then subjected to flow cytometry assay. Results Compared to collagenase-VIIIbased enzymatic digestion, collagenase-A-based digestion result ed in a higher cell yield (9. 48±1. 10) ×109 vs (4. 18±1.02)×109 and higher proportions of live cells ( 86. 36± 3. 32)% vs ( 61. 62± 10. 93)%, active CD45 + cells ( 57. 19±5. 11)% vs ( 26. 01 ± 11. 44)%, active CD3 + cells ( 8. 73 ± 2. 89)% vs ( 4. 52 ± 2. 49)%, active CD4 + cells ( 6. 19 ±2. 09)% vs (3. 22±1. 91)%, and active B220 + cells (15. 06±4. 27)% vs (5. 07±2. 20)%, providing high-quality cells for subsequent flow assays. Conclusions The collagenase A-based enzymatic digestion method is more suitable for the preparation of single-cell suspensions from the lamina propria of mouse small intestines.

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曾 勤,梁 莹,王新慧,闫 蕾,王湘鹏,杨嘉仪,余仁欢.小鼠肠道固有层单细胞悬液制备的酶消化法比较研究[J].中国比较医学杂志,2024,34(4):92~99.

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  • 收稿日期:2023-06-06
  • 在线发布日期: 2024-06-20
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