小鼠肠道固有层单细胞悬液制备的酶消化法比较研究

A comparative study of enzymatic digestion methods for the preparation of single-cell suspensions from the lamina propria of the mouse intestine

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)×10^(9) vs(4.18±1.02)×10^(9)、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。

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)×10^(9) vs(4.18±1.02)×10^(9) 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.