小鼠脂肪组织中免疫细胞分离方法的优化及亚群在肥胖小鼠中的作用

Optimization of immune cell isolation method from mouse adipose tissue and the role of subgroups in obese mice

目的尝试提供一种高效的、低损伤的分离脂肪组织免疫细胞的方式,以期为肥胖过程中免疫学机制的研究提供技术支持。方法首先分离正常小鼠附睾周围的脂肪组织,并采用机械研磨或酶消化(包括Ⅱ型/Ⅳ胶原酶、Liberase酶)的方式分离其中的免疫细胞,并结合流式细胞检测技术筛选最高效的免疫细胞分离方式。再通过高脂饮食(high fat diet,HFD)建立肥胖小鼠模型,并进一步分析肥胖过程中,小鼠脂肪组织中巨噬细胞及T淋巴细胞的比例及亚群变化。结果比较不同酶消化及机械研磨方式发现,通过高纯度的Liberase酶消化的方式获得CD45^(+)免疫细胞死亡率最低,活细胞数最多;而通过机械研磨方式获得免疫细胞死亡率最高,活细胞数最低。与正常饮食小鼠相比,肥胖小鼠脂肪组织中固有免疫细胞如中性粒细胞、巨噬细胞比例增加,尤其是M1型巨噬细胞比例增加,M2型降低,同时巨噬细胞分泌肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)增加,髓样细胞表达激发受体1(triggering receptor expressed on myeloid cells 1,TREM1)表达增加。而适应性免疫细胞中,T淋巴细胞比例增加,特别是Th1/Treg细胞的比例增加最为明显。结论建立了一种从小鼠脂肪组织中高效获取有活力的免疫细胞的方法,并证实肥胖小鼠脂肪组织中巨噬细胞、T淋巴细胞比例增加,促炎作用增强,它们在诱发脂肪组织炎症反应中发挥重要作用。

Objective To provide an efficient and low-damage method to isolate immune cells from adipose tissue to provide technical support for the study of immunological mechanisms in obesity.Methods We separated the adipose tissue around the epididymis of normal mice,and separated the immune cells by mechanical grinding or enzymatic digestion(including typeⅡ/Ⅳcollagenase,Liberase enzyme),and screened the most efficient immune cell separation method by flow cytometry technology.Then,an obese mouse model was established through high fat diet(HFD),and the proportion and subgroup changes of macrophages and T lymphocytes in mouse adipose tissue were further analyzed.Results We compared different enzymatic digestion and mechanical grinding methods and found that CD45^(+)immune cells obtained through high-purity Liberase enzyme digestion had the lowest mortality rate and the highest number of live cells.The death rate of immune cells obtained by mechanical grinding was the highest,and the number of living cells was the least.Compared with mice fed a normal diet,the proportion of innate immune cells such as neutrophils and macrophages in the adipose tissue of obese mice increased,especially the proportion of M1 macrophages,while the M2 macrophages decreased.The secretion of tumor necrosis factor-α(TNF-α)by macrophages increased,and the expression of triggering receptor expressed on myeloid cells 1(TREM1)enhanced.Among the adaptive immune cells,the proportion of T lymphocytes increased,especially the ratio of Th1/Treg cells increased the most.Conclusion We have established a method to efficiently obtain viable immune cells from mouse adipose tissue,and confirmed that the proportion of macrophages and T lymphocytes in the adipose tissue of obese mice increased,and their pro-inflammatory effect were enhanced.They played an important role in inducing inflammation in adipose tissue.