老年大鼠脑组织小胶质细胞的分离及表型的流式鉴定  

Isolation of microglia from aged rat brain tissue and phenotypic identification by flow cytometry

作　　者：刘苗苗 管美琪 余莲 杨小荣[3] 王洁[1] 李艳丽 Liu Miaomiao;Guan Meiqi;Yu Lian;Yang Xiaorong;Wang Jie;Li Yanli(Dept of Neurology,The First Hospital of Shanxi Medical University,Taiyuan030001;Dept of Pediatrics,Ministry of Education,Dept of Physiology,Shanxi Medical University,Taiyuan030001;Key Laboratoryof Cellular Physiology,Ministry of Education,Dept of Physiology,Shanxi Medical University,Taiyuan030001)

机构地区：[1]山西医科大学第一医院神经内科,太原030001 [2]山西医科大学儿科医学系,太原030001 [3]山西医科大学生理学系细胞生理学教育部重点实验室,太原030001

出　　处：《安徽医科大学学报》2024年第11期1967-1973,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金项目(编号:82101641);山西省基础研究计划项目(编号:20210302124173);细胞生理学教育部重点实验室(山西医科大学)开放基金项目(编号:KLMEC/SXMU-202010)。

摘　　要：目的建立一种优化的方法,从老年大鼠脑组织中分离纯化小胶质细胞,并对小胶质细胞表面表型进行流式鉴定。方法以年轻大鼠(3月龄)为对照,采用IV型胶原酶联合机械消化的方式制备老年大鼠脑组织单细胞悬液,用30%-37%-70%Percoll分离液分离纯化小胶质细胞,并对细胞进行荧光标记,通过流式细胞术检测老年大鼠脑组织的小胶质细胞表型。结果采用IV型胶原酶联合机械消化,并结合密度梯度离心的方法,可以获得更多数量的单细胞,且细胞存活率均大于90%。流式检测结果显示:与年轻大鼠相比,老年大鼠脑组织M1型小胶质细胞表面标志物CD86和MHC II表达均增多(P<0.01),M2型小胶质细胞表面标志物CD200R表达也增多(P<0.01)。结论采用IV型胶原酶联合机械消化,并结合密度梯度离心分离,是较好的分离纯化成年和老年大鼠脑组织小胶质细胞的方法。Objective To establish an optimized method for isolation and purification of microglia from aged rat brain tissue,and the phenotype of microglia was detected by flow cytometry.Methods With young rats(3 months old)as control,the brain tissues of aged rats were immediately processed into single cell suspensions by mechanical dissociation and enzymatic digestion using type IV collagenase.Microglia were isolated on Percoll gradients(30%-37%-70%).The cells were stained with fluorescence-labeled antibodies and the phenotype of microglia was detected by flow cytometry.Results This study developed a method that enzymatic digestion and mechanical dissociation combined with density gradient centrifugation.More single cells could be obtained by using this method.And the survival rate of cells was more than 90%.The flow cytometric analysis showed that the expression of M1 microglia marker CD86 and MHCⅡincreased(P<0.01),and the expression of M2 microglia marker CD200R increased(P<0.01)in aged rats compared with that in young rats.Conclusion The use of type IV collagenase and mechanical digestion combined with density gradient centrifugation is good for isolating and purifying microglia from adult and aged rat brain tissue.

关 键 词：衰老 神经炎症 小胶质细胞 分离纯化 流式细胞术 表面标志物 

分 类 号：R338.2[医药卫生—人体生理学]