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

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.