小胶质细胞纯化分离培养方法的改良

THE MODIFIED CULTURAL METHOD FOR ISOLATION AND PURIFICATION OF THE MICROGLIA

目的改良现有的小胶质细胞纯化分离培养方法,建立稳定简便的培养模型。方法利用盐酸利多卡因注射液代替机械振摇纯化分离小胶质细胞;利用CD11b/c(OX42)免疫细胞化学的方法对分离的小胶质细胞纯度进行鉴定,同时观察小胶质细胞形态及活化指标NFкBp65的表达情况;利用流式细胞仪,结合细胞计数及MTT细胞活力测定检测纯化分离后小胶质的增殖情况。结果改良的方法可稳定的获得1.2×106个/培养瓶(75cm2,250ml)的小胶质细胞,纯度达到98%,存活率≥95%,形态上以阿米巴样为主,继续培养3-5d,约半数细胞可转变为静止状态。NFкBp65免疫细胞化学染色为胞浆表达。流式细胞仪检测结合细胞计数及MTT细胞活力检测结果显示,体外纯化培养的小胶质细胞多位于G0/G1期,培养过程中未出现明显的增殖。结论改良的方法易于操作,产量多,纯度高。为体外小胶质细胞进一步研究提供了基础。

Objective To modify the current cultural method of rat microglia and establish a convenient and stable model of culture method for rat microglia. Methods Lidocaine hydrochloride was used to substitute mechanical shaking for isolating microglia from newborn rat cerebral cortex. The purity of isolated cells were identified by expressions of CD11b/c and NFκB p65 respectively using immunocytochemical technique. The morphology was observed by inverted phase contrast microscope, the proliferation was examined by flow cytometry, cell counting, and MTT assay. Results Present modified method steadily produced 1.2×10^6cells per flask （75cm^2, 250ml） with high purity （≥99%） and high survival rate （≥95%）. The purified microglia were in activated status according to its ameboid morphous. Pattern change, from ameboid form to resting form, appeard 3-5 days after culture in half of cell popuhion. The expression of NF-κB p65 was mainly located in cytoplasm. No significant proliferation was found in normal cultural condition. Conclusion Present modified cultural method is a stable and convenient method with higher productive rate and purity, providing a basis for further research of the microglia.