中华神经外科杂志
中華神經外科雜誌
중화신경외과잡지
Chinese Journal of Neurosurgery
2013年
9期
945-950
,共6页
叶红星%岳琪%莫链杰%张新%姚瑜%毛颖%周良辅
葉紅星%嶽琪%莫鏈傑%張新%姚瑜%毛穎%週良輔
협홍성%악기%막련걸%장신%요유%모영%주량보
人小胶质细胞%脑胶质瘤%快速分离
人小膠質細胞%腦膠質瘤%快速分離
인소효질세포%뇌효질류%쾌속분리
Human microglia%Glioma%Rapid isolation
目的 建立一种快速有效从脑胶质瘤组织中分离小胶质细胞的改良方法.方法 采用密度梯度离心法联合磁珠分选法从脑胶质瘤组织中分离小胶质细胞.用流式细胞术、Western blot 和细胞免疫荧光检测细胞纯度,流式检测免疫表型,趋化及吞噬实验检测其生物学功能;对从不同级别胶质瘤组织分离的小胶质细胞产量进行定量分析.结果 收获细胞具有小胶质细胞典型形态及Iba1染色阳性,纯度可达(95.1±4.2)％;流式细胞术检测显示其表达CD11b、HLA-DR等,并对ATP 的趋化作用呈浓度依赖性,且在体外具有良好的吞噬乳胶微球(latex beads)的功能.此外,每克胶质瘤组织分离小胶质细胞细胞产量:低级别组(n=4),(4.0±0.5) ×105;高级别组(n=8),(4.3±0.4) ×105,两组差异无统计学意义(P＞0.05).结论 利用改良的分离小胶质细胞方法能从新鲜脑胶质瘤组织中快速获得大量高纯度小胶质细胞.
目的 建立一種快速有效從腦膠質瘤組織中分離小膠質細胞的改良方法.方法 採用密度梯度離心法聯閤磁珠分選法從腦膠質瘤組織中分離小膠質細胞.用流式細胞術、Western blot 和細胞免疫熒光檢測細胞純度,流式檢測免疫錶型,趨化及吞噬實驗檢測其生物學功能;對從不同級彆膠質瘤組織分離的小膠質細胞產量進行定量分析.結果 收穫細胞具有小膠質細胞典型形態及Iba1染色暘性,純度可達(95.1±4.2)％;流式細胞術檢測顯示其錶達CD11b、HLA-DR等,併對ATP 的趨化作用呈濃度依賴性,且在體外具有良好的吞噬乳膠微毬(latex beads)的功能.此外,每剋膠質瘤組織分離小膠質細胞細胞產量:低級彆組(n=4),(4.0±0.5) ×105;高級彆組(n=8),(4.3±0.4) ×105,兩組差異無統計學意義(P＞0.05).結論 利用改良的分離小膠質細胞方法能從新鮮腦膠質瘤組織中快速穫得大量高純度小膠質細胞.
목적 건립일충쾌속유효종뇌효질류조직중분리소효질세포적개량방법.방법 채용밀도제도리심법연합자주분선법종뇌효질류조직중분리소효질세포.용류식세포술、Western blot 화세포면역형광검측세포순도,류식검측면역표형,추화급탄서실험검측기생물학공능;대종불동급별효질류조직분리적소효질세포산량진행정량분석.결과 수획세포구유소효질세포전형형태급Iba1염색양성,순도가체(95.1±4.2)％;류식세포술검측현시기표체CD11b、HLA-DR등,병대ATP 적추화작용정농도의뢰성,차재체외구유량호적탄서유효미구(latex beads)적공능.차외,매극효질류조직분리소효질세포세포산량:저급별조(n=4),(4.0±0.5) ×105;고급별조(n=8),(4.3±0.4) ×105,량조차이무통계학의의(P＞0.05).결론 이용개량적분리소효질세포방법능종신선뇌효질류조직중쾌속획득대량고순도소효질세포.
Objective To optimize a rapid and efficient method for the isolation of human microglia from glioma tissue.Methods Density separation combined with immunomagnetic bead separation was used to maximize yield and purity.Flow cytometry,western blot and cell immunofluorescence were used to identify the harvested microglia.Inmunophenotypic profile of the microglia was assessed by flow cytometry.Biological functions were assessed by chemotaxis assay and the phagocytic function test.The cellular yield of microglia from glioma tissue was assessed.Results The harvest cells had typical morphology and expressed Iba1 protein.The purity of the harvest microglia was (95.1 ± 4.2) ％.Flow cytometry showed that the microglia expressed immune molecules,such as CD11 b and HLADR,etc.Isolated human microglia migrated to ATP in a dose-dependent manner and they phagocytozed latex beads.The cellular yield from low-grade glioma tissue and high glioma tissue were (4.0 ± 0.5) × 105 cells/g wet tissue and (4.3 ± 0.4) × 105 cells/g wet tissue,respectively (P ＞ 0.05).Conclusion The protocol presented here enables rapid isolation of human microglia with typical morphology and phenotype from human fresh glioma tissue with a high purity and yield.
