小鼠脊髓微血管周细胞的体外培养及鉴定  

Isolation and identification of mouse spinal cord microvessel pericytes in vitro

作　　者：苑晓晨[1] 武清斌[1] 李宏伟[1] 荆瀛黎[1] 李炳蔚[1] 刘淑英[1] 修瑞娟[1] 

机构地区：[1]中国医学科学院北京协和医学院微循环研究所,北京100005

出　　处：《基础医学与临床》2015年第5期688-694,共7页Basic and Clinical Medicine

基　　金：2014年协和青年教师基金&中央高校基本科研业务费专项资金(33320140062)

摘　　要：目的应用周细胞培养基（PCM）分离筛选小鼠脊髓微血管周细胞（SCMP）,对其生物学功能进行评价。方法10只3周龄C57小鼠,无菌条件下取脊髓去除软脊膜,剪碎成大约1 mm×1 mm×1 mm。两次酶消化后用含20%牛血清白蛋白的DMEM离心获得微血管。用内皮细胞培养基（ECM）培养,传代2次后改用PCM培养。倒置显微镜观察细胞增殖状况。免疫细胞化学法检测血小板源性生长因子受体β（PDGFRβ）、神经元-胶质抗原2（NG2）、von Willebrand因子（v WF）和胶质纤维酸性蛋白（GFAP）的表达。流式检测CD140b、CD31、CD11b和GFAP的表达。将PCM换为10%胎牛血清（FBS）的DMEM培养基,检测细胞α-SMA表达的变化。通过周细胞-内皮细胞共培养成管实验检测细胞成管能力。结果接种48 h细胞爬出,7~9 d汇合,周细胞和内皮细胞伴随状增殖。改用PCM培养后内皮细胞减少,周细胞呈优势生长。免疫细胞化学表明PDGFRβ和NG2阳性,v WF和GFAP阴性;流式结果表明细胞PDGFRβ的阳性率为95.52%±2.55%,GFAP为0.63%±0.26%,CD31为0.80%±0.26%,CD11b为1.02%±0.35%。10%胎牛血清的DMEM促进细胞分化,α-SMA表达升高。成管实验周细胞与内皮细胞共同形成管腔样结构。结论通过PCM筛选法能够成功获得纯度较高的SCMP,所获得的细胞具备明显的周细胞的形态和功能特征。Objective To establish a method for selective culturing mouse spinal cord microvascular pericytes（ SCMP） by pericytes cell medium（ PCM） with 2% FBS. To observe the differentiation and function of the cultured cells. Methods After decapitation of 10 of 3-week-old C57 mice,the intact spinal cord was removed from the spinal column. Meninges were cleared from the spinal cord. Tissues were cut into small pieces（ approx. 1 mm× 1 mm × 1 mm）. The capillary fragments were obtained through two-step enzymatic digestion and 20% bovine serum albumin（ BSA） centrifugation. The microvessels were incubated initially under conditions optimized for endothelial cells,but after two passages switched to a medium optimized for pericyte growth. The morphology of pericytes was observed by inverted microscope. Platelet-derived growth factor receptor β（ PDGFRβ）,neuron-glial antigen 2（ NG2）,von Willebrand factor（ v WF）,and glial fibrillary acidic protein（ GFAP） were detected by immun-ofluorescence. PDGFRβ,CD31,CD11 b and GFAP were detected by flow cytometry. When switching the cells from pericyte medium into DMEM containing 10 % FBS,the α-SMA,a marker of pericyte differentiation,was detected by immunofluorescence and Western blot. The matrigel pericyte-endothelial cell co-culture was used to verify the function of cultured cells. Results The cells migrated from the attached capillary fragments after 48 h,and got confluence after 7 to 9 days. Primary cultures consisted of endothelial cells with pericytes. After switching to PCM,the pericytes grew predominantly and showed the typical rhomboid morphology. Immunofluorescence revealed that PDGFRβ and NG2 were positive,v WF and GFAP were negative. Flow cytometry showed that cells were strongly positive for PDGFRβ（ 95. 52 % ± 2. 55 %）,negative for CD31（ 0. 80 % ± 0. 26 %）,CD11 b（ 1. 02% ± 0. 35%） or GFAP（ 0. 63% ± 0. 26%）. DMEM containing 10% FBS promoted α-SMA expression,demonstrating the cells could still differentiate. In matrigel co-

关 键 词：周细胞 脊髓 微血管 细胞分离 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]