小鼠脊髓微血管周细胞的体外培养及鉴定

发布时间：2018-05-17 08:12

本文选题：周细胞 + 脊髓　；参考：《基础医学与临床》2015年05期

【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。
[Abstract]:Objective to isolate and screen mouse spinal microvascular pericyte (SCMPP) by pericyte culture medium (PCM) and evaluate its biological function. Methods the spinal cord of 10 C57 mice aged 3 weeks was removed from the spinal cord and shredded into about 1 mm 脳 1 mm under aseptic condition. Microvasculature was obtained by centrifugation with DMEM containing 20% bovine serum albumin after two enzyme digestion. Endothelial cell culture medium (ECM) was used, then subcultured for 2 times and then transferred to PCM culture. Cell proliferation was observed by inverted microscope. Immunocytochemistry was used to detect the expression of PDGFR 尾, neuron-glial antigen 2NG2An Willebrand factor (v WFV) and glial fibrillary acidic protein (GFAP) in platelet-derived growth factor receptor (尾 -PDGFR 尾) and glial fibrillary acidic protein (GFAP). The expression of CD140b, CD31, CD11b and GFAP was detected by flow cytometry. The expression of 伪 -SMA was detected by changing PCM into 10% fetal bovine serum (FBS) DMEM medium. The ability of cell tubulogenesis was measured by pericyte-endothelial cell co-culture tube-forming experiment. Results 48 h after inoculation, the cells crawled out of the cells and converged for 9 days, and the pericytes and endothelial cells were accompanied by proliferation. Endothelial cells decreased and pericytes grew preferentially after cultured with PCM. Immunocytochemistry showed that PDGFR 尾 and NG2 were positive for vWF and GFAP, and the positive rate of PDGFR 尾 was 95.52% 卤2.55%. The positive rate of GFAP was 0.63% 卤0.26 + 0.80% 卤0.26 + CD11b was 1.02% 卤0.35.10% of fetal bovine serum promoted cell differentiation, and 伪 -SMA expression increased. The endothelial cells and pericytes formed lumen-like structures. Conclusion High purity SCMP can be obtained by PCM screening method, and the obtained cells have obvious morphological and functional characteristics of pericytes.
【作者单位】：中国医学科学院北京协和医学院微循环研究所;
【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)
【分类号】：R329.2

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