| 孙廉旭,王家伟,张 莎,曹 亮,王 文.大鼠肺微血管周细胞的体外培养及鉴定[J].,2018,(6):1014-1019 |
| 大鼠肺微血管周细胞的体外培养及鉴定 |
| Isolation and Identification of Pulmonary Microvessel Pericytes in Rats |
| 投稿时间：2017-09-18 修订日期：2017-10-17 |
| DOI：10.13241/j.cnki.pmb.2018.06.003 |
| 中文关键词: 肺微血管周细胞 原代培养 体外鉴定 大鼠 |
| 英文关键词: Pulmonary microvascular pericytes Primary culture Identification Rat |
| 基金项目:国家自然科学基金项目（81373845） |
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| 摘要点击次数: 428 |
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| 中文摘要: |
| 摘要 目的：探讨SD大鼠肺微血管周细胞（rat pulmonarymicrovessel pericytes, RPMPC）的分离、培养与鉴定方法。方法：采用机械剪切、Ⅰ型胶原酶消化法和微孔过滤结合超高速离心法分离大鼠肺微血管片段，用含15%胎牛血清（FBS）的高糖培养基（DMEM）进行培养，倒置显微镜观察原代RPMPC的形态及生长特性。免疫荧光法检测神经元-胶质抗原 2 (neuron-glial antigen 2,NG2)，α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）、结蛋白( desmin )和CD31相关抗原的表达，同时应用免疫细胞化学法检测血小板源性生长因子受体β( PDGFＲ-β)的表达。CCK-8测定周细胞生长曲线。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：本方法培养获取的RPMPC纯度较高，并能连续传代。细胞48 h后爬出，呈长梭形、三角形等不规则形，8～10 d细胞汇合，呈栅栏或旋涡状生长，无接触性抑制，前期可见有少量内皮细胞伴随生长，单核偶见双核，核呈卵圆形，胞浆丰富。PDGFＲ-β、α-SMA、NG2、desmin染色阳性，CD31阴性；周细胞与内皮细胞共培养形成管腔结构。结论：通过本方法能够获得纯度较高的肺微血管周细胞，且所获细胞具有周细胞的特性及功能。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the isolation, culture and identification methods of rat pulmonary microvessel pericytes RPMPC in the lung of SD rats. Methods: Using mechanical shear, type I collagenase digestion and micropore filtration combined with ultra high speed centrifugation to separate the rat pulmonary microvascular fragments; using medium (DMEM) containing 15% fetal bovine serum (FBS) to culture the RPMPCs; and using inverted microscope to observe the morphology and growth characteristics of RPMPCs. Detecting the expression of neuron glia antigen 2 (NG2), alpha smooth muscle actin (α-SMA), desmin (desmin) and CD31 antigen by the immunofluorescent methods; Detecting the expression of platelet-derived growth factor receptor beta (PDGFR- beta) by immunocytochemistry. The growth curve of pericytes was determined by CCK-8. The tube formation ability of the cells was detected by coculture with pericytes and endothelial cells. Results: The purity of RPMPC was high and could be passaged continuously. RPMPCs climb out after 48 hours of separation, with the shape of fusiform, triangular and irregular form. After 8 ~ 10d, the cells converge and grow as a palisade or vortex, without contact inhibition. In the early stage, a few endothelial cells were found, which were mononuclear or binuclear and rich in cytoplasm. PDGFR- beta, alpha -SMA, NG2 and desmin staining were positive, and CD31 staining was negative. The co culture of pericytes and endothelial cells leads to the formation of luminal structures. Conclusion: The pulmonary microvascular pericytes of higher purity can be obtained by this method, and the acquired cells have the characteristics and functions of pericytes. |
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