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Abstract：
Culturing cerebrovascular smooth muscle cells (CVSMCs) in vitro can provide a model for studying many cerebrovascular diseases. This study describes a convenient and efficient method to obtain mouse CVSMCs by enzyme digestion.
Mouse circle of Willis was isolated, digested, and cultured with platelet-derived growth factor-BB (PDGF-BB) to promote CVSMC growth, and CVSMCs were identified by morphology, immunofluorescence analysis, and flow cytometry. The effect of PDGF-BB on vascular smooth muscle cell (VSMC) proliferation was evaluated by cell counting kit (CCK)-8 assay, morphological observations, Western blotting, and flow cytometry.
CVSMCs cultured in a PDGF-BB-free culture medium had a typical peak-to-valley growth pattern after approximately 14 days. Immunofluorescence staining and flow cytometry detected strong positive expression of the cell type-specific markers alpha-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain 11 (SMMHC), smooth muscle protein 22 (SM22), calponin, and desmin. In the CCK-8 assay and Western blotting, cells incubated with PDGF-BB had significantly enhanced proliferation compared to those without PDGF-BB.
We obtained highly purified VSMCs from the mouse circle of Willis using simple methods, providing experimental materials for studying the pathogenesis and treatment of neurovascular diseases in vitro. Moreover, the experimental efficiency improved with PDGF-BB, shortening the cell cultivation period.
Mouse circle of Willis was isolated, digested, and cultured with platelet-derived growth factor-BB (PDGF-BB) to promote CVSMC growth, and CVSMCs were identified by morphology, immunofluorescence analysis, and flow cytometry. The effect of PDGF-BB on vascular smooth muscle cell (VSMC) proliferation was evaluated by cell counting kit (CCK)-8 assay, morphological observations, Western blotting, and flow cytometry.
CVSMCs cultured in a PDGF-BB-free culture medium had a typical peak-to-valley growth pattern after approximately 14 days. Immunofluorescence staining and flow cytometry detected strong positive expression of the cell type-specific markers alpha-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain 11 (SMMHC), smooth muscle protein 22 (SM22), calponin, and desmin. In the CCK-8 assay and Western blotting, cells incubated with PDGF-BB had significantly enhanced proliferation compared to those without PDGF-BB.
We obtained highly purified VSMCs from the mouse circle of Willis using simple methods, providing experimental materials for studying the pathogenesis and treatment of neurovascular diseases in vitro. Moreover, the experimental efficiency improved with PDGF-BB, shortening the cell cultivation period.
摘要：
背景：体外培养脑血管平滑肌细胞（CVSMCs）可以为研究许多脑血管疾病提供模型。本研究描述了一种通过酶消化获得小鼠CVSMCs的便捷高效方法。
方法：分离Willis小鼠圈，消化，并与血小板衍生生长因子-BB（PDGF-BB）一起培养以促进CVSMC生长，和CVSMC通过形态学鉴定，免疫荧光分析，和流式细胞术。用细胞计数试剂盒(CCK)-8法评价PDGF-BB对血管平滑肌细胞(VSMC)增殖的影响,形态学观察，西方印迹,和流式细胞术。
结果:在无PDGF-BB的培养基中培养的CVSMC在大约14天后具有典型的峰谷生长模式。免疫荧光染色和流式细胞术检测到细胞类型特异性标志物α-平滑肌肌动蛋白（α-SMA）的强阳性表达，平滑肌肌球蛋白重链11（SMMHC），平滑肌蛋白22（SM22），Calponin,和Desmin。在CCK-8检测和蛋白质印迹中,与不含PDGF-BB的细胞相比,用PDGF-BB孵育的细胞具有显著增强的增殖.
结论：我们使用简单的方法从Willis小鼠圈中获得了高度纯化的VSMC，为体外研究神经血管疾病的发病机制和治疗提供实验材料。此外，PDGF-BB提高了实验效率，缩短细胞培养时间。
方法：分离Willis小鼠圈，消化，并与血小板衍生生长因子-BB（PDGF-BB）一起培养以促进CVSMC生长，和CVSMC通过形态学鉴定，免疫荧光分析，和流式细胞术。用细胞计数试剂盒(CCK)-8法评价PDGF-BB对血管平滑肌细胞(VSMC)增殖的影响,形态学观察，西方印迹,和流式细胞术。
结果:在无PDGF-BB的培养基中培养的CVSMC在大约14天后具有典型的峰谷生长模式。免疫荧光染色和流式细胞术检测到细胞类型特异性标志物α-平滑肌肌动蛋白（α-SMA）的强阳性表达，平滑肌肌球蛋白重链11（SMMHC），平滑肌蛋白22（SM22），Calponin,和Desmin。在CCK-8检测和蛋白质印迹中,与不含PDGF-BB的细胞相比,用PDGF-BB孵育的细胞具有显著增强的增殖.
结论：我们使用简单的方法从Willis小鼠圈中获得了高度纯化的VSMC，为体外研究神经血管疾病的发病机制和治疗提供实验材料。此外，PDGF-BB提高了实验效率，缩短细胞培养时间。
