CAJ | 학술논문

目的探讨大鼠脑微血管周细胞的分离、培养和鉴定方法。方法10只3周龄Wstar 大鼠，无菌分离脑组织，采用2次酶消化和1次密度梯度离心法分离脑微血管片段，接种于35 mm培养皿进行原代培养。采用相差显微镜观察细胞形态，免疫荧光法鉴定α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）、神经元-胶质抗原2（neuron-glial antigen 2，NG2）、von Willebrand因子(von Willebrand factor，vWF)和胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）相关抗原，甲基噻唑基四唑法测定细胞生长曲线。结果周细胞从贴壁的脑微血管片段周围爬出，呈多角性，12～14 d后细胞融合95％。免疫荧光染色显示，周细胞分子标志物α-SMA和NG2相关抗原呈双阳性，vWF和GFAP 相关抗原呈双阴性，证实培养细胞为脑微血管周细胞。原代细胞初始生长速度较慢，传代细胞36～60h进入对数生长期，72～108 h生入平台期。结论该方法能成功分离出纯度较高的大鼠脑微血管周细胞。
목적탐토대서뇌미혈관주세포적분리、배양화감정방법。방법10지3주령Wstar 대서，무균분리뇌조직，채용2차매소화화1차밀도제도리심법분리뇌미혈관편단，접충우35 mm배양명진행원대배양。채용상차현미경관찰세포형태，면역형광법감정α-평활기기동단백（α-smooth muscle actin,α-SMA）、신경원-효질항원2（neuron-glial antigen 2，NG2）、von Willebrand인자(von Willebrand factor，vWF)화효질섬유산성단백（glial fibrillary acidic protein，GFAP）상관항원，갑기새서기사서법측정세포생장곡선。결과주세포종첩벽적뇌미혈관편단주위파출，정다각성，12～14 d후세포융합95％。면역형광염색현시，주세포분자표지물α-SMA화NG2상관항원정쌍양성，vWF화GFAP 상관항원정쌍음성，증실배양세포위뇌미혈관주세포。원대세포초시생장속도교만，전대세포36～60h진입대수생장기，72～108 h생입평태기。결론해방법능성공분리출순도교고적대서뇌미혈관주세포。
Objective To explore the method of primary isolation, cultivation and identification of rat brain microvessel pericytes. Methods The brain tissue of 10 3 week-old Wistar rats was separated sterilely. The brain microvessel fragments were separated using two-step enzyme digestion and one-step gradient centrifugation and were seeded in 35-mm dishes for primary culture. The cell morphology was observed by phase contrast microscopy; the immunofluorescence assay was used to identify the associated antigns, such as the α-smooth muscle actin (α-SMA), neuron-glial antigen 2 (NG2), von Willebrand factor (vWF), and glial fibrillary acidic protein (GFAP). Methyl thiazolyl tetrazolium was used to determine the cell growth curve. Results Pericytes climbed out from the adherent brain microvascular fragments around,showing polygonal, and the cell fusion was 95％ after 12-14 days. Immunofluorescence staining revealed that the molecular markers of the pericytes α-SMA and NG2 related antigens showed double positive, while the vWF and GFAP related antigens showed double negative and the cultured cells were confirmed as brain microvascular pericytes. The growth rate of primary cells was slower. The passage cells entered into logarithmic growth phase after 36 to 60 hours and entered into plateau phase after 72 to 108 hours. Conclusions This method may successfully isolate rat brain microvascular pericytes with higher purity.