兔脊髓微血管内皮细胞的体外培养及鉴定

Culture and identification of primary rabbit spinal cord microvascular endothelial cells in vitro

中文关键词:  脊髓微血管内皮细胞  酶促消化法  Ⅷ因子相关抗原  免疫细胞化学染色法  日本大耳白兔

英文关键词:Microvesselsendelial cells of spinal cord  Enzymatic digestion method  Factor Ⅷ related antigen  Immunocytochemistry  Japanese large ear white rabbit

中文摘要:

  【摘要】 目的：建立一种高效的兔原代脊髓微血管内皮细胞（spinal cord microvascular endothelial cells，SCMECs）的体外分离培养方法。方法：选取2周龄日本大耳白兔15只，雌雄不限。在无菌条件下剪开背部皮肤肌肉层，充分暴露脊柱，沿其纵轴方向剪开椎管，获得脊髓组织。剪碎脊髓，用牛血清白蛋白密度梯度离心去髓鞘杂质、筛网过滤获得脊髓微血管段。采用酶促消化法，予0.1% Ⅱ型胶原酶消化微血管段20min后，将其接种于培养皿中，并置于CO2培养箱内静止培养72h。上述操作每次取5只，重复3次，以第3次培养的目的细胞为观察、鉴定对象。通过倒置相差显微镜观察目的细胞生长情况及其形态特征、免疫细胞化学染色法检测细胞第Ⅷ因子相关抗原表达情况，鉴定所培养的目的细胞。结果：接种后的脊髓微血管段镜下呈短棍样或分支状；微血管段培养24h后有短梭形细胞从其周围迁移爬出，呈“出芽式”生长；48h后“岛屿状”的细胞集落形成，增殖迅速；72h后细胞铺满皿底，呈典型的单层、“铺路石样”、镶嵌式排列生长。免疫细胞化学染色显示，99%以上的细胞胞质呈现棕红色，Ⅷ因子相关抗原表达为阳性，苏木素衬染胞核显示为蓝色。结论：酶促消化法可以成功分离培养出活性好、纯度高的日本大耳白兔脊髓微血管内皮细胞。

英文摘要:

  【Abstract】 Objectives: To establish an efficient in vitro culture method for spinal cord microvascular endothelial cells(SCMECs) of rabbits. Methods: 15 Japanese large-ear white rabbits(2-week-old, either sex) were selected. Under sterile conditions, the spinal cord was obtained by incising the back skin, muscle layers and vertebral canal along its longitudinal axis. The spinal cord was minced, and myelin impurities were removed by density gradient centrifugation using bovine serum albumin. The spinal cord microvascular segments were obtained by filtration through a sieve. The enzymatic digestion method was used, with 0.1% type Ⅱ collagenase digesting the microvascular segments for 20min, after which, the segments were seeded into culture flasks, and then placed in a CO2 incubator for static culture for 72h. The above procedures were performed with five rabbits at a time, repeated three times, and the target cells from the third culture were taken as the objects of observation and identification. The growth and morphological characteristics of the cells were observed under an inverted phase-contrast microscope. Immunocytochemical staining was used to detect the expression of factor Ⅷ-related antigen to identify the cultured SCMECs. Results: Microscopically, the segments showed short-rod or branched shapes post-seeding. After 24h of culture, short spindle-shaped cells migrated out from around the microvascular segments and grew in a "budding" manner. After 48h, "island-like" cell colonies formed. After 72h, the cells covered the bottom of the dish, growing in a typical monolayer, "paving stone-like" mosaic pattern. Immunocytochemical staining showed that over 99% of the cells had brownish-red cytoplasm, with positive expression of factor Ⅷ-related antigen, hematoxylin stained the nuclei and was shown in blue. Conclusions: Enzymatic digestion can successfully isolate and culture SCMECs of Japanese large-ear white rabbits with good activity and high purity.