改良免疫磁珠结合克隆法培养SD大鼠阴茎海绵体内皮细胞

Culture of rat corpus cavernosal endothelial cells using modified immunomagnetic beads and cloning

目的:探索大鼠阴茎海绵体内皮细胞(CCECs)分离、纯化和培养的方法,观察其生长特性,并为勃起功能障碍的研究提供种子细胞。方法:采用0.1%弹性蛋白酶消化SD大鼠阴茎海绵体组织,分离原代SD大鼠CCECs,然后采用免疫磁珠纯化,进一步扩增,利用克隆环筛选出单克隆CCECs,并观察其形态及增殖特性,免疫荧光染色鉴定CCECs血管假性血友病因子(VWF)、免疫组化染色鉴定CCECs CD31分子,流式细胞仪测定CCECs纯度,CCK-8法检测CCECs细胞增殖并绘制生长曲线。结果:经克隆环筛选纯化培养1周后倒置相差显微镜下观察到CCECs生长汇合呈铺路石样改变。生长曲线显示CCECs最初1~2 d处于潜伏期,生长缓慢,3~4 d处于对数生长期,生长迅速,第6天进入平台期。免疫荧光染色检测CCECs VWF呈阳性,荧光显微镜下胞质内可见大量绿色荧光。免疫组化检测CCECs CD31分子呈阳性,倒置显微镜下胞质内可见大量黄褐色颗粒。流式细胞仪检测经磁珠联合克隆环纯化VWF标记的CCECs细胞阳性率可达(91.9±3.75)%。结论:免疫磁珠联合克隆环纯化法可成功培养出纯度高SD大鼠CCECs,并可在内皮细胞培养液中稳定生长及传代。

Objective： To search for the methods of isolating, purifying and culturing corpus cavernosal endothelial cells （CCECs） from SD rats, observe their growth characteristics, and providing seed cells for the study of erectile dysfunction （ED）. Methods： The corpus cavernosal tissue from the SD rat was digested with 0.1% elastase, followed by purification of CCECs with im- munomagnetic beads. After further amplification, monoclonal CCECs were sorted out with the cloning cylinder and their morphological and proliferative characteristics were observed. The von Willebrand factor （VWF） in the CCECs was identified by immunofluorescence staining, the CD31 molecule detected by immumohistochemistry, proliferation of the cells measured with CCK-8 and growth curves. the purity of the CCECs determined by flow cytometry, and the Results ： After 7 days of purification and culture, the CCECs were fused into a monolayer under the inverted phase-contrast microscope, arranged like fagstones. The growth curves showed that the CCECs were in latency with a low growth rate at 1 - 2 days, in the logarithmic growth phase with a rapid rate at 3 - 4 days, and into the platform phase around the 6th day. VWF was positively expressed in the CCECs with much green fluorescence, and so was CD31 with a large number of brownish particles. The positive rate of the CCECs which were labelled with the VWF purified with magnetic beadscombined with cloning cylinders was up to （91.9 ± 3.75 ） %. Conclusion ： High-purity rat CCECs can be cultured successfully using immunomagnetic beads combined with cloning cylinders, with stable proliferation and passage in the endothelial cell medium.