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

发布时间：2019-02-15 20:37

【摘要】：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。
[Abstract]:Aim: to explore the method of isolation, purification and culture of (CCECs) from rat cavernous endothelial cells, observe its growth characteristics, and provide seed cells for the study of erectile dysfunction. Methods: the penile cavernous tissue of SD rats was digested with 0.1% elastase. The primary SD rat CCECs, was isolated and purified by immunomagnetic beads. The monoclonal CCECs, was screened by clone ring. The morphology and proliferation characteristics were observed, the CCECs CD31 molecules were identified by (VWF), immunohistochemical staining of CCECs angiophilic factor, the purity of CCECs was detected by flow cytometry, the proliferation of CCECs cells was detected by CCK-8 method and the growth curve was plotted. Results: the CCECs growth confluence was found to be a paving stone change under inverted phase contrast microscope after 1 week of culture by clone ring screening and purification. The growth curve showed that the CCECs was in the incubation period for 1 ~ 2 days, and the growth was slow, while for 3 ~ 4 days it was in the logarithmic growth period, and it grew rapidly. On the 6th day, the CCECs entered the plateau stage. Immunofluorescence staining showed that CCECs VWF was positive, and a large amount of green fluorescence could be seen in cytoplasm under fluorescence microscope. Immunohistochemical staining showed that CCECs CD31 molecules were positive, and a large number of yellow brown granules could be seen in the cytoplasm under inverted microscope. The positive rate of VWF labeled CCECs cells was (91.9 卤3.75)% detected by flow cytometry. Conclusion: high purity SD rat CCECs, can be successfully cultured by immunomagnetic beads combined with clone ring purification method, and can grow and subculture stably in endothelial cell culture medium.
【作者单位】：西南医科大学附属医院泌尿外科;
【基金】：四川省科技厅基金(14JC0803) 四川省教育厅基金(15ZA0164) 四川省人力资源和社会保障厅回国人员科研基金(川人社办发[2016]64号)~~
【分类号】：R698

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