兔阴茎海绵体内皮细胞的培养和鉴定

Culture and Identification of Rabbit Cavernosal Endothelial Cells

本研究旨在探讨兔阴茎海绵体内皮细胞体外培养技术,为构建组织工程化阴茎海绵体提供种子细胞。无菌获取兔阴茎海绵体组织后,分别采用IV型胶原酶消化法和弹性蛋白酶消化结合机械挤压法分离内皮细胞,比较两种分离方法所获内皮细胞的纯度;进一步在添加生长因子的内皮细胞生长液中培养扩增,观察细胞形态及增殖情况,并对传代细胞进行免疫组化染色鉴定。结果显示采用弹性蛋白酶消化结合机械挤压组织法能有效分离内皮细胞且保留基质的完整性,避免基质细胞混杂,明显优于IV型胶原酶消化法;培养的内皮细胞呈典型的鹅卵石形态,内皮细胞特异性标志CD31、第Ⅷ因子相关抗原(von Willebrand factor,vWF)表达阳性。结果提示采用弹性蛋白酶消化结合机械挤压组织法能快速有效地分离纯度较高的内皮细胞,进一步在内皮细胞生长液中培养能稳定传代扩增,可为阴茎海绵体组织工程提供种子细胞。

The objective of this study was to establish a protocol for isolating, culturing, proliferating cavernosal endothelial cells, and to obtain large number of autologous cavernosal endothelial cells for tissue-engineered substitutes. Cavernosal tissues were either minced into 1 to 2 mm3 pieces and incubated with 0.02% collagenase IV for 2h or first minced into 20 mm3 then incubated with 0.05% elastase for 1h, 2h or 3h, respectively;the undigested tissue blocks were repeatedly squeezed with a spatula for 2 min....