[Culture and identification of rabbit cavernosal endothelial cells].
[Culture and identification of rabbit cavernosal endothelial cells].
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发表时间：
2008-08
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通讯作者：
Yun Wei;Lei Wei
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作者：
Yun Wei;Lei Wei
本研究旨在建立海绵体内皮细胞的分离、培养、扩增方法，为组织工程替代物的制备提供大量的自体海绵体内皮细胞。将海绵体组织切碎成1至2 mm 3的块，并与0.02%胶原酶IV孵育2 h，或首先切碎成20 mm 3，然后分别与0.05%弹性蛋白酶孵育1 h、2 h或3 h;用刮刀反复挤压未消化的组织块2 min。收集细胞，并与补充的内皮细胞生长培养基一起培养。观察细胞形态及增殖情况。免疫组织化学方法检测细胞类型特异性蛋白CD 31和vWF。结果表明，0.05%弹性蛋白酶分离细胞2 h后再挤压残余组织不仅能有效分离细胞，而且能防止组织被基质细胞污染，优于IV型胶原酶分离细胞。海绵体内皮细胞呈典型的鹅卵石样形态，可传代7代。免疫组化显示内皮特异性蛋白CD 31和vWF阳性。结果表明，海绵体内皮细胞的分离方法简单有效，细胞增殖快，易于传代。这种新的培养方法将有利于阴茎组织工程的发展。
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. Cells were collected and cultured with supplemented endothelial cell growth medium. Morphology and expansion of the cells were observed. Cell-type specific proteins CD31 and vWF were analyzed by immunohistochemical methods. The results revealed that cell isolation with 0.05% elastase for 2h followed by additional squeezing of residual tissue was effective not only for cell isolation, but also for preventing the tissue from getting contaminated by stromal cells, which was better than cell isolation with collagenase IV. Cavernosal endothelial cells were found with a typical cobblestone morphology and could be passaged 7 passages. Immunohistochemistry showed that endothelial specific proteins CD31 and vWF were positive. These results suggest that the isolation of cavernosal endothelial cells is simple and effective, the cells proliferate rapidly and they can be passaged easily. This method of new cultures will be of benefit to penile tissue engineering.