A Simple and Nonenzymatic Method to Isolate Human Corpus Cavernosum Endothelial Cells and Pericytes for the Study of Erectile Dysfunction
A Simple and Nonenzymatic Method to Isolate Human Corpus Cavernosum Endothelial Cells and Pericytes for the Study of Erectile Dysfunction
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DOI：
10.5534/wjmh.180091
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发表时间：
2020-01-01
影响因子：
4.8
通讯作者：
Suh, Jun-Kyu
中科院分区：
文献类型：
--
作者：
Yin, Guo Nan;Ock, Jiyeon;Suh, Jun-Kyu
目的：建立一种简单的非酶法分离人阴茎海绵体组织中内皮细胞和周细胞的方法，并评价人阴茎海绵体内皮细胞和周细胞的血管生成能力，为高糖诱导的血管病的研究奠定基础。对于原代人海绵状周细胞培养，通过重力将海绵状组织沉降到培养皿中。我们进行免疫细胞化学和蛋白质印迹，以确定表型和形态学的变化，从第1至第5代。结果：成功分离出高纯度的人阴茎海绵体EC和周细胞。原代培养的EC显示血管性血友病因子的高阳性染色，周细胞显示NG2和血小板衍生生长因子受体β的阳性染色。原代培养的EC和周细胞保持其细胞特性，直到第2或第3代。然而，我们观察到它们的典型表型从第4代和形态学特征从第3代的显着变化。人海绵状EC或周细胞形成组织良好的毛细血管样结构在正常葡萄糖条件下，而严重受损的管形成检测在高糖condition.Conclusions：这项研究提供了一个简单的和非酶的方法，为原代培养人海绵状EC和周细胞。我们的研究将有助于我们了解糖尿病勃起功能障碍的病理生理学，也是确定候选治疗靶点疗效的有价值的工具。
Purpose: To establish a simple and nonenzymatic technique to isolate endothelial cells (ECs) and pericytes from human corpus cavernosum tissue and to evaluate the angiogenic ability of the human cavernous EC or pericytes for the study of high glucose-induced angiopathy.Materials and Methods: For primary human cavernous EC culture, cavernous tissues were implanted into Matrigel in dishes. For primary human cavernous pericyte culture, cavernous tissues were settled by gravity into dishes. We performed immunocytochemistry and Western blot to determine phenotype and morphologic changes from passage 1 to 5. The primary cultured cells were exposed to a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition, and then tube formation assay was done.Results: We successfully isolated high-purity EC and pericytes from human corpus cavernosum tissue. Primary cultured EC showed highly positive staining for von Willebrand factor, and pericyte revealed positive staining for NG2 and platelet-derived growth factor receptor-beta. Primary cultured EC and pericytes maintained their cellular characteristics up to passage 2 or 3. However, we observed significant changes in their typical phenotype from the passage 4 and morphological characteristics from the passage 3. Human cavernous EC or pericytes formed well-organized capillary-like structures in normal-glucose condition, whereas severely impaired tube formation was detected in high-glucose condition.Conclusions: This study provides a simple and nonenzymatic method for primary culture of human cavernous EC and pericytes. Our study will aid us to understand the pathophysiology of diabetic erectile dysfunction, and also be a valuable tool for determining the efficacy of candidate therapeutic targets.