【摘要】 【目的】探讨应用Percoll非连续密度梯度离心法从人前列腺增生组织中纯化上皮细胞的可行性。【方法】无菌条件下用胶原酶消化前列腺增生组织碎块,获得细胞悬液经两种不同质量密度溶液(!A=1.070g/mLPercoll和!B=1.035g/mLPercoll)的梯度柱离心后,以注射器于两种Percoll溶液界面之间抽出前列腺上皮细胞,以HBSS液洗涤细胞后置于37℃、体积分数5%CO2孵箱中连续培养。观察细胞形态及生长,台盼蓝染色计算活细胞率,细胞角质蛋白免疫组织化学染色鉴定上皮细胞。【结果】经Percoll非连续比重梯度离心法,能将上皮细胞与成纤维细胞和其他基质细胞分离,纯度达95%以上。台盼蓝染色计算活细胞率达95%。纯化得到的细胞呈上皮细胞特有的铺路石样外观,可连续传3～5代。细胞角质蛋白CK7/8染色阳性证实细胞为上皮源性。【结论】Percoll非连续密度梯度离心法简便可行,能获得纯度较高、活力良好的前列腺上皮细胞。更多还原

【Abstract】 [Objective] To evaluate the feasibility of culturing epithelial cells from human benign prostatic hyperplasia (BPH) tissue by discontinuous Percoll gradient centrifugation. [Methods] Prostate tissues from BPH men were cut into small pieces and digested as single cell suspension by collagenase in sterile conditions. The cell suspension was added into discontinuous Percoll gradient (mass density, ?籽A = 1.035 g/mL and ρB = 1.070 g/mL) and centrifuged. The cells were sucked out between the interface of the two Percoll solutions by a syringe, washed by HBSS solution and cultured in 37℃??渍= 5%CO2 condition. Trypan blue staining was used to calculate the cell viability. The cell-morphology was observed by light microscope and the cell characteristics were identified by immunohistochemistry staining of cytokeratin. [Results] The epithelial cells were isolated from fibroblasts and other stroma cells, the purity could be achieved as 95% . Trypan blue staining result showed 95% of the cells were viable. The cultured epithelial cells had typical cobblestone shape and could be maintained up to 3-5 passages. The cells were stained positively by cytokeratin 7/8 antibody.[Conclusions ] The method of discontinuous Percoll gradient centrifugation is a convenient and feasible method to enrich pure and viable prostatic epithelial cells.更多还原