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【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。
【Objective】 To investigate the feasibility of purifying epithelial cells from human benign prostatic hyperplasia by Percoll discontinuous density gradient centrifugation. 【Methods】 Prostate hyperplasia tissue fragments were digested with collagenase under aseptic conditions, and then the cell suspension was centrifuged through gradient columns with two different mass density solutions (! A = 1.070g / mL Percoll and! B = 1.035g / mL Percoll) Prostate epithelial cells were extracted from the interface between two Percoll solutions with a syringe, washed with HBSS solution, and then cultured at 37 ° C in a 5% CO2 incubator at a constant volume fraction. Cell morphology and growth were observed. Trypan blue staining was used to calculate the percentage of viable cells. Epithelial cells were identified by immunohistochemical staining of keratin. 【Result】 Percoll non-continuous gravimetric gradient centrifugation could separate epithelial cells from fibroblasts and other stromal cells with a purity of more than 95%. Trypan blue staining calculated viable cell rate of 95%. Purified cells were epithelial cell-specific appearance of paving stones, continuous 3 to 5 generations. Cytokeratin CK7 / 8 staining positive cells confirmed epithelial origin. 【Conclusion】 Percoll discontinuous density gradient centrifugation is simple and feasible, and can obtain prostatic epithelial cells with high purity and good vitality.