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发表时间：2018-01-01

发表刊物：沈阳农业大学学报

所属单位：化工学院

卷号：49

期号：1

页面范围：27-33

ISSN号：1000-1700

摘要：In order to build an optimal in vitro cell model for studying pesticide poisonous nephropathy, we systematically investigated a series of critical factors, optimized and combined the conditions, and finally developed a simple and reliable protocol to isolate and culture the primary renal tubular epithelial cells. We characterized the tissue digestion by observing the forms of renal tubules with collagenase Ⅰ, collagenaseⅡ, collagenase Ⅳ, collagenase Ⅴ for 15 min and 3h, with the time of collagenaseⅡdigestion, including 0 min, 8 min, 15 min, 40 min, 1 h, and 3h. To investigate the dish coating conditions for cell proliferation, the rat tail collagen I, laminin and basement membrane extractant were contrasted by MTT method. We used MTT method to investigate the effects of different concentrations of serum on cell proliferation and activity maintaining, and used cell immunofluorescence to investigate the expression of Na+/K+ATPase in renal epithelial cells cultured with different concentrations of serum in the third day and fourteenth day. We found that digesting the tissues with collagenaseⅡfor 15min could lead to tubular sections with good shape and suitable size, and the basement membrane extractant was better than others. In the proliferative stage, compared to 0% and 5% serum, 1%, 2% and 4% serum had more positive effect. In the plateau, compared to 0% and 1% serum, 2%, 4% and 5% could maintain better cell viability. In the third day, Na+/K+ATPase expression with 2% serum was the highest, and in the fourteenth day, Na+/K+ATPase expressions with 1% and 2% serum were better than others. Finally, we chose 2% serum as the optimal serum concentration. With optimal isolation and culture conditions, the cells expressed CK-18 and alkaline phosphatase. Moreover, compared with existing methods, our method reduced the period of cells growth from 4-7 days to 2-3 days, and the culture medium could be used in long time culture of tubular epithelial cells and maintained their physiological functions for 14 days.

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