原代肾小管上皮细胞的提取和培养方法的优化  

Isolation and Culture of Primary Renal Tubular Epithelial Cells

作　　者：曲玥阳 陈旭[2] 岳喜庆[2] 罗勇[1] 赵伟杰[1] 林炳承[1] QU Yue-yang;CHEN Xu;YUE Xi-qing;LUO Yong;ZHAO Wei-jie;LIN Bing-cheng(Department of Chemical Engineering/State Key Laboratory of Fine Chemicals,School of Pharmaceutical Science and Technology,Dalian University of Technology,Dalian Liaoning 116023,China;College of Food Science,Shenyang Agricultural University,Shenyang Liaoning 110161,China)

机构地区：[1]大连理工大学制药科学与技术学院/精细化工重点实验室,辽宁大连116023 [2]沈阳农业大学食品学院,沈阳110161

出　　处：《沈阳农业大学学报》2018年第1期27-33,共7页Journal of Shenyang Agricultural University

基　　金：国家自然科学基金项目(21675017);中央高校基本科研项目(DUT17LK25)

摘　　要：为建立理想的研究农药中毒性肾病体外细胞模型,系统考察了原代肾小管上皮细胞提取和培养的一系列关键因素,经过优化组合,提出了一种更为高效实用的原代肾小管上皮细胞提取和培养方法。通过显微镜明场观察肾小管节段组织形态和纯度,研究0.1%的Ⅰ型胶原酶、Ⅱ型胶原酶、Ⅳ型胶原酶、Ⅴ型胶原酶在15min和3h的时候对组织的消化情况以及Ⅱ型胶原酶作用0min,8min,15min,40min,1h,3h时组织消化情况。利用MTT比色法考察了鼠尾I型胶原,层粘连蛋白,基质胶对培养皿的包被情况对细胞增殖速率的影响,利用MTT比色法考察不同浓度的血清对细胞增殖速率以及细胞活性维持的影响,细胞免疫荧光法考察不同浓度血清对肾小管上皮细胞的Na^+/K^+ATPase表达的影响。结果表明:利用Ⅱ型胶原酶,消化15min可以获得较好的肾小管节段;利用基质胶包被培养皿是性价比最高的促进肾小管上皮细胞增殖的条件;观察到在细胞增殖期内,1%,2%,4%的血清浓度较0%和5%有较好的促进细胞增殖作用;在平台期内,2%,4%,5%的血清浓度较0%和1%有较好的细胞活性维持作用;第3天时,2%血清浓度培养的肾小管上皮细胞的Na^+/K^+ATPase表达最强;第14天时,1%和2%血清浓度培养的细胞的Na^+/K^+ATPase表达较好,最终确定血清最优浓度为2%。在优化条件下所获得的原代肾小管细胞的角蛋白18和碱性磷酸酶的表达呈阳性。经过上述系统优化,细胞生长周期由传统方法的4~7d缩短到2~3d,并且实现了传统方法无法实现的原代肾小管上皮细胞长期培养,时间长达14d,从而为农药中毒性肾病的深入研究奠定了坚实的基础。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 collagenaseⅠ,collagenaseⅡ,collagenaseⅣ,collagenaseⅤfor 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.

关 键 词：肾小管上皮细胞 原代培养 长期培养 

分 类 号：R962.6[医药卫生—药理学]