中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (50): 9358-.doi: 10.3969/j.issn.1673-8225.2010.50.012

• 组织构建实验造模 • 上一篇    下一篇

小鼠肾脏近端小管上皮细胞原代培养条件优化及功能测试

臧路平1，吴新荣1，2   

  1. 1华南理工大学轻工与食品学院，广东省广州市   510640；2解放军广州军区广州总医院药学部，广东省广州市  510010
  • 出版日期:2010-12-10 发布日期:2010-12-10
  • 通讯作者: 吴新荣，博士生导师，教授，主任药师，华南理工大学轻工与食品学院，广东省广州市 510640；解放军广州军区广州总医院药学部，广东省广州市 510010 gzwxrong@msn.com
  • 作者简介:臧路平★，女，1985年生，山东省济宁市人，汉族，华南理工大学在读硕士，主要从事药物筛选及治疗机制研究。 zangluping1985@163.com

Optimization of primary culture and functional test of mouse renal proximal tubular epithelial cells

Zang Lu-ping1, Wu Xin-rong 1,2   

  1. 1 College of Food and Bioengineering, South China University of Technology, Guangzhou  510640, Guangdong Province, China; 2 Department of Pharmacy, General Hospital of Guangzhou Military Area Command of Chinese PLA, Guangzhou  510010, Guangdong Province, China
  • Online:2010-12-10 Published:2010-12-10
  • Contact: Wu Xin-rong, Doctoral supervisor, Professor, Chief Pharmacist, College of Food and Bioengineering, South China University of Technology, Guangzhou 510640, Guangdong Province, China; Department of Pharmacy, General Hospital of Guangzhou Military Area Command of Chinese PLA, Guangzhou 510010, Guangdong Province, China gzwxrong@msn.com
  • About author:Zang Lu-ping★, Studying for master’s degree, College of Food and Bioengineering, South China University of Technology, Guangzhou 510640, Guangdong Province, China zangluping1985@ 163.com

PDF

724

被引次数

5

摘要：

背景：好的肾脏近端小管上皮细胞原代培养方法能够为药物筛选、机制研究等提供模型基础。
目的：探索小鼠肾脏近端小管上皮细胞原代培养的最佳处理条件，并对其进行尿酸吸收功能测试。
方法：无菌取小鼠肾脏，去除外膜及脂肪组织，切取近端皮质部分，机械破碎，Ⅰ型胶原酶消化，依次过50目，100目筛网过滤，Percoll密度梯度离心法分离肾小管节段，接种至一次性细胞培养瓶。制备细胞爬片，SP法染色鉴定细胞。在1 500 μmol/L 尿酸浓度下，以不同浓度梯度的丙磺舒和苯溴马隆作为指示药物，检测细胞尿酸吸收及作为药物作用体外模型功能。
结果与结论：经机械破碎的肾脏近端皮质组织在400 U胶原酶处理20 min时，肾小管节段贴壁率达到最高为54.5%；首次换液最佳时间为72 h；细胞在原代培养第5天生长达到对数期，在第10天状态开始有所下降，折光率低，出现部分漂浮死亡细胞。在1 500 μmol/L尿酸浓度的吸收培养基中，吸收实验进行30 min时，其对尿酸吸收速率达到最大。指示药物丙磺舒和苯溴马隆在不同的浓度下，对于尿酸吸收的抑制率不同，其中苯溴马隆的抑制率要高于丙磺舒。提示在上述分离培养条件下能够培养出数量多、活性高的目的细胞，并且具有良好的尿酸吸收功能，在一定的尿酸和药物浓度下，能够作为研究药物作用的体外细胞模型。

关键词: 小鼠, 肾脏, 近端小管, 上皮细胞, 原代培养, 尿酸吸收

Abstract:

BACKGROUND: An excellent primary culture of renal proximal tubular epithelial cells provides basis for drugs screening and mechanism research.
OBJECTIVE: To establish a modified method for the primary culture of mouse renal proximal tubule epithelial cells, and to test it urate uptake function.
METHODS: Kidneys were harvested and the cortices were dissected, minced, and digested using collagenase type Ⅰ. Fragments of the tissues were filtered with 50 and 100 mesh filter. Then the deposition was dissociated by gradient centrifugation with percoll. The isolate was implanted in the cell culture flasks. The bred cells were sub-cultured and identified by SP immunocytochemistry. Uric acid uptake function was tested by a range of probenecid and benzbromarone concentrations in transport medium contained 1 500 μmol/L uric acid.
RESULTS AND CONCLUSION: The adherent rate of segments of renal proximal tubules reached 54.5% after digested using 400U collagenase type I for 20 minutes; the most suitable time for the first change of the culture medium was 72 hours; Log phase began from the fifth day and the cell’s growth conditions become bad in the tenth day. In the transport medium contained    1 500 μmol/L urate acid, the 30-min uptake of uric acid was the highest and was taken as a measure of an initial rate. Different urate uptake inhibitions were appeared from different concentrations of the indicate drugs probenecid and benzbromarone. The inhibitions of benzbromarone were higher than probenecid. The mouse renal proximal tubular epithelial cells cultured by the modified method will yield rich and homogeneous harvest, and present well function of urate acid uptake. The study offers a model in vivo for pharmacological studies.

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