课题背景：本实验设计思路是首先通过时间依赖实验以初步筛选出比较科学合理的时间点，再在此基础上，进行剂量依赖性的观察，以观察转化生长因子β1对大鼠近端肾小管上皮细胞中的基质细胞衍生因子1的影响。

应用要点：实验通过证实在大鼠近端肾小管上皮细胞中转化生长因子β1对基质细胞衍生因子1的调节作用，为揭开基质细胞衍生因子与肾间质纤维化间的关系提供实验基础。

术语解析：基质细胞衍生因子1是一种细胞因子，属CXC(Cys-X-Cys)类趋化蛋白，系统命名CXCL12（CXC chemokine ligand-12），是已知唯一能与受体CXCR4结合并激活的天然趋化因子。基质细胞衍生因子1 和CXCR4广泛地表达于多种细胞和组织中，包括免疫细胞、脑、心脏、肾、肝、肺和脾，在免疫系统、循环系统及中枢神经系统的发育中起着至关重要的作用。


摘要
目的:已知转化生长因子β1与肾脏组织纤维化形成有密切关系。拟进一步探讨转化生长因子β1对大鼠近端肾小管上皮细胞（NRK52E）中基质细胞衍生因子1表达的影响。
方法：实验于2006-03/2007-05在四川大学生物治疗国家重点实验室神经分子生物实验室完成。①实验材料：大鼠近端肾小管上皮细胞株NRK52E，由澳大利亚Monash医学中心肾内科实验室提供；转化生长因子β1由cytolab公司提供。②实验分组：将大鼠近端肾小管上皮细胞分为正常对照组：无转化生长因子β1 干预；实验组：又分为在同一转化生长因子β1浓度(2 μg/L)下，培养6，12，24 h；在不同的转化生长因子β1浓度(2，5，10 μg/L)下，培养24 h。③利用免疫细胞化学技术对同一转化生长因子β1浓度(2 μg/L)干预不同时间后的大鼠近端肾小管上皮细胞中基质细胞衍生因子１的蛋白表达进行半定量分析，选择出最佳的作用时间点；通过反转录-聚合酶链反应、Western-Blotting以检测大鼠近端肾小管上皮细胞中基质细胞衍生因子1在不同转化生长因子β1浓度干预下培养24 h后的mRNA、蛋白表达变化情况。
结果：①培养12，24 h时,大鼠近端肾小管上皮细胞中基质细胞衍生因子1的蛋白表达比0 h增高(P < 0.05)；24 h时表达略高于12 h (P > 0.05)。提示，基质细胞衍生因子1的表达到达一平台期，24 h为最佳的作用时间点。②从mRNA水平和蛋白水平均证实，2 μg/L转化生长因子β1干预24 h后的大鼠近端肾小管上皮细胞中基质细胞衍生因子1的表达高于正常对照组 (P < 0.05)；随着转化生长因子β1的浓度增大，表达呈下降趋势，10 μg/L时表达低于2 μg/L时的表达(P < 0.05)。
结论：基质细胞衍生因子1在正常的大鼠近端肾小管上皮细胞中呈低表达状态，对转化生长因子β1的干预表现出一定的时间、剂量依赖性，基质细胞衍生因子1可能参与了肾间质纤维化的发生、发展。
关键词：转化生长因子β；趋化因子；肾小管，近端／细胞学；组织构建

邹杨，樊均明.转化生长因子β1对大鼠肾小管上皮细胞中基质细胞衍生因子1表达的影响[J].中国组织工程研究与临床康复，2008，12(11):2029-2033 [www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2029(ps).pdf]

Effect of transforming growth factor-beta 1 on stromal cell-derived factor 1 expression in rat renal tubular epithelial cells

Abstract

AIM： It has been proved that transforming growth factor-β1 (TGF-β1) has a close correlation with kidney fibrosis. This study investigated the effects of TGF-β1 on stromal cell-derived factor 1 (SDF-1) in rat renal tubular epithelial cells (NRK52E).
METHODS: The experiment was carried out in the Division of Molecular Neurobiology, State Key Laboratory of Biotherapy, Sichuan University from March 2006 to May 2007. ①Rat renal tubular epithelial cells (NRK-52E, Monash Medical Center, Australia) were divided into normal control group, which were not treated with TGF-β1 (Cytola), and experimental group, which were subdivided into time-dependent groups: NRK-52E cells were incubated with TGF-β1 at the same concentration (2 μg/L) for 6, 12, and 24 hours, and dose-dependent groups: NRK-52E cells were treated with TGF-β1 at different concentrations (2, 5, and 10 μg/L) for 24 hours. ②The semi-quantitative calculation of SDF-1 protein expression in the cells treated with TGF-β1 at the concentration of 2 μg/L for different time was accessed by immunocytochemistry to find the best time point. Then the variances of the mRNA and protein expression level in cells treated with TGF-β1 at different concentrations for 24 hours were detected by RT-PCR and Western-Blotting.
RESULTS: ①The protein expression level of SDF-1 in NRK-52E cells treated with TGF-β1 for 12 and 24 hours was higher than for 0 hour (P < 0.05), and the level for 24 hours was slightly higher than for 12 hours (P > 0.05). This indicated the SDF-1 expression reached a flat stage, and 24 hour was the bets time to study the dose dependence. ②Both the mRNA and protein expression level analyses demonstrated that the expression of SDF-1 in NRK-52E cells treated with 2 μg/L TGF-β1 for 24 hours was significantly higher than that in normal control group (P < 0.05), and the expression was declined as the concentration of TGF-β1 increased. Moreover, the expression with 10μg/L was lower than with 2 μg/L (P < 0.05).
CONCLUSION: SDF-1 is weakly expressed in normal NRK-52E cells, and its expression shows a time- and dose-dependence to TGF-β1. It suggests that SDF-1 may play a significant role in the occurrence and development of renal fibrosis.

Zou Y, Fan JM. Effect of transforming growth factor-beta 1 on stromal cell-derived factor 1 expression in rat renal tubular epithelial cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(11):2029-2033(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2029(ps).pdf]