摘要
目的 探讨小鼠肾小管上皮细胞是否存在淋巴细胞特异蛋白质酪氨酸激酶（ｌｃｋ）基因表达及ｌｃｋ／ｐ３８细胞分裂原活化蛋白信号传递分子经ＩＬ－１２、ＩＬ－２、脂多糖（ＬＰＳ）等刺激时在肾小管上皮细胞的变化。方法无菌分离培养ＢＡＬＢ／Ｃ小鼠肾小管上皮细胞，采用地高辛标记的ｌｃｋ寡核苷酸探针原位杂交检测肾小管上皮细胞中ｌｃｋ基因表达；应用放射自显影方法观察ｌｃｋ活性变化，利用兔疫印迹分析ｌｃｋ蛋白表达及ｐ３８磷酸化。结果 ＩＬ－１２、ＩＬ－２、ＬＰＳ刺激肾小管上皮细胞活化，ｌｃｋ ｍＲＮＡ表达较对照组明显增强；经上述物质刺激后ｌｃｋ活性、ｐ３８磷酸化分别于５ ｍｉｎ、１５ ｍｉｎ最强，其中ＬＰＳ刺激最显著而ｌｃｋ、ｐ３８蛋白水平则无明显变化。加入ｌｃｋ抑制剂ＰＰ１，ＩＬ－１２刺激时未发现ｐ３８磷酸化，而ＩＬ－２、ＬＰＳ刺激只有轻度抑制。结论ＩＬ－１２、ＩＬ－２、ＬＰＳ促进肾小管上皮细胞中ｌｃｋ基因表达，并只有ＩＬ－１２唯一通过ｌｃｋ诱导ｐ３８磷酸化，它们皆可通过ｌｃｋ／ ｐ３８信号传递途径参与对肾小管上皮细胞发挥生物学作用。
Objective To investigate whether lymphocyte-specific protein-tyrosine kinase (lck) expression exists in murine renal tubular epithelial cells and to observe the variations of lck/p38 mitogen-activated protein kinase signaling pathways molecules in renal tubular epithelial cells stimulated with IL-12, IL-2 and LPS, respectively. Methods Renal tubular epithelial cells were obtained by separation from BALB/C mice and cultured in conditioned medium. The mRNA expression of lck in renal tubular epithelial cells was measured by in situ hybridyzation with digoxin-labeled lck oligonucleotide prob. Lck activity was determined by autoradiography, and expression of lck protein and phosphorylation of p38 by western blotting analysis. Results The level of lck mRNA was significantly enhanced in renal tubular epithelial cells stimulated with IL-12, IL-2 or LPS compared to control groups. The maximal effect on lck activity and p38 phosphorylation were present 5 and 15 minutes after stimulation as indicated above. In addition, the effect of LPS was the most obvious among the three stimulations, while the expression level of lck and p38 protein was not increased. IL-12 did not stimulate p38 phosphorylation in renal tubular epithelial cells pre-incubated with lck inhibitor PP1, while the inhibitory effects were mild in renal tubular epithelial cells stimulated with IL-2 or LPS. Conclusion IL-12, IL-2 and LPS may promote lck gene express in renal tubular epithelial cells and only IL-12 can induce p38 phosphorylation through lck uniquely, and they can play biologic role in renal tubular epithelial cells via lck/ p38 signaling pathways.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2000年第6期367-370,共4页
Chinese Journal of Nephrology
基金
卫生部基金资助项目!(94-1-105)
关键词
肾小管间质性肾炎
肾小管上皮细胞
信号传递
LCK
Renal tubule
Epithelial cells
Signal transduction
Lymphocyte-specific protein-tyrosine kinase
p38
