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，利用免疫印迹分析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，lckmRNA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5min、15mln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。

Role of Ick/ p38 signaling pathways in murine renal tubular epithelial cells

LI Qinggang ,LI Youji ,ZHENG Xunhua,et al.

(Department of Nephrology,Key Clinical Kidney Research Institute of Ministry of Health,First Affiliated Hospital,Sun Yat-sen University of Medical Sciences,Guangzhou 510080,China)

Abstract：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 lek 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.

Keywords：Renal tubule;Epithelial cells;Signal transduction;Lymphocyte-specific protein-tyrosine kinase;p38

基金项目：卫生部基金资助项目(94-1-105)

参考文献：

［1］Healy E,Brady HR.Role of tubular epithelial cells in the pathogensis of tubulointerstitial fibrosis induced by glomerular disease.Curr Opin Nephrol Hypertens,1998,7:525-530.

［2］Gabbai FB,Boggiano C,Peter T,et al.Inhibition of inducible nitric oxide synthase intensifies injury and functional deterioration in autoimmune interstitial nephritis.J Iummunol,1997,159:6266-6275.

［3］Fan X,Oertil B,Wuthrich RP.Up-regulation of tubular epithelial interleukin-12 in autoimmune MRL-Fas(lps) mice with renal injury.Kidney Int,1997,51:79-86.

［4］Banu N, Meyers CM,IFN-gamma and LPS differenially modulate class Ⅱ MHC and B7-1 expression on murine renal tubular epithelial cells.Kidney Int,1999,55:2250-2263.

［5］Schafer PH,Wang L,Wadsorth SA,et al.T cell activation signals up-regulate p38 mitogen-activated protein kinase activity and induce TNF-IL Pha production in a manner distinct from lps activation of monocytes.J Immumol,1999,162:659-668.

［6］Pignata C,Prasad KV,Hallek M,et al.Phosphorylation of Src family lck tyrosine kinase following interleukin-12 activation of human natural killer cells.Cell Immunol,1995.165:211-216.

［7］Chung SD,Alabi N,Livingston,et al.Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hornmnally defined medium.J Cell Biol,1982,95:118-126.

［8］Detisac C J,Sews MA,Garvin A J,et al.Tissue culture of human kidney epithelial cells of proximal tubule origin.Kidney Int,1984,25:383-390.

［9］Kroshian VM,Seridan AM,Lieberthal W.Functional and cytoskeletal changes induced by sublethal injury in proximal tubular epithelial cells.Am Physio Soc,1994,F21-F27.

［10］Tinkle CW,Lipschitz D,Ponnappan U.Decreased association of P56lck with CD4 may account for lowered tyrosine kinase activity in mitogen-activated human T lymphocytes during aging.Cell Immunol,1998,186:154-160.

［11］Hanke JH,Gardner JP,Dow RL,et al.Discovery of a novel,potent,and Src family-selective tyrosine kinase inhibitor,study of Lck-and FynT-dependent T cell activation.J Biol Chem,1996,271:695-701,

［12］李晓玫,唐嘉薇,王海燕.白细胞介素1对肾系膜细胞分裂活化蛋白激酶不同亚类的作用.中国免疫学杂志,1998,14:300-303.

［13］Amundatottir LT,Leder P.Signal transduction pathways activated and required for mammary carcinogenesis in response to specific oncogenes.Oncogene,1998,16:737-746.

［14］张宗梁,林明群,王妙珠,等.巨噬细胞免疫变信号:Raf-1,MAPK PH, MAPK P42和p38 MAPK的研究.实验生物学报,1997,30:73-79.

［15］Zhang F,Nakamura T,Aune TM.TCR and IL-12 receptor signal cooperate to activate an individual response element in the IFN gamma promoter in effector Th cells.J Immunol,1999,163:728-735.

收稿日期：2000-01-18