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目的 探讨肠三叶因子 (ITF)对肠上皮细胞增殖能力的影响及可能的信号转导机制。 方法 (1)分离、提取Wistar大鼠的肠上皮细胞膜。分别用浓度为 0 .0 1、0 .10、1.0 0、10 .0 0 μg/ml的ITF刺激细胞膜 ,以膜结合法测定膜上ITF受体酪氨酸蛋白激酶 (TPK)活性的变化 ,以ITF受体TPK活性基础值作正常对照。 (2 )体外培养肠上皮细胞株IEC 6,部分细胞作为正常对照 ;部分细胞用 1.0 0μg/ml的ITF进行刺激 ;部分细胞用丝裂素活化蛋白激酶 (MAPK)家族的 3种阻断剂———PD0 980 5 9、SB2 0 2 190、SB2 0 2 4 74分别进行预处理后 ,加入 1.0 0 μg/ml的ITF。采用氚标记胸腺嘧啶脱氧核苷 (3 H TdR)掺入法 ,观察上述各种方法处理后 ,IEC 6的DNA合成率及MAPK活性的变化。 结果 与正常对照相比 ,在ITF的刺激下 ,ITF受体的TPK活性、IEC 6的MAPKs活性及DNA合成率均明显增高 (P<0.0 1)。使用PD0 980 5 9后 ,能明显阻断ITF的后两种作用 (P <0.0 1);使用SB2 0 2 4 74能部分降低该作用 ,而使用SB2 0 2 190效果不明显。 结论 提示ITF主要通过细胞外信号调节激酶 (ERKs)途径传递胞外信号、促进细胞增殖
Objective To investigate the effect of intestinal trefoil factor (ITF) on the proliferation of intestinal epithelial cells and the possible signal transduction mechanism. Methods (1) The Wistar rat intestinal epithelial cell membrane was isolated and extracted. The cell membrane was stimulated with ITF at concentrations of 0.010, 1.010, 10.0 and 10.0 μg / ml respectively, and the changes of ITF receptor tyrosine protein kinase (TPK) activity on the membrane were measured by membrane binding assay. Based on the ITF receptor TPK activity as a normal control. (2) The intestinal epithelial cell line IEC 6 was cultured in vitro. Some cells were used as normal control. Some cells were stimulated with ITF 1.0 0μg / ml. Some cells were treated with three blockers of mitogen-activated protein kinase (MAPK) - PD0 980 5 9, SB2 0 2 190, SB2 0 2 4 74, respectively, after pre-treatment, add 1.0 0 μg / ml of ITF. Tritium labeled thymidine incorporation method was used to observe the changes of DNA synthesis rate and MAPK activity of IEC 6 after the above treatments. Results Compared with the normal control group, ITF receptor TPK activity, IEC 6 MAPKs activity and DNA synthesis rate were significantly increased (P <0.0 1). The use of PD0 9805 9 significantly blocked the latter two effects of ITF (P <0.0 1); SB2 0 2 4 74 partially reduced this effect, whereas the effect of SB2 0 2 190 was insignificant. The results suggest that ITF mainly transmit extracellular signals via extracellular signal-regulated kinase (ERKs) pathway and promote cell proliferation