肠三叶因子对肠上皮细胞增殖的影响及其信号转导机制的实验研究

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

Experimental study on the effects of intestinal trefoil factor on intestinal epithelial proliferation and its signal transduction mechanism

OBJECTIVE: To explore the effects of intestinal trefoil factor (ITF) on intestinal epithelial proliferation and its possible signal transduction mechanism. METHODS: 1). The intestinal epithelial cytoplasmic membrane was isolated and harvested from Wistar rats, and it was treated with various doses of ITF in the concentration of 0.01, 0.10, 1.00 and 10.00 micro g/ml. The tyrosine protein kinase (TPK) activity from cytoplasmic membrane ITF receptor was determined by membrane-bound method. 2). The intestinal epithelial cells 6 (IEC-6) cultured in vitro were employed in the study. Some of the cells were used as normal control, while a group of cells were stimulated by 1 micro g/ml ITF, and others were treated by PD098059, SB202190 and SB202474, respectively. The last three agents were inhibitors of three members of mitogen-activated protein kinase family (MAPKs), i.e. extracellular signal regulated protein kinases (ERKs), protein kinase p38 (p38), and stress-activated protein kinase (SAPK). They were used before the addition of 1 micro g/ml of ITF. The changes in DNA synthetic rate and the MAPK activity of IEC-6 after being treated by above agents were assessed by (3)H-TdR incorporation method. RESULTS: ITF receptor possessed TPK activity. TPK activity of ITF receptor, MAPKs activity and DNA synthetic rate of IEC-6 were increased obviously under the stimulation of ITF (P < 0.01). The above ITF effects could be evidently blocked by PD098059 and partially attenuated by SB202474. But SB202190 showed no effect in this respect. CONCLUSION: ITF could promote intestinal epithelial proliferation and transmit extracellular signals mainly by means of ERKs signalling pathway.