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目的 探讨蛋白质酪氨酸磷酸酶(SHP2)及丝裂原活化蛋白激酶磷酸酶5 (MKP5 )在P2Y嘌呤受体激活人不同转移潜能的前列腺癌细胞系MAPKs信号通路中的调节机制及其与细胞侵袭能力的相关性。方法 构建野生型和突变型SHP2及野生型MKP5表达质粒并分别稳定转染入1E8(高转移)和(或)2B4(不转移)细胞。应用免疫沉淀法检测细胞SHP2的酪氨酸磷酸化的程度,免疫印迹法检测ERK1 /2、p38的磷酸化,用Matrigel穿膜实验检测细胞的体外侵袭能力。结果 ATP可刺激细胞的SHP2发生磷酸化,且在1E8中的激活程度高于2B4。野生型SHP2转染可使2B4细胞中ATP对ERK1 /2的激活时效明显延长;而突变型SHP2延迟并降低1E8细胞中ATP对ERK1 /2的激活水平;两者对细胞p38的活性均无明显影响。ATP刺激可使不转移2B4细胞穿膜数明显增多(比对照组增加72 2% ±14 0% ),野生型SHP2转染可使经ATP刺激的细胞穿膜数进一步增加18 7%。ATP刺激可使高转移1E8细胞穿膜数进一步增加(112 8% ±32 0% ),而转染突变型SHP2可部分(40 9% )抑制ATP的促细胞侵袭作用。转染野生型MKP5在明显抑制p38磷酸化的同时,也能部分抑制ATP的促侵袭作用(抑制率在1E8和2B4分别为22 4%和28 7% )。结论 SHP2正性调节P2Y嘌呤受体介导的ERK活化,并可促进前列腺癌细胞的侵袭能力。而MKP5
Objective To investigate the regulatory mechanism of protein tyrosine phosphatase (SHP2) and mitogen-activated protein kinase phosphatase 5 (MKP5) in the MAPKs signal pathway of P2Y purine receptor activating human metastasis potential Correlation of invasiveness. Methods Wild-type and mutant SHP2 and wild-type MKP5 expression plasmids were constructed and stably transfected into 1E8 (high metastasis) and / or 2B4 (non-metastatic) cells respectively. The level of tyrosine phosphorylation of SHP2 was detected by immunoprecipitation. The phosphorylation of ERK1 / 2 and p38 was detected by immunoblotting and the invasion ability of cells was tested by Matrigel transmembrane assay. Results ATP stimulated the phosphorylation of SHP2 in cells, and its activation in 1E8 was higher than that in 2B4. The wild-type SHP2 transfection could significantly prolong the activation of ERK1 / 2 by ATP in 2B4 cells, while the mutant SHP2 delayed and decreased the activation of ERK1 / 2 by ATP in 1E8 cells; influences. The number of transmembrane of untransfected 2B4 cells increased significantly (by 72 2% ± 140% compared with the control group) by ATP stimulation. The transfection of wild-type SHP2 could further increase the number of ATP-stimulated cells by 18 7%. ATP stimulation increased the number of transplanted cells in the highly metastatic 1E8 cells (112 8% ± 32 0%), while transfected mutant SHP2 partially (40 9%) inhibited the pro-cellular invasion of ATP. Transfection of wild-type MKP5 partially inhibited the pro-invasive effect of ATP while p38 phosphorylation was significantly inhibited (inhibition rates of 22 4% and 28 7% at 1E8 and 2B4, respectively). Conclusion SHP2 positively regulates P2Y purinergic receptor-mediated ERK activation and promotes invasiveness of prostate cancer cells. MKP5