蛋白激酶C对肥大细胞活化信号转导的影响

目的 :观察蛋白激酶 C (PKC)对肥大细胞 RBL - 2 H 3活化时酪氨酸磷酸化及 c- fos和 c- jun表达等信号转导的影响。方法 :采用流式细胞术和酶联免疫吸附试验测定活化的 RBL - 2 H 3细胞在佛波酯 (PMA)作用后 ,酪氨酸磷酸化和组胺浓度的改变 ;用 RT- PCR观察 PKC对 RBL - 2 H3细胞 c- fos和 c- jun基因 m RNA表达的影响。结果 :致敏 RBL - 2 H3细胞 ,在 PMA作用 48h后 ,抗原活化时磷酸化酪氨酸的平均荧光强度和组胺浓度分别为 (2 .79± 0 .0 7) %和 (6 0 .75± 1.38) nm ol/L ,都明显低于对照组的 (4 .47± 0 .0 3) %和 (10 4.47± 1.31) nm ol/L (P<0 .0 5 ) ;c- fos和 c- jun m RNA的表达量分别减少 91%和 82 %。结论 :PKC的活化可调节肥大细胞酪氨酸蛋白激酶活性 ,上调 c- fos和 c- jun m RNA的表达

Effect of protein kinase C on signal transduction in antigen activated mast cells

Objective: To investigate the effect of protein ki nase C on signal transduction such as tyrosine phosphorylation, c-fos and c-ju n mRNA expression in antigen activated mast cells. Methods: RBL-2H3 cells either untreated or treated with phorbol 12-myristate 13 -acetate (PMA) were sensitized with anti-DNP IgE, and activated with DNP-BSA, histamine release and tyrosine phosphorylation were quantitatively measured by ELISA and flow cytometry, respectively. The effect of PKC on the ex pression of c-fos and c-jun in serum-deprived RBL-2H3 cells activated by DNP-BSA detected by ethidium staining of PCR-amplified cDNA, the amplified cDNA products were subjected to Southern blot hybridization using specific prob es to determine the veracity of amplification. Results: Tyr osine phosphorylation and histamine release were significantly reduced from (4.4 7±0.03)% to (2.79±0.07)% and (104.47±1.31) nmol/L to (60.75±1.38) nm ol/L, respectively, 45 min after DNP-BSA stimulation in sensitized cells pre treated with PMA for 48 h. Bands of the size predicted for the amplified cDNA we re obtained: 299 bp for c-fos, and 651 bp for c-jun, a decrease of 91% and 82% , respectively, for c-fos and c-jun mRNAs was observed in antigen stimulated c ells pretreated with PMA for 48 h. Conclusion: PKC plays an impo rtant role in modulating the tyrosine phosphorylation and histamine release resp onses and may upregulate the expression of c-fos and c-jun in antigen activate d mast cell.