蛋白激酶及DDPH对HECCM促PASMC增殖的影响

Effects of DDPH and Protein Kinases on the Proliferation of PASMC by HECCM

应用猪肺血管的内皮细胞进行常氧 /低氧培养 ,制备常氧 /低氧内皮细胞条件培养基 (Norm oxic/ Hypoxial en-dothelia cell conditional m edium,NECCM/ HECCM) ,分别观察蛋白激酶 A(protein kinase A,PKA)、蛋白激酶 C(proteinkinase C,PKC)和受体酪氨酸蛋白激酶 /胞浆酪氨酸蛋白激酶 (recepror tyrosine protein kinase/ plasma tyrosine protein ki-nase,RTK/ PTK)及 DDPH对 HECCM促肺动脉平滑肌细胞 (pulmonary arterial smooth m uscle cell,PASMC)增殖的影响。探讨这些蛋白激酶在促 PASMC增殖中的作用 ,以及 DDPH抑制这种促增殖的机理。结果提示 :PKC是 HECCM促 PASMC增殖的关键性效应分子 ,但其被激活的上游信号转导分子除了 RTK/ PTK外 ,还有其它非 RTK/ PTK介导的信号转导方式 ;DDPH对 HECCM促 PASMC增殖的抑制点在 PKC上游 ,可能直接作用于 RTK/ PTK,或者阻断 Ca2

Normoxic/hypoxic culture of endothelia from porcine pulmonary vessels was performed to prepare normoxic/hypoxic endothelial cell conditioned medium (NECCM/HECCM) in order to observe the effect of DDPH, and PKC, PKA and RTK/PTK on the proliferation and \[Ca 2+ \]i of pulmonary arterial smooth muscle cells (PASMCs) induced by HECCM. By using primary cell culture, PASMC proliferation was measured by MTT colorimetry and intracellular free calcium determined by Fura 2/Am fluroscence. The results revealed that HECCM could induce PASMC proliferation and increase its \[Ca 2+ \]i markedly as compared with NECCM or control group ( P <0.01); DDPH (200 ng/ml), Staurosporine (20 nmol/L), Genstein (25 μmol/L) and complete down regulation PKC with PMA(1 μmol/L) could inhibit PASMC proliferation induced by HECCM, while the inhibitive action of Staurosporine was stronger than Genstein's; DDPH and Genstein could decrease the \[Ca 2+ \]i of PASMC induced by HECCM, while Staurosporine had no significant effect on it; The proliferation level of PASMC in the inhibitor of PKA groups were higher than that in control group with the difference being not statistically significant between them ( P >0.05). The results suggest that HECCM can induce PASMC proliferation, which can be suppressed by DDPH; PKC plays a major role in the proliferation of PASMC induced by HECCM. Besides RTK/PTK, maybe there is uncertain way which can make PKC active.PKA is not important in the proliferation of PASMC induced by HECCM. DDPH can decrease \[Ca 2+ \]i of PASMC induced by HECCM.