Effect Of PKCδ To Permeability Injury Of Rat Pulmonary Microvascular Endothelial Cells Monolayer Induced By LPS

Objective : To study the expression of protein kinase C(PKC) subtype PKCδ in rat pulmonary microvascular endothelial cell (RPMVEC).Through observing the change of the PKC8 on LPS- induced RPMVEC injury , and the effect of Rottlerin(inhibitor of PKC8) on RPMVEC monolayer permeability injury, to find the function of PKC subtype on the LPS-induced RPMVEC injury. Methods RPMVEC from Wistar rat was isolated and cultured in vitro. Both Western blot and immunohistochemistry were used in vitro to identify the expression of PKC8 and the change of PKC8 in LPS-induced RPMVEC injury. Micro-infiltrator was used to measure the changes of cultured RPMVEC monolayer permeability coefficient(Kf) after exposure to LPS and LPS+Rottelrin.Result: 1 We succeeded in isolating and culturing RPMVEC;through the method of morphology, immunohistochemistry (VIII factor-related antigen), the binding experiment of FITC-BSA, confirmed the cell is RPMVEC. We found a new method of add EDTA into trypase , decurtated the time of coction and passage, decreased the injury of trypase to RPMVEC during coction and passage. Freezing and anabiosising the RPMVEC, made the fundament of test in future . 2 Immunohistochemistry was used at first to identify that PKC8 was widely expressed in cytoplasm. By the method of Western blot analysis,we found single band of protein blotting, molecular massand was 77KD, which confirmed the existenced of PKC8 in RPMVEC. We also found that LPS resulted in the high expression value of PKC8 in RPMVEC. 3 After treatment with LPS 10μg/ml, Rottlerin 5μM/L for15min,30min ,60min,90min,120min,the Kf were increased significantly in LPS group, and which had no different in Rottlerin group. Rottlerin+LPS group all value were...