The Molecular Mechanisms Of Notch/Delta Signaling In The Effects Of Neuropeptide Substance P On The Prolafication Of The Type â…¡ Alveolar Epithelial Cells Exposed To Hyperxia

PART 1 TO CULTURE AND IDENTIFY THE TYPE II ALVEOLAR EPITHELIAL AND ESTABLISH THE MODEL OF THE HYPEROXIA LUNG INJURYObjective to culture and identify the primary type II alveolar epithelial and establish the model of hyperoxia lung injury successfully. Methods the primary type II alveolar epithelial removed from the Sprague-Dawley which pregnanted only 18 days was cultured by the differential centrifugal and differential attachment method.The hyperoxia lung injury model was established by the hyperoxia airtight installment which was ventilated 95%oxygen. Morphologic change and cell number were observed respectively by the light microscope and transmission electron microscopy(TEM). The expression of the surfactant protein C(SP-C) was observed by the immunofluorescence. Results Under the light microscope the type II alveolar epithelial which was cultured under normal circumstance grew following the volmer-weber(VW) mode and formed cellular joins. The cellular nuclei was enlarged and the cytoplasm contained a large number of dense granules. While under the transmission electron microscopy(TEM)the microvilli on the cell membrane and neurosecretory granules in the cytoplasm were commonly seen.The surfactant protein C(SP-C)expressing a lot was detected by immunofluorescence. To the opposited,the cell exposed to the hyperoxia grew with intercellular spaces dilatation and increased nucleus under the light microscope. The microvilli on the cell membrane disappeared and adipose hollow formed. The surfactant protein C(SP-C) expression detected by immunofluorescence reduced.Conclusions The type II alveolar epithelial grew well and the hyperoxia lung injury model established successfully.PART2 EFFECT OF NEUROPEPTIDE SUBSTANCE P REGULATES NOTCH/DELTA SIGNAL PATHWAY ON THE PROLIFERATION OF THE TYPE II ALVEOLAR EPITHELIAL CELLS EXPOSED TO HYPERXIAObjective To investigate the relationship of neuropeptide substance P(SP)and Notch/Delta signal pathway on the regulatory mechanism of the proliferation of the type II alveolar epithelial cells(AECIIs) in lung injury induced by hyperxia. Methods The AECIIs of primary premature rats were isolated and purified. The AECIIs were divided into six groups randomly which were intervened with 21%air, 95% oxygen,substance P(SP),SP antagonist(L703.606) and the blocker of Notch signal pathway(MW167) respectively. Morphologic change were observed by the light microscope.Cell number was counted at 6h,12 h,18h and 24 h time point after intervened.The proliferation rate was measured by Cell Counting Kit8(CCK8).Cell cycle was detected by flowcytometry.The expression of Dll1,Dll4 and Hes1 m RNA were detected by Real-time PCR. Results The cell number and the proliferation rate in the hyperoxia groups decreased significantly compared to the air group while increased remarkbly after the intervention of SP. The expression of Dll1 and Dll4 and Hes1 decreased significantly in the hyperoxia groups. While the expression of Dll1 and Dll4 and Hes1 increased remarkbly after the intervention of SP.Conclusion SP could increased the cell viability and proliferation and decreased the injury of the AECII exposed to hyperxia which might be associated with the activation of Notch signal pathway.