Proliferation And Transdifferentiation Of TypeⅡ Alveolar Epithelial Cells In Premature Rats

Part 1 Effects of Hyperoxia on Proliferation and Transdifferentiation of TypeⅡAlveolar Epithelial Cells from Premature Delivery RatsObjective To establish a hyperoxia-exposed type II alveolar epithelial cells ( AECIIs ) injury model to investigate effects in vitro of hyperoxia on the proliferation and transdifferentiation of AECIIs in premature delivery rats.Methods AECIIs were gained by primary culture from 19 d fetal rat lung. After purified and adherence to the culture flask for 15 h, AECIIs were randomly assigned to two groups: air control groups and hyperoxia groups. The hyperoxia groups were filled with 95 % oxygen-5 % CO2 at 3 L/min for 10 min, and then incubated at 37°C in a CO2 incubator, while the air control groups were incubated at 37°C in a CO2 incubator directly. After cultured for 24, 48 and 72 h, cells were harvested. The morphological change of cells was observed under inverted phase contrast microscope and transmission electron microscope. AECII–specific protein-surfactant protein C ( SP-C ) and typeⅠalveolar epithelial cells ( AECⅠ)-specific protein aquaporin5 ( AQP5 ) were detected by immunocytochemical staining. The expression levels of SP-C and AQP5 mRNAs and their proteins were observed by RT-PCR and flow cytometry. Cell number was counted every 24 h with a hemacytometer. Cell viability was determined by Trypan blue dye exclusion. Flow cytometry were used as assays for cell cycle. The expression of Ki67 was dected by immumofluorescence method and flow cytometry.Results Cell number increased in cultures exposed to room air. Compared with air control groups, Cells exposed to hyperoxia for 48 and 72 h had reduced cell number and cell viability. The expression rate of Ki67 in positive cells and fluorescence index(FI) decreased markedly after exposure to hyperoxia for 24,48 and 72 hours. Hyperoxia increased the percentage of cells in G1 phase and decreased the percentage in S and G2/M phases of the cell cycle ( P<0.05 or P<0.01 ). At the same time, after exposure to hyperoxia, primarily cultured AECIIs rapidly lost their characteristics and gained some AECⅠ-like outward appearance. Cuboidal AECIIs spreaded and flattened, lost lamellar bodies and microvilli. With these morphological changes, AECIIs stopped expression of AECII–specific protein SP-C and expressed AECⅠ-associated protein AQP5. Compared with air control groups, the expression rate of SP-C mRNA in positive cells and fluorescence index ( FI ) decreased markedly in 3 groups of exposure to hyperoxia for 24,48 and 72 hours ( P<0.05 or P<0.01 ),but the AQP5 expression increased significantly in 2 groups of exposure to hyperoxia for 24 and 48 hours ( P<0.05 or P<0.01 ). The expression of AQP5 began decreasing and had no notable difference compared with exposure to air for 72 hours ( P﹥0.05 ).Conclusion (1) Hyperoxia could inhibite proliferation of primary cultured AECIIs from fetal rat lung in G1 phase of the cell cycle and decrease the expression rate of Ki67. This proliferation arrest may be associated with the pathogenesis of chronic lung disease of premature infants. (2) Although transdifferentiation of AECIIs soccured spontaneously in vitro, it was accelerated on exposure to hyperoxia. Hyperoxia-induced AECIIs transdifferentiation may play a key role in the repairation of alveolar epithelial cell injury in the premature delivery rat lung. Part 2 Proliferation and Transdifferentiation Characteristics of TypeⅡAlveolar Epithelial Cells Co-cultured with Lung FibroblastsObjective To set up a co-culture model of typeⅡalveolar epithelial cells ( AECIIs ) with lung fibroblasts ( LFs ) from fetal rat lung, observe the proliferation and transdifferentiation characteristics of AECIIs, and to further study the interaction between AECII and LF in lung development and injury in vitro.Methods AECIIs and LFs were isolated and purified. A co-cultured model of AECIIs and LFs was set up by PCF Millicell culture plate inserts and six-well cluster dishes. AECIIs were plated into the wells of six-well cluster dishes at a density of 5×105/mL. LFs were plated into the Millicell culture plate inserts at a density of 1×106/mL. Control groups consisted of AECIIs cultured with Millicell culture plate inserts containing no LF, or AECIIs cultured by themselves. After incubated for 2 and 4 days, the morphological change and growth of cells were observed under inverted phase contrast microscope. Cell number was counted with a hemacytometer, cell viability was determined by Trypan blue dye exclusion. The SP-C and AQP5 expressions in mRNA and protein levels were observed by RT-PCR and flow cytometry. Flow cytometry was also used to detect the cell cycle and Ki67 expression of AECIIs.Results (1) A co-cultured model of AECⅡs and LFs was set up well, and this coculture model constructed in vitro mimiced microenvironment in vivo. (2) AECIIs cultured alone for 4 days, AECIIs grew scatteredly with flattened nuclei, greaten volum, decreased chromatospherite and transdifferentiated partly into AECⅠ-like cells. Co-culture of AECIIs with LFs for 4 days, AECIIs also grew aggregates with rounded nuclei and had no obvious change in cell volum. Compared with the control groups, the SP-C expression in mRNA and protein levels increased markedly(P <0.01)after cocultured with LFs for 2 and 4 days, while the AQP5 expression decreased(P <0.05 or P <0.01). The cell morphology and immun marker results indicated that AECIIs could keep its morphology well, and the transdifferentiation of AECIIs to AECⅠs was decreased when co-cultured with LFs. (3) There was no notable difference between coculture groups and control groups on 2 days in cell number and viability of AECIIs. After co-clutured with LFs for 4 days, AECIIs also kept its morphology, cell viability. Proliferation of AECIIs increased significantly than that of AECIIs cultured by themselves, and (95.2±4.9)% cells retained the ability to exclude dye. Cocultured with LFs for 2 and 4 days, the percentage of cells in G0/G1 phases decreased ( P <0.01 ), while the percentage of cells in S and G2/M phases increased ( P <0.01 or P <0.05 ), and the expression rate of Ki67 in positive cells and fluorescence index(FI) also increased ( P <0.01 or P <0.05 ), these suggested that cocultured with LFs could promote the proliferation of AECIIs.Conclusion The coculture model constructed in vitro mimics microenvironment in vivo. Differentiation and proliferation function of AECⅡcan be maintained in this coculture model, this can be used to study the the interaction between AECⅡand LF in lung development and injury in vitro.Part 3 Effects of Hyperoxia on the Dynamic Expression of SP-C and AQP5 in Premature Rats Lung DevelopmentObjective To observe the dynamic expressions of surfactant protein C ( SP-C ) and Aquaporin5 ( AQP5 ) in premature rats and after inhaling hyperoxia and investigate roles of SP-C and AQP5 in lung development and hyperoxia lung injury.Methods Gestation 21 day ( term=22 day ) Sprague-Dawley ( SD ) fetuses were randomly assigned to air control group and hyperoxia group within 12～24 h after birth. Hypreoxia group was exposed to about 85% oxygen and air group in room air. After 1 to 14 days of exposure, lung tissue was extracted. Immunohistochemistry was used mainly for the location of the SP-C and AQP5 expressions in lung tissue, SP-C and AQP5 mRNA were detected by reverse transcription polymerase chain reaction ( RT-PCR ). SP-C and AQP5 expressions in protein levels were detected by western-blot. Results (1) The expression of SP-C could be found at various time after birth in premature rats, and the positive staining was restricted to the AECⅡ. At day 1 after birth, the expression of SP-C was highest and began to decrease at day 4. Compared with air control group, the SP-C mRNA and protein decreased significantly in hyperoxia group of day 1 ( P <0.01 ). After exposure to hyperoxia for 7 to 10 days, the SP-C expression increased remarkedly ( P <0.01 or P <0.05 ) but decreased again by 14 days of hyperoxia ( P <0.05 ). (2) The expression of lung AQP5 in premature rats increased persistently after birth, and the positive staining was restricted to the AECⅠ. Compared with the air control group, the AQP5 mRNA in hyperoxia group of day 1 increased significantly ( P <0.05 ), but after exposure to hyperoxia for 4 to 14 days, the AQP5 expression decreased remarkedly in mRNA and protein levels ( P <0.05 or P <0.01 ).Conclusions SP-C and AQP5 may have participated the physiological and pathological process in premature rats lung development and hyperoxia lung injury. Hyperoxia exposure lead to a down regulation or functional impairment of the SP-C and AQP5 expression , this may be an important factor for the development of hyperoxia lung injury.