Effects Of Different Volume Fraction Of Oxygen On The Growth Status And Intracellular Ca²⁺ Levels Of The Primary AEC â…¡

Chapter OneThe Primary Culture and Identification of Type Ⅱ Alveolar Epithelial CellsObject To explore the method of separation、purification and identification of type Ⅱ alveolar epithelial cells(AECII). Methods We used trypsin and IV collagenase to digest the lung tissue, then purified and flitered it with the culture dishs coated by 1gG. The cells were identified by inverted microscope and laser confocal microscopy.Results After selection and purification, the obtained cell purity was very high. The cells could be observed adhere to the bottom of the culture dishs after had been cultured for 8 hours. And the cells were distributed like islands. We could obviously observe the cell nucleus. The cell volume increased obviously than before after 48 hours, the neighboring islands connected together, liking paving stones. Under the electronmicroscope, the cells were scattered, their karyotheca were complete, there were many lamellar bodies, endoplasmic reticulum, cytolysosome and mitochondrium in the cytoblastema, whose structures were clear. The superficial microvilli arranged orderly. The green fluorescence could be observed by laser confocal microscopy in the cytoblastema. Conclusion The activity and purification of type II alveolar epithelial cells was high, obtained by digestion of trypsin and collagenase and purlfed by culture dishs coated with IgG. The method could be put into practice as the routine way of primary culture of type II alveolar epithelial cells, and both electron microscope and immunofluorescence could identify the cells.Chapter TwoEffects of different state of oxidative stress on the growth status of primary alveolar type Ⅱ epithelial cellsObject To observe the growth status of the primary alveolar type Ⅱ epithelial cells in the environment of different volume fractions of oxygen. Methods Primary culture the alveolar type Ⅱ epithelial cells, to establish the oxygen volume fraction 0.95 group (95%O2), oxygen volume fraction 0.75 group (75%O2), oxygen concentration 0.50 group (50%O2), oxygen volume fraction 0.25 group (25%O2) and the control group (5%CO2), five kinds of cell models, each one exposured 12h,24h and 48h, detected the growth condition of cells with light microscopy, detected the apoptotic of cells by flow cytometry, and detected the levels of intracellular oxidative stress indicators-SOD and MDA. Results Growth condition:When given oxygen as long as 24h, the number of the cells of the experimental group decreased, comparing with the control group, the number of the 95% and 75% group decreased too, comparing with the previous time point, and the differences were statistically significant; when 48h, the number of the cells were reduced, comparing the experimental group with the normal group at the same time as so as the previous time point in the experimental group, having a significant difference, and the structure of cells was damaged, many visible cell debris appeared. Cell apoptosis:Compared with the normal group, the apoptosis rate of each experimental groups at each time point of were increased. The 95% group was the most significant, and the time longer, the more obvious increased. Oxidative stress:The SOD of each experimental group were relatively lower, MDA were increased to varying degrees, compared with the control group at the same time point. Conclusion AECII hyperoxia may cause damage through oxidative stress pathways, or even apoptosis.Chapter ThreeEffects of different state of oxidative stress on the intracellular calcium levels of primary alveolar type Ⅱ epithelial cellsObject To observe the intracellular calcium levels of the primary alveolar type Ⅱ epithelial cells in the environment of different volume fractions of oxygen. Methods Primary culture the alveolar type Ⅱ epithelial cells, to establish the group models the same as the chapter two, each group exposured 5min,10min,15min and 30min, then stop giving oxygen, use the live cells confocal microscopy workstation to observe the intracellular calcium dynamics. Results Give the AECⅡ hyperoxia stimulation, [Ca2+]i rose to a certain extent and then slowly declined, and maintained that level. Stop giving oxygen, [Ca2+]i rose again and then gradually stabilized. However, giving oxygen for 10min,15min and then stopped, the rebound was not obvious, and no significant changes when stopped after giving for 30min. Conclusion Hyperoxia could affect the calcium level within AECⅡ, may cause or worse oxidative stress through calcium, leading to cell damage.