In Asthmatic Airway Smooth Muscle Cell Proliferation Of The Primary Cycle And Cyclin D1 Expression In The Study

Part one Murine model of AsthmaPurpose To build stable and reproductive model of asthmatic mice.Methods Male Balb/c mice (n=16) were randomly divided intoasthmatic group(n=8) and control group(n=8). Asthmatic mice weresensitized with injections of 10mg OVA and 10mg aluminum hydroxidein 0.1ml saline intraperitoneally on the day 0, day 7 and day 14.From day15 to day 67(6 weeks), mice were challenged twice or three times a week,each challenge being 30 min in a chamber, with free-breathing mice beingexposed to 1% OVA aerosol mist produced by a Nebulizer. The mice ofcontrol group were sensitized and challenged with equal amounts ofsaline instead of OVA. All the mice were killed in 24 hours after the lastchallenge. Take the trachea of mice for the primary culture of murineairway smooth muscle cells(ASMC), and the left lobus medius pulmonisfor histological HE staining.Results1. Asthematic mice gave appearance as visible itch on the head and face,deep and fast breath, nodding gasp, calm, bow, obvious skin rash on thepalmar surface and so on. Mice of control group had no appearance 2. Histological HE staining indicated that eosinophils infiltration, ciliumloss, formatin of mucous plug and smooth muscle cell layer thickeningwere observed in asthma group, while histological HE staining indicatedas normal in control group.Conclusions Asthmatic mice were successfully established and couldbe used in further study.Part Two Primary Culture of airway smooth muscle cells from mouseObjective To establish a method for primary culture of mouse airwaysmooth muscle cells (ASMC) for the related research.Methods The primary and transfer culture were done by tissue-pieceinoculation. The cells were purified by several methods, includingnatural passage transfer and differential attachment. The cultured cellswere identified by morphological characteristics andimmunocytochemistry.Results Survive of tissue pieces is good. The purity of the third passageASMC was over 95%. Immunocytochemical staining with specific mAbagainst mouse SM-α-actin demonstrated more than 90% cells as positive.Conclusion It is a simple and reliable method for obtaining highlypurified and satisfactory ASMC. Part Three Difference of cell proliferation and the expression of cyclin D1 in theairway smooth muscle cells of murine asthmatic modelObjective To investigate the cell proliferation and cell cycle and theexpression of cyclin D1 in the primary cultred airway smooth musclecells(ASMC) in murine model of asthma.Methods The murine asthmatic model and the primary ASMC werecultured. The primary ASMC of normal mice were chosen as controlgroup. The cell proliferation was detected by MTT. The cell cycle wasmeasured by FCM, and the expression ofcyclin D1 was measured byFCM.Results1. The cell proliferation was detected by MTT. Compared with thenormal ASMC line, the cell proliferation was significantly increased(P＜0.05).2. The cell cycle was measured by FCM. Compared with the normalASMC line, the percentage of S phase of asthmatic ASMC line wassignificantly increased.3. The expression of cyclin D1 was observed by immunofluorescence.Laser Focus microscope indicated that Cyclin D1 was expressed in aii the ASMC, mainly in intracytoplasm.4. The expression of cyclin D1 was measured by FCM. The expression ofcyclin D1 in the asthmatic ASMC was significantly increased comparedwith the normal cell line.Conclusion The proliferation of ASMC is increased in the progressionof airway remodeling in asthma and cyclin D1 may contribute to thisprogression.