The Role Of IL-1β In ICAM-1Expression Of Lung Fibroblasts

Objective: This study is to observe how the pro-inflammatory cytokines(such IL-1β) influence lung fibroblasts to ICAM-1, by culturing the lungfibroblasts of Wistar rat in vitro, and using two methods. One isimmunocytochemical staining method for the determination of intercellularadhesion molecule-1(intercellular adhesion molecule-1and ICAM-1)expression in lung fibroblast parts, and the other is the method of RT-PCR todetermine the ICAM-1mRNA expression.Methods: A tissue explant method is adopted to culture rat embryo lungfibroblasts. The method is as following: the Wister parturient pregnant ratswere anaesthetized with ethylether. And immediately the fetal rat was takenout and then its lung tissue. The DMEM containing20%fetal bovine serumwas added into the lung tissue1mm×1mm×1mm in size. About a week later,its cells covered the bottom of the bottle. The experiment object was thecultured lung fibroblasts of5to6generations; whose shape, structure, and thegrowth state are consistent. Those were divided into three groups:(1)control group: simply to culture lung fibroblasts for6hours with DMEM;(2)Different concentration of IL-1β group:①to culture lung fibroblasts withIL-1β (0.5ng/ml) DMEM;②to culture lung fibroblasts with IL-1β(5ng/ml)DMEM;③to culture lung fibroblasts with IL-1β(15ng/ml) DMEM. All aboveculturing time is6hr.(3)Groups of the same concentration with different culturing times: lungfibroblasts were cultured within certain range of concentrations(IL-1β15ng/ml)with different time(2h、4h、6h、12h、24h), and were dividedinto control group,2h group,4h group,6h group,12h group,24h group.The expression localizations of ICAM-1mRNA in lung fibroblasts wereobserved with the method of immunocytochemical staining. And RT-PCR was used to measure the expression level of ICAM-1mRNA in lung fibroblasts.All the groups were for6times.Results:1The primary lung fibroblasts of Wistar labor pregnant rat were culturedsuccessfully. In this experiment, the lungfibroblast cells of Wistar laborpregnant rat were cultured for5~10h through the application of tissue slabmethod, and then observed in the inverted microscope. Round cells werefound outside from the tissue. After about one week later, the round cellsgradually became oval, diamond, and into the radial shape, i.e. foot longer.The cells were fused with the adjacent ones. Not only large round but brightnuclei were formed by interconnection. After trypsogen digestion for2~3times, the legacy organizations, red blood cells, epithelial cells were graduallycleared. The rest were the ideal experimental models whose form, structure,growth state were consistent with each other (consistent cell morphology, fullcell body, uniform cytoplasm, clear nucleolus, visible mitotic, good growthcondition).2The expression locations of ICAM-1in the lung fibroblastsThe ICAM-1expression showed weakly positive in normal lungfibroblasts, mainly in membrane, occasionally in cytoplasm, and no in nucleus.The ICAM-1was stained brown in the membrane of lung fibroblasts. With theintervention of IL-1β(15ng/mL), the positive granules in the membraneincreased and the color became noticeably darker.3To measure the expression level of ICAM-1mRNA in lung fibroblasts withRT-PCRThere was a specific ICAM-1mRNA belt in the234bp among all thegroups, and specific belt, i.e. included Rat β-action, appeared in576bp.Through the density scanning of electrobands of ICAM-1mRNA, it wasdenoted that there were certain expressions of ICAM-1mRNA in normal lungfibroblasts, but very low(0.112±0.02). Different concentrations ofIL-1β(0.5ng/mL,5ng/mL,15ng/mL)were added into cell-culture media for6hr.And the expression levels of ICAM-1mRNA were0.368±0.02、0.736±0.03、 0.805±0.02. The levels were significantly elevated compared with the controlgroup(P<0.01), and the levels were elevated at the same time while theconcentration of IL-1β was increased. The difference among different groupswas significant for statistics(P<0.01).With the stimulation of the same concentration of IL-1β(15ng/mL), theexpression levels of ICAM-1mRNA were0.137±0.013、0.466±0.01、0.672±0.09、0.872±0.03、0.678±0.021、0.502±0.07in0h、2h、4h、6h、12h、24h respectively. Despite the concentration of IL-1β was same, the expressionlevels of ICAM-1mRNA were different in the different time. The resultdenoted that the level increased for2h(P<0.01), reached a peak for6h(P<0.01),decreased for8h, and then never increased, but the level was higher for24hthan the control group. The difference among different groups had a greatstatistical significance（P<0.01）.Conclusion：1The study gained primary cultured cells sucessfully, using tissue explantculture. This method is simple and easy to accomplish, which avoids theexcess digestion of pancreatin. It is one of the better methods to achieve lungfibroblasts in vitro.2The expression level of ICAM-1mRNA was very low in the normal lungfibroblasts. The expression of ICAM-1was mainly located at the membrane oflung fibroblasts.3IL-1β raised the expression levels of ICAM-1mRNA at transcriptional level.4IL-1β induced the expression level of ICAM-1mRNA of rat lung fibroblastsby the model of concentration and time.