Expression And Significance Of Igf-Ⅱr In Free Silica Dustinduced Lung Myofibroblast

Silicosis is a system disease with a wide range of nodular fibrosis in lung,which is induced by inhaling much free silica dust for a long time.The increasing of inflammatory cells,such as pulmonary alveolar macrophages,neutrophils and lymphocytes,is the main behaviour in the prophase of pulmonary fibrosis.And proliferation of mesenchymal cell,increasing of matrix and accumulation of matrix collagen are in the later stage.Most of the proliferation cells are fibroblasts besides the alveolar epithelial cells.At present,it is believed that macrophage is the direct effect cells of free silica dust at Silicosis,Macrophages could secrete much cytokines after stimulated,such as interleukin,transtbrming growth factorβ,tumor necrosis factor,fibronectin,etc. These cytokines could stimulate the proliferate of fibroblasts and increase the synthesize of reticulum fiber and collagen fiber.The pathological changes of the lung tissue was difficult to eliminate.The pulmonary fibrosis is a chronic,progressive pathological process,so exploring the molecular mechanisms of pulmonary fibrosis to delay or block the development of fibrosis is still important.The process that fibroblasts transform into myofibroblasts plays a key role in the development of fibrosis.Myofibroblast is an atypical fibroblast with the characteristics of both fibroblasts and smooth muscle cells in ultrastructure and function,which express a-smooth muscle actin distinctively.Large number of studies have proved that transforming growth factor-βplays an important role in the process of pulmonary fbrosis.Most of organizations in mammalian can express Insulin-like growth factorⅡreceptor,which can be combined to the pro-transforming growth factorβand stimulate it cleavage and activate.Some researches suggested that IGF-Ⅱmay amplify signal to promote cells proliferate continuously and accelerate cancer cells to grow spontaneously,by combining with IGF-ⅡR on itself or surrounding cells.In this study,the rat enterocoelia macrophages and lung fibroblasts in vitro were cultrured.Then the lung fibroblasts were exposed to the macrophages culture supematant which was stimulated by different concentration of flee silica dust. Analyze the lung fibroblasts whether transformate to myofibroblasts or not,Study the changes of mRNA and protein levels of IGF-ⅡR in myofibroblasts,to explore the significance of IGF-ⅡR,to provide reference for the mechanisms of occurance and development and the treatment measure of Silicosis,further.Methods1.Establish the primary culture model of rat enterocoelia macrophage and fibroblast in vitro.The enterocoelia macrophages were obtained by lavaging the Mature SD rat and the lung tissue was digested by trypsin,then the cells were separated and puriflcated and followed by cultured at the 37℃,5%CO₂ conditions. MTT method was used to describe the growth curve of lung fibroblast.2.Identify the transformation of lung fibroblast.The serum-free DMEM medium was used for preparation of the free silica dust fluid.The final concentrations of free silica dust were 0μg/ml,25μg/ml,50μg/ml,75μg/ml,100μg/ml,respectively.The free silica dust fluid was used to stimulate M_φfor 24h,then the culture supernatant was collected.After that,the fibroblast was stimulated by culture supernatant for 24h,then the expression of theα-SMA protein was detected by using immunohistochemistry.3.The changes of the IGF-ⅡR in gene transcription and protein expression after stimulation.(1)The total RNA extracted from flbroblasts was undergone the reverse transcription polymerase chain reaction(RT-PCR),with GAPDH as the internal reference to detect the expression level of IGF-ⅡR of fibroblasts.(2) The expression of theα-SMA protein was camed out by using immunohistochemistry.4.Statistical analysis.SPSS12.0 was used to statistically analyze the datas.A difference at P＜0.05 was considered statistically significant.Result1.Peritoneal macrophages and lung fibroblasts isolated from rat for primary culture grew well,and can be used in vitro experiment.2.Identifing the transformation of lung fibroblast:The fibroblasts was stimulated by 0μg/ml,25μg/ml,50μg/ml,75μg/ml,100μg/ml culture supematant after free silica dust stimulating the macrophages.The difference ofα-SMA expression was significant between high concentration group and control group(P＜0.05).With the concentration of free silica dust increasing,the expression ofα-SMA was also increased.3.Expression of IGF-ⅡR mRNA in myofibroblasts:After the supernatant of M_φwhich stimulated by different concentrations of free silica dust working on lung fibroblasts for 24h,the expression of IGF-ⅡR mRNA in all of experimental groups were higher than that of control group.The differences of IGF-ⅡR mRNA expression were significant between 75μg/ml,100μg/ml concentration groups and control group (P＜0.05).4.Expression of IGF-ⅡR protein in myofibroblasts:The supernatant of M_φwhich stimulated by different concentrations of free silica dust worked on lung fibroblasts for 24h.The results showed that with the concentration of free silica dust increasing,the expression of IGF-ⅡR was also increased.Conclusion1.The culture supernatant of M_φstimulated by free silica dust can induce the lung fibroblasts transformate into myofibroblasts.2.Expression of IGF-ⅡR was increased in myofibroblasts induced by free silica dust.