In Vitro Experiment Of The Effect Of Free SiO₂ Dust To Rat Circulating Fibrocytes

The traditional source of fibroblasts is tissue in wound repair, and the source of peripheral blood had also been confirmed. Circulating fibrocytes can simultaneously express markers of hematopoietic cells and synthesis of a variety of extracellular matrix proteins, type-Ⅰ, type-Ⅲcollagen and a-SMA was identified as the main ones. Because of its synthesis of a variety of extracellular matrix proteins, cytokines, wound contraction, the ability to promote angiogenesis, participating in skin wound, diabetic wound repair, circulating fibrocytes play the role of immune-presenting cells and promoting wound healing fibrosis. The fact that the blood circulation system has a few circulating fibrocytes has been confirmed, and the resurch of the effect of free silica to circulating fibrocytes has not been reported.So in this experiment, the changes of mRNA and protein levels of collagen-Ⅰ, collagen-Ⅲand alpha-Smooth muscle actin (a-SMA) of circulating fibrocytes at different days was analyzed by PCR and ELISA. And the effect of free silica to circulating fibrocytes was preliminarily explored.Objective:To explore the effect of free silica to circulating fibrocytes, provide clues and experimental basis for the mechanisms of pneumoconiosis with cFb for further research.Method:1 Establish the primary culture model of rat enterocoelia macrophage and circulating fibrocytes in vitro. MTT method was used to describe the growth curve of circulating fibrocytes. Identifing circulating fibrocytes by immunohistochemical method at different concentrations and days. The serum-free DMEM medium was used for the free silica fluid. The concentrations of free silica were 0,20,40,60,80μg/ml respectively. The free silica fluid was used to stimulate macrophage for 24h, then collecting the culture supernatant. Then the circulating fibrocytes was stimulated by culture supernatant for 24h.2 The reverse transcription polymerase chain reaction (RT-PCR) was used to detected the mRNA, and immunohistochemical method and Enzyme-linked immunosorbent adsorption test (ELISA) was used to detected the protein levels of collagen-Ⅰ, collagen-Ⅲand a-SMA in circulating fibrocytes.Result:1. Circulating fibrocytes which were not stimulated by supernatant and were stimulated by supernatant which were not stimulated by free silica started to express Collagen-Ⅰ, Collagen-Ⅲat sixth day, and a-SMA at the nineteenth day. Circulating fibrocytes which were stimulated by supernatant which were stimulated by free silica started to express Collagen-Ⅰand Collagen-Ⅲat third day, and a-SMA at the thirteen day.2. The expression of mRNA and protein of the three indexes at different concentrationscompared to the control group, the changes at the dose of 20,40,60,80μg/ml were statistically significant (P<0.05), the expression increased from 0μg/ml to 80μg/ml gradually.3. Circulating fibrocytes were stimulated by supernatant which were stimulated by 80μg/ml silica at different days①The expression of mRNA of Collagen-Ⅰand Collagen-Ⅲ:Compared to the third day, the ninth day,the twelfth day, the fifteenth day and the eighteenth day were statistically significant (P<0.05), the expression sincreased from the ninth day to the eighteenth day.②The expression of mRNA ofα-SMA:Compared to the thirteenth day, the nineteenth day, the twenty second day, the twenty fifth day and the twenty eighth day were statistically significant (P<0.05), the expression increased from the nineteenth day to the twenty eighth day.③The expression of protein of Collagen-Ⅰand Collagen-Ⅲ:Compared to the third day, the ninth day, the twelfth day, the fifteenth day and the eighteenth day were statistically significant (P<0.05), the expression sincreased from the ninth day to the eighteenth day.④The expression of a-SMA:Compared to the thirteenth day, the nineteenth day, the twenty second day, the twenty fifth day and the twenty eighth day were statistically significant (P<0.05), the expression increased from the nineteenth day to the twenty eighth day. Conclusion:1. The time in which supernatant stimulated by silica can promote lymphocytes transform into circulating fibrocytes advances.2. After stimulated by the culture supernatant of macrophages which were stimulated by free silica, circulating fibrocytes can significantly increase the expression of mRNA and protein of Collagen-Ⅱand Collagen-Ⅲwith concentrations and days increasing.