Thy-1 Expression In The Pathogenesis Of Pulmonary Fibrosis Rat Induced By Bleomycin

[Purpose] Idiopathic pulmonary fibrosis(IPF) is a most frequent kind of interstitial lung diseases with increasing incidence. IPF characterized by an insidious onset, quick progress and worse prognosis still lacks effective clinical treatment at present. The pathogenesis of IPF is uncertain yet, but it is certain that fibroblasts play a very important role in IPF. Fibroblasts have increased proliferative capacity and secretory matrix-productive function in the development of lung fibrosis, in which fibroblast and many proinflammatory factors interact on each other. The heterogeneous of fibroblasts have been confirmed, and there are many differences in morphs, responses to inflammation factors and secretion. We can divide fibroblasts into two types: Thy-1-/Thy-1+ according to the expression of Thy-1. It is reported that Thy-1's expression reduces during the formation of lung fibrosis. In other words, the normal fibroblasts are Thy-1+ cells. There are no relative report about the relationship between Thy-1 and pulmonary fibrosis at present. The researches abroad mainly focus on vitro culture, while no relative report in vivo culture has been found. Furthermore, the pathogenesis of Thy-1's change have not been identified. Our experiment uses rat model induced by bleomycin, and measures the content of hydroxyproline in lung during the fibrosis formation. We use the immunohistochemistry method to measure the expression of Thy-1 and Thy-1 mRNA, ELISA to measure the content of cytokine in BALF, in order to observe the tendency of Thy-1, the relationship between the expression of Thy-1 and the synthesis of mRNA, and the relation between collagenation and cytokine. So we can approach the pathogenesis of pulmonary fibrosis in fibroblast direction, and provide a new research direction.[Methods] 1. The rats were randomly divided into BLM group and NS control group, and the blank control group. After the rats were anesthetized, we exposed the pars cervicalis tracheae, infusing BLM 0.5mg/0.125ml/100g through tracheae into the BLM group and infusing the same dose NS into the control group NS. We kill the rats separately on the 1^st , 3^rd , 7^th, 14^th, 28^th day, to take the upper and middle lobe of right lung to do HE staining, immunohistochemistry, and hybiridization in situ, to take inferior lobe of right lung for hydroxyproline measurement, to take left lung to do BALF with its supernate fluid used for ELISA. 2. Observe histopahological change by HE staining. 3. Observe the change of Thy-1's expression in fibroblast at different time point(1^st ,3^rd ,7^th ,14^th ,28^thday) after drug theapy. 4. Measure the expression of Thy-1 mRNA at different time point(1^st ,3^rd ,7^th ,14^th ,28^th day) by hybridization in situ. 5. Measure the content of HYP in lung by paradimethylaminobenzaldehyde method. 6. Measure IL-1 in BALF by mouse thymocyte method and TNF by genitian violet. 7.Analyze the relation between the expression of Thy-1 and the generation of Thy-1 mRNA during the formation of lung fibrosis, and relation between collagen secretion and cytokine. 8. Adopt SPSS11.5 medical statistics software to analyze, and variances wererepresented by the form of (?)SD. We adopted randomized blocks analysis of variance for the compare of each interclass mean, and adopted linear correlation for the two interfactors' correlation analysis. There was statistical significance by mean of P<0.05.[Results] 1. On the 1st, 3rd day after medication, the rats in NS control group had light reactive inflammation, low grade inflammation cells effusion in the alveoli but no dropsical alvelolar septum, and fibrocyte cellular proliferation and fibrosis were not found either. On the 7th day, the inflammation disappeared completely. Per contra, we could find obvious inflammation in the rats of BLM group, with high grade inflammation cells effusion in the alveoli and low grade dropsical alvelolar septum on the 1st and 3rd day. On the 7th day , fibrocyte cellular proliferation and fibrosis could be found. The 14^th day saw the alveolar wall thickening, fibroblast cellular proliferation , the obvious multiplication of base material, collagen fibers accumulation, disorganization of alveoli and patches of fibrosis. Onthe 28^th day, the alveolar wall obviously thickened and some alveolar space disappeared substituted by collagen fibers, collagenoblast and macrophagus. All of this indicated the success of our BLM modeling. 2.Most of the collagenoblast, about 88.01±4.12%, in the interstitial was Thy-1+ in normal lung tissue. But in the BLM group, since the 7^th day, the expression of Thy-1 in collagenoblast decreased obviously. On the 28^th day, it reached its lowest level. We could not find Thy-1 in fibioblast focus. 3. Fibroblasts Thy-1mRNA BLM expression in the experimental group gave no obvious downward trend in the treatment time points, with no statistical significance (P> 0.05); In the fibroblasts lesions of Thy-1- expression Thy-1mRNA coloring could be found. 4. HYP in the lung tissue homogenate started to rise seven days from the start. 5.BALF IL-1 in volume at each time point had no significant difference. TNF began to increase on the first three days, reached its peak on the 7^th day, began to fall after that, but remained at a relatively high level; 6. Fibroblast Thy-1 expression in lung tissue and the amount of HYP showed a negative correlation, the correlation coefficient r=-0.921;Thy-1 protein and mRNA expression and the expression of IL-1, TNF levels had no significant correlation.[Discussion] A common pathological feature of interstitial lung fibroblasts in the interstitial lung disease is gathered proliferation, collagen and other extracellular matrix deposition lot. Fibroblast is the main source of extracellular matrix , it can be activated by a variety of pro-inflammatory cytokines and the activated fibroblasts leads to a lot of collagen synthesis, and the release of a variety of cytokines, to accelerate and facilitate the process of fibrosis. Different Thy-1 expression is of great significance to the biological characteristics of different fibroblast cell. Thy-1- fibroblasts showed high cell activity, accelerated proliferation and increased secretion of collagen under the effect of a variety of pro-inflammatory cytokines, but could release a variety of cytokines, further promoting the formation of pulmonary fibrosis; Thy-1+ cell is a relatively static state of the cells, and is weak in interacting on cytokines and in the release of extracellular matrix. The results show Thy-1 had a high rate of expression in normal lung tissues but the expression gradually reduced in the process of forming pulmonary fibrosis. Thy-1 expression was rare in fiber lesions especially in sections with obvious fibrosis, namely Thy-1-fibroblasts increased gradually in the process of pulmonary fibrosis. Such changes and the expression of interstitial lung collagen content were closely related. In the process of pulmonary fibrosis, collagen content grew gradually, with obvious increase on the seventh day, reaching its peak on the 28^th day, negatively correlated with Thy-1 expression. The results suggest Thy-1- fibroblast is the main source of collagen.Experiments in vitro abroad demonstrated that cytokines such as IL-1, TNF, TGF were related to the reduction in the Thy-1 expression. This experiment measured in BALF the change of TNF and IL-1 in the process of pulmonary fibrosis, and found no significant correlation with Thy-1 expression, leading to the consideration that the change in vivo expression of Thy-1 was the result of a multi-factor, and that other factors were involved in this process in addition to the cytokines mentioned above. The mechanism of the reduction of Thy-1 in pulmonary fibrosis process is still not clear, and in this respect little research has been done. The simultaneous determination of the experimental Thy-1mRNA fibroblasts indicated no corresponding decrease in Thy-1mRNA with the decrease of Thy-1 suggesting its reduction other than in the mRNA level. In vitro study we found that Thy-1 shed from the cell surface, appeared in the corresponding cell culture medium, hinting that the reduction in lung fibroblast Thy-1 expression might be caused by the shedding of protein for a variety of reasons. Determination of changes of Thy-1 in BALF will be our aim of work for the next step, and it'll be of great significance to the explanation of the changing mechanism in Thy-1.[Conclusions] 1. In normal lung we found fibroblast expression Thy-1, while no expression in the conspicuous domain of pulmonary fibrosis. 2.The reduction of Thy-1 expression and that of Thy-1mRNA synthesis is not closely related. 3.Thy-1-firoblast are the main cells of collagen synthesis.4. The reduction of Thy-1 is not obviously correlated with IL-1 and TNF, which may be acomposite factor effect.