Investigation Of Clinical And Basic Research In Interstitial Lung Disease With Connective Tissue Disorders

BackgroudConnective tissue diseases are sorts of chronic autoimmune disease with diverse clinical manifestation. Lung contains rich collagen fiber and blood vessels, and has the immune adjustment, metabolic, hormonal, and other functions, so it is common organ easily involved by all sorts of connective tissue disease. Interstitial lung disease caused by connective tissue diseases is called interstitial lung disease with connective tissue disorders (CTD-ILD).Its early pathological change can be exudative alveolar inflammation lesions primarily, as the disease progresses, the collagen deposition, which eventually led to the widespread irreversible lung fibrosis and pulmonary function damage.The pathological type of CTD-ILD is very complex, because all the pathological type can exist in CTD-ILD, and also can exist side by side on the same patients.2002 ATS-ERS recommended interstitial lung disease classification standards about current pathological type of CTD-ILD is accepted. CTD-ILD has the high incidence,the high mortality rate, and lack of standard treatment method, the routine treatment of glucocorticoid and cyclophosphamide easily lead to complications such as infection, so it becomes the difficult and hot in current clinical research. In this study we focus on the pathological type of CTD-ILD, explore the correlation between the pathological type and original diseases, imaging, treatment plans, pulmonary function, inflammation factors, in order to provide the basis to fully recognize and treatment of CTD-ILD.In recent years, some studies have shown that caveolin-1 and pulmonary fibrosis are relevant.Caveolin-1 is the size of 21-24Kda embedded in the cell membrane, is the main component of the caveolin, with various biological functions such as cell membrane transhipment, cholesterol transportation, lipid stability and signal transduction. so we research on the expression of caveolin-1 in CTD-ILD, and its regulation in human lung fibroblasts (HLF) in induced by transforming growth factor-β1(TGF-β1), in order to help to understand further pathogenesis of CTD-ILD and find new therapeutic targets.Recent research has shown that the pathologic features between Idiopathic Pulmonary Fibrosis(IPF) and CTD-ILD were similar that both had fibroblast foci with mainly consisted of myofibroblats (MF). MF has many functions, such as secreting cytokines, resulting to the abnormal deposition of extracellular matrix, and inducing the aleolar epithelial cell apoptosis.TGF-β1 can transfer the human lung fibroblasts differentiation to myofibroblats.And at present, CTD-ILD patients were lack of definite effective treatment method. Sou-Medrol(MP) and cyclophosphamide(CTX) were the most commonly used drugs in treatment of CTD. Therefore, we research on the influence of MP and CTX in HLF proliferation and HLF differentiation to MF induced by TGF-β1, in order to provide the references on clinical treatment.Objective1. Search the relationship between the pathological types and original diseases, imaging, treatment plans, pulmonary function, inflammation factors.2.Observe the expression of caveolin-1 in CTD-ILD, and research its regulation in human lung fibroblasts induced by transforming growth factor-β1 in vitro.3.Explore the Sou-Medrol and cyclophosphamide can inhibit the human lung fibroblasts transfer differentiation to myofibroblats induced by TGF-β1 in vitro.Methods1. We retrospectively analyzed 45 patients who were diagnosed as CTD-ILD and had CT guided percutaneous lung biopsy from January 2003 to December 2010 in Guangdong General Hospital. We classified the pathological types into usual interstitial pneumonia (UIP),nonspecific interstitial pneumonia(NSIP) and other type. We divided the treatments into CTX<12g,CTX≥12g,non-CTX. We treated the effects into improving, stable, deterioration and unable to define. We recorded the HRCT performance of the puncture site, treatment plans and ESR, c-reactive protein (CRP),platelets(PLT),forced vital capacity (FVC),forced expiratory volume in one second(FEV1),diffusion capacity for carbonmonoxide of lung(DLCO.), oxygen partial pressure (PO2), carbon dioxide partial pressure(PCO2), oxygen saturation and pulmonary infection situation before and after treatment. Analyze the relationship between the pathological types and original diseases, HRCT performance of the puncture site, treatment plans, pulmonary function, inflammation factors.2.Lung tissue of CTD-ILD/normal stained by HE,caveolin-1 and TGF-β1 expressions were explaned by confocal microscope. Normal human lung fibroblasts(HLF) process in primary culture, indentification,adding 10μg/L TGF-β1 induced by 24hours, expression of caveolin-1 was compared between before and after induction. The number 11-20 generations of HLF were exposed to TGF-β1 with different concentrations (0,1,3,5,10,15μg/L),and to 10μg/L TGF-β1 with different times(O,6,12,24,48h), caveolin-1 mRNA and protein expression were detected by quantitative PCR and western blotting.3.The original lung fibroblasts were cultured and generated in vitro, the 11-20 generation cells are selected to the following experiment:HLF were transferred differentiation to MF induced by 10μg/L TGF-β1. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) colorimetric method to detect the proliferation inhibition rate of Sou-Medrol and cyclophosphamide, we choose the IC50 value as each drug concentration. Based on this, we divided the trials into 5 groups: PBS control group,TGF-1 group,TGF-β1+MP group, TGF-β1+CTX group,TGF-β1+MP+CTX group. After HLF were exposed to each group for 24 hours, we detected the expression of a-smooth muscle actin using the western blot method.4.Statistical analysis:the test of significance is worked out by software package of SPSS13.0. Description of quantitative data with mean±tandard deviation, description of classified information with proportion and the corresponding number. A p value of<0.05 was considered significant.Results1.The group patients include 7 SLE,11 pSS,3 PM,5 DM,8 SSc,3 RA,4 WG,2 MTCD,1 ASS and 1 RP. The pathological types were constructed by different original diseases (P=0.013), SLE, pSS, PM, DM corresponded pathologic types mainly NSIP, ASS, MCTD, RA corresponded pathologic types mainly UIP, SSc, RP, WG corresponded pathologic types mainly other type. The pathological types related to the performance HRCT of the puncture site(r=0.545, P=0.043), UIP corresponded-the performance HRCT of the puncture site mainly grid shadow and ground-glass appearance, NSIP mainly corresponded spot piece shadow, honeycomb shade and nodular shadow mainly performed other type, real variable the shadow displayed in three pathological type all. Three pathological types of systemic inflammation index had different rate increases, but the differences between them was no statistically significant; In addition, the differences between different pathological types in lung function and arterial blood gas analysis were all no statistically significant. In therapy, between different pathological types the therapeutic effect was not statistically significant (P=0.849),and the chances of lung infection were almost equal (P=0.863).But the differences between different therapeutic trentments in therapeutic effect have statistical significant(P=0.005),and according to the better and stable percent, the group of CTX≥12g was superior to the group of CTX<12g or non-CTX. During follow-up, patients with pulmonary infection protocols were 51.1%,3 patients including 2 cases of NSIP,1 case of not defined type died because of this.2. The HE results had shown that in the group of normal control lung tissue, the alveoli and lung interval were clear, inflammatory cells infiltration and alveolar septal thickening abnormal changes were not present; but in the lung tissues of patients with CTD-ILD had pathological changes,such as lung interval break or thickening, inflammatory cells infiltration, alveolar lumen secretions. The confocal microscope results presented Caveolin-1 and TGF-β1 co-expression in lung interstitial, and compared with normal lung tissue,CTD-ILD patients had increased in the expression of TGF-β1,but decreased in caveolin-1.Caveolin-1 mainly localized in the membrane of HLF, its expression decreased after the induction of TGF-β1, with trend of depending on concentration and time. Compared to 0μg/L group, caveolin-1 mRNA expressions in 5,10,15μg/L groups were all reduced,with statistical significance(all P<0.01);but 10,15μg/L groups caveolin-1 protein were decreased, with statistical significance(all P<0.01).And also compared to 0h group, caveolin-1 mRNA expressions in 6h,12h,24h,48h groups were all reduced,with statistical significance(all P<0.01);but the caveolin-1 protein expressions in 24h and 48h groups were decreased,with statistical significance(all P<0.01).3. The Sou-Medrol and cyclophosphamide can inhibit the proliferation of human lung fibroblasts, with concentration dependent.We respectively choose 10-3mg/ml as MP and 10-5mg/ml as CTX trail drug concentrations. Results show that compared to PBS control group,α-SMA protein expression increased by cells exposed to TGF-β1 (10μg/L) for 24h, with statistical significance(P<0.001). 10μg/L TGF-β1 and MP,CTX co-cultured 24h, we found that compared to PBS control group, the a-SMA protein expressions all increased, with statistical significance(P=0.003,0.020); but compared to TGF-β1 group, theα-SMA protein expressions all decreased, with statistical significance(P=0.024,0.003); compared to combined drug group, the a-SMA protein expressions also had statistical significance(P<0.001,0.001).The difference in two alone drug group has no statistical significance(P=0.327). Compared to combined drug group, PBS control group has no statistical significance(P=0.207), TGF-β1 group,TGF-β1+MP group,TGF-β1+CTX group all had statistical significance (respectively P<0.001,<0.001,0.001)。Conclusion1.The pathological types related to original diseases and the performance HRCT of the puncture site, between different pathological types it has no statistically significant differences in inflammation index, pulmonary function, arterial blood gas analysis, the treatment effect and lung infection occurrence probability. The differences between different therapeutic trentments in therapeutic effect have statistical significant, and the group of CTX≥12g was superior to the group of CTX <12g or non-CTX.2.CTD-ILD patients had increased in the expression of TGF-β1, but decreased in caveolin-1.Caveolin-1 mainly localized in the membrane of HLF, TGF-β1 induced low expression of caveolin-1.The reduced caveolin-1 may be involved in the occurrence and development of CTD-ILD.3.The Sou-Medrol and cyclophosphamide can inhibit the human lung fibroblasts transfer differentiation to myofibroblats induced by TGF-β1, two medicine combined application may be better than the two drugs used alone.