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Background connective tissue disease (connective tissue disorders, CTDs) is a class of diseases that cause damage to the body through its own immune response, these diseases can involve almost the various organs of the body. Lungs are rich in collagen fibers and blood vessels, and immune regulation, metabolic, endocrine function, it is easier for a variety of connective tissue disease organ violated. Manifestation of lung damage caused by connective tissue disease, which is more common interstitial lung disease, known as connective tissue disease-related pulmonary interstitial disease (interstitial lung disease with connective tissue disorders, CTD-ILD). Pathological changes early exudative lesions mainly alveolitis, with the progress of the disease, widely collagen deposition, and ultimately lead to the irreversibility of pulmonary fibrosis and lung function impairment. Complex type of connective tissue disease-related pulmonary interstitial pathological lesions, pathological type of all interstitial lung disease can be present in the CTD-ILD, and can coexist in the same patient, pathological type of CTD-ILD is recognized using 2002 years recommended by the ATS and ERS interstitial lung disease classification standards. CTD-ILD high incidence and high mortality, lack of a unified standard for the treatment, the most commonly used hormones and cyclophosphamide treatment method is easy to secondary infection, the complications of clinical research difficult and hot. The experiment focuses on the connective tissue disease due to interstitial lung disease pathology analysis, to explore the relationship between the pathological type of the original disease, imaging, treatment options, lung function, inflammatory markers and other factors, to a comprehensive understanding of the clinical and provide the basis for the treatment of CTD-ILD; while part of the mechanism of pulmonary fibrosis in vitro. Caveolin -1 (caveolin-1) is a 21-24Kda the size of the cell membrane embedded proteins in the body is the main components of the caveolin involved in membrane transporter, cholesterol transport, lipid stability and cellular signal transduction and many other biological functions. Studies have shown that caveolin-1 and pulmonary fibrosis, so the experiment caveolin-1 expression in the interstitial connective tissue disease-related pulmonary lesions in TGF-β1-induced human lung fibroblasts the regulation of expression of a preliminary study, to a better understanding of its role in the pathogenesis of connective tissue disease-related pulmonary interstitial disease, and help to find new therapeutic targets. Recent years, studies have shown that the CTD-ILD and idiopathic pulmonary fibrosis (idiopathic pulmonary fibrosis, IPF) has a similar pathological features, namely the presence of a large number by myofibroblasts (myofibroblast, MF), composed of fibroblast foci MF secrete a variety of cytokines, resulting in the abnormal deposition of extracellular matrix, as well as the induction of apoptosis in alveolar epithelial cells. TGF-β1-induced human lung fibroblast cells (human lung fibroblast, HLF) transformed into myofibroblasts, visible role in promoting pulmonary fibrosis. On the clinical treatment of CTD-ILD is the most commonly used drug is the methylprednisolone (Sou-Medrol, MP) and cyclophosphamide (cyclophosphamide, CTX), but its mechanism is not yet clear, the experiment to be adopted by the in vitro observation MP and CTX on HLF proliferation of TGF-β1-induced the HLF differentiation as the impact of the MF, explore the MP and CTX on the mechanism of action of CTD-ILD, provide a theoretical basis for the clinical treatment of CTD-ILD. Purpose and meaning. Understanding of the relationship between the pathological type of CTD-ILD primary disease, the puncture site imaging findings, lung function, treatment options, inflammation indicators. Learn caveolin-1 expression in the lung tissue of patients with CTD-ILD, and to explore in vitro for its role in pulmonary fibrosis. 3 by in vitro experiments to explore the impact of methylprednisolone and cyclophosphamide HLF TGF-β1-induced transformation to MF. Research methods of January 2003 to December 2010, the Guangdong Provincial People's Hospital rheumatology hospital diagnosis of CTD-ILD parallel retrospective analysis of 45 patients after percutaneous lung biopsy under CT guidance. The pathological types of usual interstitial pneumonia (usual interstitial pneumonia, UIP), nonspecific interstitial pneumonia (nonspecific interstitial pneumonia, NSIP) and type three, the treatment program is divided into CTX lt; 12g, CTX ≥ 12g, non-CTX group, the treatment effect into improved stability, deterioration and can not determine the four groups, record the puncture site of the high-resolution CT (high resolution computerized tomography HRCT) imaging findings, treatment programs and before and after treatment ESR (erythrocyte sedimentation rate, ESR), C-reactive protein (C-reactive protein, CRP), platelets (platelets, PLT), FVC (forced vital capacity, FVC), one second forced expiratory volume (forced expiratory volume in one second, FEV1 ), carbon monoxide diffusing capacity (diffusion capacity for carbonmonoxide of lung DLCO), partial pressure of oxygen (partial pressure of oxygen, PO2), carbon dioxide partial pressure (partial pressure of carbondioxide, PCO2), oxygen saturation and lung infection happens. Analyze the relationship between the pathological type of CTD-ILD primary disease, the puncture site of HRCT, inflammatory markers, lung function, arterial blood gas analysis, treatment. Patients and normal lung tissue frozen sections 2.CTD-ILD HE staining, line caveolin -1, TGF-β1 immune double fluorescent staining; normal lung tissue in primary fibroblast culture, identification, adding 10μg / L TGF-β1-induced HLF 24 hours, the expression of caveolin-1 fluorescence compared before and after induction; selection of the 11-20 generation HLF final concentration were 0,1,3,5,10,15 μg / L TGF-β1 induced 24h, and concentration of 10μg / L TGF-β1-induced 0,6,12,24,48 h, real time PCR and Western Blot method detection of caveolin-1mRNA and the protein expression. Cultured in vitro primary HLF and passaged with 11-20 cells following experiment: 10μg/LTGF-β1 HLF Induced MF transdifferentiation. Application of the the four MTT assay methylprednisolone and cyclophosphamide on HLF proliferation inhibition rate, the IC50 values ??of the two drugs as single drug concentration of cells derived using SPSS software; this basis is divided into: PBS control group ( the negative control group), TGF-β1 group (10μg / L) (positive control group), TGF-β1 A Strong Long Group (MP group), TGF-β1 in the cyclophosphamide group (CTX group), expression of TGF = β1 methylprednisolone dragon ± cyclophosphamide group (combination group). The groups role HLF 24h, application of the Western Blot method to detect the expression of α-smooth muscle actin (α-SMA). 4 Statistical analysis: SPSS13.0 software for statistical analysis. Clinical research: the description of the quantitative data are expressed as mean ± standard deviation, a description of the segment information; groups were compared using one-way ANOVA, two sets of quantitative factors before and after the level differences using a paired t-test, multiple constituent ratio and the corresponding number of classification factors than with differences compared by chi-square test; does not meet the parametric test statistical analysis using the Kruskal Wallis non-parametric tests, the degree of association of two categorical variables with correlation coefficient column associated, classified information corresponding relationship with correspondence analysis. Basic research: the groups were compared using ANOVA analysis of variance by the Levene test of homogeneity, the homogeneity of variance using LSD method for multiple comparisons; Welch approximation for unequal variances analysis of variance and Dunnett's T3 multiple comparisons. P lt; 0.05 was considered statistically significant. Results: 45 patients enrolled, including systemic lupus erythematosus (systemic lupus erythematosus, SLE) 7 patients with primary Sjogren's syndrome (primary Sjogren syndrome, pSS) 11 patients with polymyositis (polymy olitis, PM ) 3, dermatomyositis (dermatomylsitis, DM) 5 cases of systemic sclerosis (systemic sclerosis, SSc) 8 cases of rheumatoid arthritis (rheumatoid arthritis, RA) 3 cases of Wegener's granulomatosis (Wegener's granulomatosis WG) 4 cases, mixed connective tissue disease (mixed connective tissue disease, MCTD) 2 cases of anti-synthetase antibody syndrome (anti-synthetase syndrome ASS) and recurrent multi polychondritis (relapsing polychondritis, RP) and 1 case . Pathological types of primary disease constitute different (P = 0.013), SLE, PSS, PM, DM corresponding pathological type NSIP ASS, MCTD, RA pathological type mainly for UIP, SSc, RP, WG corresponding pathological The main types of the other type. The pathological type puncture site HRCT imaging findings related (r = 0.545, P = 0.043), UIP corresponding puncture site of HRCT, ground glass main grid shadow NSIP corresponding patches shadow, honeycombing knot the Festival Film pathological type mainly for the other type, the the opacities pathological type of performance are available in three kinds. Three types of pathological inflammation indicators have different degrees, but no statistically significant differences between them, lung function and arterial blood gas analysis results showed no significant difference between the different types of pathology. Treatment, no significant difference in treatment effect between the different types of pathology (P = 0.849), pulmonary infection is basically the same (P = 0.863). Statistically significant (P = 0.005), but the differences in treatment effect between the different treatment options, improved and stable percentage, CTX ≥ 12g the group superior CTX lt; 12g and non CTX group. 51.1% of patients with lung infection during follow-up, three patients died, including 2 cases of NSIP were not stereotypes. 2.HE staining showed that: alveoli of the lung tissue of normal control group, lung interval structure clear, no inflammatory cell infiltration, alveolar septal thickening abnormal pathological changes; patients with CTD-ILD group the visible pulmonary intervals fracture or thickening, inflammatory cell infiltration , the alveolar cavity secretions oozing pathological change. Immunohistochemical double staining results showed that: caveolin -1, TGF-β1 co-expression in the lung interstitium, compared with the control group, CTD-ILD lung tissue in patients with increased expression of TGF-β1, caveolin-1 expression weakened . Caveolin-1 expression on the membrane of the HLF, it decreased expression of TGF-β1-induced, and presented a certain concentration and time dependent manner. Decreased compared with TGF-β10μg / L (control group), caveolin of-1mRNA expression in 5,10,15 μg / L TGF-β1 group differences were statistically significant (P lt; 0.01); while caveolin-1 protein expression only in 10,15 μg / L TGF-β1 group reduced the difference was statistically significant (P lt; 0.01). Oh group to the role of TGF-β1 in control, the expression of caveolin-1mRNA role of TGF-β1 6h, 12h, 24h, 48h group were decreased, the differences were statistically significant (P lt; 0.01); while the small Wo protein-1 protein expression in TGF-β1 for 24h, 48h group decreased, the difference was statistically significant (P lt; 0.01). Methylprednisolone and cyclophosphamide can inhibit the proliferation of HLF, and show a certain concentration dependency, single drug concentration of the two drugs respectively to take 10-3mg/ml and 10-5mg/ml. The results show that the 10μg / L TGF-β1-induced HLF 24h, a-SMA protein expression compared to PBS control group increased, and the difference was statistically significant (P lt; 0.001). TGF-β1 with single-agent MP or CTX were cultured for 24 hours, and found: a-SMA expression, two single-drug group were increased compared with the PBS negative control group, the difference was statistically significant (P = 0.003,0.020); but compared to the positive control group, TGF-β1, the the two monotherapy group a-SMA expression decreased, the difference was statistically significant (P = 0.024,0.003); compared with the combined treatment group, found that the two monotherapy group a- SMA expression decreased significantly less than the combined treatment group, and the difference was statistically significant (P = lt; 0.001,0.001). A-SMA expression differences between the MP and CTX alone treatment group was not statistically significant (P = 0.327). A-SMA expression in the combination group and PBS control group, the difference was not statistically significant (P = 0.207) with the control group of TGF-β1, TGF-β1 MP group, TGF-β1 CTX group compared to both a significant decline in the difference were statistically significant (P = lt; 0.001, lt; 0.001,0.001). Conclusion: 1. CTD-ILD patients with pathological type associated with the primary disease, the puncture site HRCT findings of inflammatory markers between the different types of pathology, pulmonary function, blood gas analysis, the treatment effect, as well as lung infection incidence were not statistically significance; statistically significant differences in treatment effect between the different treatment options is superior to CTX CTX ≥ 12g group lt; 12g and non CTX group. In TGF-β1 expression in the lung tissue of patients 2.CTD-ILD enhanced caveolin-1 expression reduce; TGF-β1-induced caveolin-1 expression was decreased HLF membrane can, and show a certain degree of concentration and time dependent trend, lags behind the mRNA expression and protein expression, indicating that TGF-β1-induced caveolin-1 expression in the decline may be involved in the occurrence and development of CTD-ILD. Methylprednisolone and cyclophosphamide can be suppressed TGF-β1-induced HLF conversion to MF, the inhibitory effect of the two drugs in combination is superior to the two drugs alone.
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