Objective To explore the role of chronic ethanol ingestion in pulmonary fibrosis. Methods Twenty SD rats were randomly divided into a control group (n=10) and an ethanol group ( n=10) , and fed with quantitative non-ethanol and ethanol Lieber-DeCarli liquid diet every day respectively. All rats were sacrificed after 8 weeks. The morphological changes and collagen deposition of lung tissue were observed under light microscope by HE and Masson staining. Levels of glutathione (GSH) and hydroxyproline (HYP) in lung tissues were measured by colorimetric method. The content of connective tissue growth factor (CTGF) in lung tissue was detected by ELISA. Results Compared with the control group, varied degrees of alveolar and alveolar septal infiltration of inflammatory cells can be shown in the ethanol group, and also some alveolar wall damage or collapse.Masson staining showed that the ethanol group has more significant deposition of collagen fibers in alveolar interstitumthan the control group. The content of GSH in rat lung tissue reduced, but the contents of HYP and CTGF increased in the ethanol group compared with the control group [ GSH( mg/g) :0.08±0.04 vs. 0.22±0.14, HYP(mg/g) : 0.57±0.15 vs. 0.40 ± 0.09, CTGF(ng/mL) :306.57±46.86 vs. 134.02±79.82, Plt;0.05] . Conclusions Lieber-DeCarli ethanol liquid diet can establish a rat model of chronic ethanol ingestion. Lung injury and pulmonary fibrosis in rats can be induced by chronic ethanol ingestion. Ethanol may be one of the causes of the pulmonary fibrosis.
Objective To investigate pathogenesis of dry eye and applied value in diagnosis of dry eye with connective tissue disease(CTD) by apoptosis detection, using impression cytology flow cytometry (ICFC) in conjunctiva epithelial cells. Methods A total of 60 patients (120 eyes) with CTD, after asked case history and measured the basal Schirmer’s test (S-I-T), Break-Up Time (BUT), fluorescent Staining (FL), were divided into 4 groups: the first group without Sjögren syndrome or dry eye (NSS1), the second group without Sjögren syndrome but dry eye (NSS2), the third group with Sjögren syndrome and non-dry-eye (SS1) and the fourth group with Sjögren syndrome and dry eye (SS2). And apoptosis of conjunctiva epithelial cells was detected by ICFC. Results The apoptosis rate of conjunctiva epithelial cells was statistically significant (Plt;0.001) between every two groups, except that between NSS1 group and SS1 group (P=0.998). And apoptosis rate was a positive correlation with FL (r=0.926, Plt;0.001), but negatively with S-I-T and BUT (r= –0.712, r= –0.818, Plt;0.001). Dye eye and Sjögren-syndrome both affected the apoptosis level of conjunctiva epithelial cell and there was an interaction between them. Conclusion Apoptosis plays an important role of ocular damage and apoptosis detection helps with diagnosis of dry eye with CTD. Dye eye and Sjögren-syndrome increase apoptosis level. Apoptosis detection by ICFC in conjunctiva epithelial cells is a minimally invasive and effective way to detect ocular apoptosis.
ObjectiveTo compare the clinical characteristics,high-resolution computed tomography (HRCT) manifestations,pulmonary function results,serum autoantibodies and treatment modality of connective tissue diseases related interstitial lung diseases (CTD-ILDs) and idiopathic interstitial pneumonias (ⅡPs). MethodsPatients explicitly diagnosed with CTD-ILDs and ⅡPs were retrospectively selected from Nanjing Drum Tower Hospital between January 2004 and December 2012.The clinical features were abstracted,including age,gender,symptoms,signs,serum autoantibody results,HRCT findings,and treatment.Patient characteristics were compared between CTD-ILDs and ⅡPs using a Pearson's χ2 test for categorical variables,and a Student's t test for continuous variables. ResultsA total of 692 patients with complete data were included in this study,with 240 CTD-ILDs cases and 452 ⅡP cases.CTD-ILDs could exist in different types of CTDs,which were mainly shown in Sjogren's syndrome,rheumatoid arthritis,and dermatomyositis/polymyositis.Age,gender,connective tissue diseases related characteristics such as dry eyes,dry mouth,and arthralgia,and several autoantibodies such as ANA,SSA,SSB all showed significantly difference between CTD-ILDs and ⅡPs (P<0.05).However there were no significant differences in cough,dyspnea after exertion,velcro crackles on auscultation,or finger clubbing between two groups (P>0.05). The HRCT manifestations of CTD-ILDs were reticular opacities,patchy consolidation,band-like shadows,and pleural thickening.Pulmonary function tests commonly showed restrictive lung function and decreased diffusing capacity.The histopathologic findings of lung biopsies of CTD-ILDs were mostly chronic inflammatory cell infiltration,as well as hyperplasia of fibrous tissue and septal thickness.The finding of chronic inflammatory cell infiltration showed significant difference between CTD-ILDs and ⅡPs (P<0.05),while the HRCT manifestations,pulmonary function results or other histopathologic findings did not(P>0.05).The current treatment modality was corticosteroids plus immunosuppressants. ConclusionDespite the similarities,CTD-ILDs show distinct clinical,laboratory and imaging features from from ⅡPs in clinical practice.
Objective To investigate the expression and clinical significance of connective tissue growth factor (CTGF) in colorectal cancer. Methods The expressions of CTGF in 62 patients’ colorectal cancer tissues and their corresponding adjacent tissues were detected by SP immunohistochemical method. The results were statistically analyzed. Results The positive rates of CTGF in colorectal cancer and adjacent tissues were 61.3% and 19.4% respectively， and the difference between them was significant (P＜0.05). The expression of CTGF was related to degree of differentiation, depth of infiltration and lymph node metastasis or not (P＜0.05）, namely the lower degree of differentiation, the deeper depth of infiltration and the more lymph node metastasis, the corresponding positive expression rates were lower (P=0.030, P=0.032 and P=0.017 respectively), but correlation with gender was not significant (Pgt;0.05）. Conclusion CTGF may play an important role in the occurrence of colorectal cancer， which contributes a lot to guide clinical treatment and prognosis.
ObjectiveTo explore the expression of periostin in bronchoalveolar lavage fluid (BALF) of patients with dermatomyositis-related interstitial lung disease (DM-ILD) and rheumatoid arthritis-related interstitial lung disease (RA-ILD).MethodsA total of 44 patients with DM-ILD and 28 patients with RA-ILD were underwent bronchoalveolar lavage. Cells in BALF were collected and analyzed by absolute different cell counts. The level of periostin and Krebs von den Lungen-6 (KL-6) were tested by enzyme linked immunosorbent assay. Results of high resolution CT of patients were scored. Thirty patients without interstitial lung disease (ILD) served as a control group.ResultsLevels of periostin and KL-6 were significantly increased in BALF of patients with DM-ILD and RA-ILD compared with control group (all P<0.05). Levels of periostin were positively correlated with lymphocyte counts and levels of KL-6 in BALF (allP<0.05). Furthermore, levels of periostin were significantly correlated with high resolution CT scores (P<0.05).ConclusionsLevels of periostin are increased in patients with DM-ILD and RA-ILD. Periostin might be served as an indicator of CTD-ILD.
ObjectiveTo investigate the effect of transforming growth factor β1 (TGF-β1) induced proliferation of ligamentum flavum cells and ligamentum flavum hypertrophy and its effect on connective tissue growth factor (CTGF) expression.MethodsThe ligamentum flavum tissue in lumbar intervertebral disc herniation was extracted and the ligamentum flavum cells were isolated and cultured by collagenase pre-digestion method. Morphological observation, immunofluorescence staining observation, and MTT assay were used for cell identification. The 3rd generation ligamentum flavum cells were divided into 5 groups. The cells of groups A, B, C, and D were respectively sealed with 3 ng/mL TGF-β1, 50 ng/mL CTGF, 3 ng/mL TGF-β1+CTGF neutralizing antibody, and 50 ng/mL CTGF+CTGF neutralizing antibody. Serum free DMEM was added to group E as the control. MTT assay was used to detect the effects of TGF-β1 and CTGF on the proliferation of ligamentum flavum cells. Western blot was used to detect the expression of CTGF protein. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of collagen type Ⅰ, collagen type Ⅲ, and CTGF genes.ResultsThe morphological diversity of cultured ligamentum flavum cells showed typical phenotype of ligamentum flavum fibroblasts; all cells expressed collagen type Ⅰ and vimentin, and some cells expressed collagen type Ⅲ; MTT identification showed that with the prolongation of culture time, the absorbance (A) value of each generation of cells increased gradually, and the A value of the same generation of cells at each time point was significantly different (P<0.05), there was no significant difference in A value between the cells of each generation at the same time point (P>0.05). After cultured for 24 hours, MTT assay showed that the A value of cells in groups A and B was significantly higher than that of group E (P<0.05). After adding CTGF neutralizing antibody, the A value of cells in groups C and D decreased, but it was still higher than that of group E (P<0.05). There were also significant differences among groups A, C and groups B, D (P<0.05). Western blot analysis showed that the relative expression of CTGF protein in groups A and B was significantly higher than that in group E (P<0.05), while the relative expression of CTGF protein in groups C and D was significantly lower than that in group E (P<0.05), and the difference between groups A, C and groups B, D was also significant (P<0.05). qRT-PCR detection showed that the mRNA relative expression of CTGF, collagen type Ⅰ, and collagen type Ⅲ in group A was significantly higher than that in group E (P<0.05). After adding neutralizing antibody, the mRNA relative expression of genes in group C was inhibited and were significantly lower than that in group A, but still significantly higher than that in group E (P<0.05). The mRNA relative expressions of collagen type Ⅰ and collagen type Ⅲ in group B was significantly higher than that in group E (P<0.05), but the mRNA relative expression of CTGF was not significantly different from that in group E (P>0.05); after neutralizing antibody was added, the mRNA relative expression of collagen type Ⅰ and collagen type Ⅲ in group D was inhibited and was significantly lower than that in group B, but still significantly higher than that in group E (P<0.05); there was no significant difference in the mRNA relative expression of CTGF between group D and groups B, E (P>0.05).ConclusionTGF-β1 can promote CTGF, collagen typeⅠ, collagen type Ⅲ gene level and protein expression in ligamentum flavum cells, and TGF-β1 can synergistically promote proliferation of ligamentum flavum cells through CTGF.
Objective To study the effects of connective tissue growth factor (CTGF) on retinal Müller cells based on transcriptome analysis of RNA-seq technology.MethodsRetinal Müller cells were divided into the control group and the CTGF treatment group which was continuously cultured with 10 ng/ml of CTGF for 24 h. The influence of CTGF on the migration of Müller cells were tested by scratching experiments. The RNA transcriptome analysis was applied to complete transcriptome sequencing, differentially expressed genes and functional enrichment analysis of the two groups of cells. HiSeq sequencing technology was used to sequence the whole transcriptome of the two groups of cells to obtain biological big data, and analyze the differentially expressed miRNAs on this basis. The functions and signal pathways of differential miRNAs were analyzed through gene annotation (GO) functional significance enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway significant enrichment analysis. Based on transcriptome data, genes with differential expression multiples in the top ten between the two groups were screened out, and the expression of bone morphogenetic protein 4 (BMP4) gene was verified by real time fluorescence quantification PCR (qRT-PCR), immunofluorescence and Western blot.ResultsAfter CTGF stimulation of Müller cells, cell viability and mobility which compared with the control group were significantly increased, with statistically significant differences (t=3.453, P＜0.05). The differential gene expression profile of CTGF induced Müller cells was obtained by RNA transcriptome analysis. Comparing the sequencing results of the two groups, it was found that 325 differentially expressed genes included 152 up-regulated genes and 173 down-regulated genes. The results of GO functional significance enrichment analysis showed that the functions of differential miRNA were mainly divided into three categories: biological processes, cellular components, and molecular functions. These differentially expressed genes were involved in signaling between nervous systems, adhesion between cells, and the interaction between cytokines and their receptors. These differentially expressed genes were involved in different metabolic pathways and biological processes such as tissue inflammation and fibrosis. BMP4 gene was seected for verification through immunofluorescence, qRT-PCR and western blot. The results showed that the expression of BMP4 was significantly higher than that in the control group, and the difference was statistically significant (t=39.490, 10.110, 5.470; P=0.004, 0.001, 0.006).ConclusionCTGF promotes cell proliferation and migration by up-regulating the expression of BMP4 in Müller cells, leading to tissue fibrosis and inducing inflammation.
Objective To investigate the effects and significance of nerve growth factor (NGF) and its high affinity receptor of tyrosine kinase A (TrkA) expressions on proliferative connective tissue of bile duct in rats after bile duct ligation (BDL). Methods Forty-six female Sprague-Dawley rats were randomly divided into two groups: control group ( n =6) and BDL group ( n =40). The model of obstructive jaundice in rat was made by bile duct ligation, then duodenohepatic ligament was taken and treated with anti-NGF and anti-TrkA receptor antibody. Expressions of NGF and TrkA receptor in connective tissue of bile duct were investigated by immunohistochemistry, blood specimens were collected from left ventricle to detect serum total bilirubin (TB) and alanine aminotransferase (ALT). Results After BDL, TB level obviously elevated in the third day, and continued until the fourteenth day, then descended. By day 21 and 28, it returned to normal level. Compared with normal bile duct, due to bile stasis, an increased thickness of the bile duct wall was observed by microscope which correlated with the proliferation and differentiation of connective tissue cell. NGF and TrkA were expressed by the cell membrane and the cytoplasm of connective tissue cell and inflammatory infiltration cell after BDL. The trend between their expressions and bilirubin levels was similar. Conclusion NGF and its receptor TrkA regulate the proliferate and differentiation of connective cell in bile duct. They may play a key role in the formation of bile duct scar, which seems to be hardly reversed by relief of bile stasis in a short time.
ObjectiveTo investigate the role of transforming growth factor β1(TGF-β1) and connective tissue growth factor (CTGF) in pathogenesis and progression of human intervertebral disc degeneration by detecting the expressions of these two factors in different degrees of degenerative discs. MethodsThe lumbar intervertebral discs were collected from 33 patients with lumbar disc herniation and 12 patients with lumbar vertebral fracture between November 2012 and April 2013.All samples were observed under the microscope after HE staining,and then were divided into different subgroups according to the degenerative degree.The expressions of TGF-β1 and CTGF were detected by Western blot. ResultsAccording to the pathological features,10 discs were defined as normal discs,10 as mild degenerative discs,9 as moderate degenerative discs,and 16 as severe degenerative discs.The histological observation showed that rounded nucleus pulposus cells with similar size evenly distributed in the cartilage-like matrix,and no hyperplastic collagenous fiber was seen in normal discs;mild degenerative discs characterized by slightly larger nucleus pulposus cells in the matrix,but cells did not decrease,a small quantity of inflammatory cells infiltrated in the matrix,hyperplasia of collagenous fiber was not seen;most of the nucleus pulposus cells became bigger,some showed a bulb form,the number of nucleus pulposus cells was significantly reduced,low grade hyperplasia of collagenous fiber emerged in the matrix,new vessels and inflammatory cells were both found in some specific areas of discs in moderate degenerative discs;there was no nucleus pulposus cells in the matrix of severe degenerative discs,the hyperplasia of collagenous fiber was obvious.The relative expression of TGF-β1 in 3 degeneration discs was significantly higher than that in normal discs (P<0.05),and the expression of TGF-β1 was significantly higher in severe degenerative discs than in moderate and mild degenerative discs (P<0.05),but no significant difference between moderate and mild degenerative discs (P>0.05).The relative expression of CTGF in moderate and severe degeneration discs was significantly higher than that in normal discs (P<0.05);and the expression of CTGF in mild degenerative discs was higher than that in normal discs,but there was no significant difference (P>0.05);and significant difference in CTGF expression was found among 3 degeneration discs (P<0.05). ConclusionThe expressions of TGF-β1 and CTGF are closely related to the degree of human lumbar disc degeneration,these two factors may play an important role in promoting lumbar intervertebral disc degeneration.
Objective To investigate the expression of connective tissue growth factor（CTGF）in human proliferative membranes of proliferative vitreoretinopathy（PVR），and the relationship among CTGF，transforming growth factor-beta; receptor（TGF-beta;R）and extracellular matrix（ECM）. Methods Immunohistochemistry method of streptavidin-biotin-peroxidase complex（SABC）was used to detect the expression of CTGF，TGF-beta;RⅡ，fibronectin（FN），collagen Ⅰ，and collagen Ⅲ protein in43periretinal membranes（PRM）of PVR obtained by vitrectomy，and the correlations of the expression of CTGF，TGF-beta;RⅡ and ECM were analyzed by statistics. Results CTGF and TGF-beta;RⅡ protein highly expressed in PRM of PVR and most of the CTGF-positive cells were epithelial cells.The result of immunohistochemistry showed that the positive rates of CTGF and TGF-beta;RⅡ protein were 70.6% and 76.5%in PVR C membranes，and 73.9% and 69.6%in PVR D membranes respectively.Relationship between positive expression and membranesprime; grades appeared no statistical correlation(P＞0.05).Statistical analysis showed that there was a correlation between the expression of CTGF and TGF-beta;RⅡ，FN，and collagen Ⅰ and Ⅲ protein，respectively. Conclusions The expression of CTGF and TGFbeta;RⅡ protein is up-regulated in PRM of PVR，which suggests that the activation of TGF-beta;RⅡ is involved in the production of CTGF，and CTGF is closely related to the production of ECM and play an important role in the pathogenesis of PVR. (Chin J Ocul Fundus Dis, 2006, 22: 192-195)