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)
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.
ObjectiveTo construct the connective tissue growth factor (CTGF) recombinant interference vector (shRNA) and observe its inhibitory effect on the expression of endogenous CTGF in retinal vascular endothelial cells. Methods The human CTGF shRNA was constructed and the high-titer CTGF shRNA lentivirus particles was acquired via three-plasmid lentivirus packaging system to infect retinal vascular endothelial cells. The optimal multiplicity and onset time of lentivirus infection were identified by tracing down the red florescent protein in interference vector. The cells were classified into three groups: blank control group, infection control group and CTGF knockdown group. The differences in cells migrating ability was observed through Transwell allay. The mRNA and protein expression of CTGF, fibronectin, α-smooth muscle actin (α-SMA) and collagen Ⅰ (Col Ⅰ) were quantified through real-time PCR testing and Western blot system. Data between the three groups were examined via one-way analysis of variance. ResultsThe result showed that an optimal multiplicity of 20 and onset time of 72 hours were the requirements to optimize lentivirus infection. Transwell allay result showed a contrast in the number of migrated cells in the CTGF knockdown group and that in the blank control group and infection control group (F=20.64, P=0.002). Real-time PCR testing showed a contrast in related gene expression (CTGF, fibronectin, α-SMA and Col Ⅰ) in the CTGF knocked-down group and that in the blank control group and infection control group (F=128.83, 124.44, 144.76, 1 374.44; P=0.000, 0.000, 0.000, 0.000). Western blot system showed the statistical significance of the contrasted number of related protein expression (CTGF, fibronectin, α-SMA and Col Ⅰ) in the knockdown group and that in the blank control group (F=22.55, 41.60, 25.73, 161.68; P=0.002, 0.000, 0.001, 0.000). ConclusionThe success in producing CTGF shRNA lentivirus particle suggests that CTGF shRNA lentivirus can effectively knock down CTGF expression.
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 expression of connective tissue growth factor (CTGF) in the chronic sciatic nerve compression injury and to explore the effect of rhodiola sachalinensis on the expression of CTGF. MethodsForty-five adult male Sprague Dawley rats were randomly divided into groups A, B, and C:In group A (sham-operated group), only the sciatic nerve was exposed; in group B (compression group), sciatic nerve entrapment operation was performed on the right hind leg according to Mackinnon method to establish the chronic sciatic nerve compression model; and in group C (compression and rhodiola sachalinensis group), the sciatic nerve entrapment operation was performed on the right hind leg and rhodiola sachalinensis (2 g/mL) was given by gavage at a dose of 0.5 mL/100 g for 2 weeks. The nerve function index (SFI) was observed and neural electrophysiology was performed; histology, transmission electron microscope, real-time fluorescent quantitative PCR, and Western blot were performed to observe the morphological changes of the compressed nerve tissue and to determine the mRNA and protein levels of CTGF, collagen type I, and collagen type Ⅲ at 2, 6, and 10 weeks after operation. ResultsAt 6 and 10 weeks after operation, SFI of groups A and C were significantly better than that of group B (P < 0.05), but there was no significant difference between groups A and C (P > 0.05). The nerve function test showed that the nerve motor conduction velocity (MCV) and the amplitude of compound muscle action potential (CMAP) of group B were significantly lower than those of groups A and C, and distal motor latency (DML) was significantly prolonged in group B (P < 0.05), but there was no significant difference between groups A and C (P > 0.05). Histology and transmission electron microscope observations showed that myelinated nerve fibers degenerated and collagen fiber hyperplasia after sciatic nerve chronic injury in group B, and rhodiola sachalinensis could promote the repair of nerve fibers in group C. At 2 weeks postoperatively, the number of myelinated nerve fibers in groups B and C were significantly less than that of group A (P < 0.05), and the myelin sheath thickness of groups B and C were significantly larger than that of group A (P < 0.05). At 6 and 10 weeks postoperatively, the number of myelinated nerve fibers in groups B and C were significantly more than that of group A (P < 0.05); the myelin sheath thickness of group B was significantly less than that of groups A and C (P < 0.05). The effective area of nerve fiber had no significant difference among groups at each time point (P > 0.05). Real-time fluorescent quantitative PCR and Western blot results showed that the mRNA and protein expressions of CTGF, collagen type I, and collagen type Ⅲ in group B were significantly higher than those in groups A and C at each time point (P < 0.05), but there was no significant difference between groups A and C (P > 0.05). ConclusionSciatic nerve fibrosis can be caused by chronic nerve compression. The increased expression of CTGF suggests that CTGF plays an important role in the process of neural injury and fibrosis. Rhodiola sachalinensis can significantly reduce the level of CTGF and plays an important role in nerve functional recovery.
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 Sj?gren syndrome or dry eye (NSS1), the second group without Sj?gren syndrome but dry eye (NSS2), the third group with Sj?gren syndrome and non-dry-eye (SS1) and the fourth group with Sj?gren 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 Sj?gren-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 Sj?gren-syndrome increase apoptosis level. Apoptosis detection by ICFC in conjunctiva epithelial cells is a minimally invasive and effective way to detect ocular apoptosis.
Objective To investigate the role of IFN-γ in suppressing bleomycin-induced pulmonary fibrosis in rats.Methods Seventy-five SD rats were randomly divided into five groups (15 rats in each group)，ie.a normal group，a bleomycin-induced pulmonary fibrosis model group，a dexamethasone-treated group，a high-dose IFN-γ-treated group (150 000 U/kg) and a low-dose IFN-γ-treated group (50 000 U/kg).Five rats in each group were randomly killed in 7th day，14th day and 28th day after relative treatment respectively，and lung tissue samples were harvested for histopathology study.HE and Masson staining were used to determine the extent of alveolus inflammation and pulmonary fibrosis respectively.Histoimmunochemical method were adapted to determine protein levels of TGF-β1，CTGF，type Ⅰcollagen and type Ⅲ collagen in pulmonary tissues.Results Histopathological study showed that treatment with either dexamethasone or IFN-γ (both high dose and low dose) remarkably meliorated the extent of alveolus inflammation and suppressed pulmonary fibrosis (compared with model group，all Plt;0.05).Histoimmunochemical study suggested that both dexamethasone and IFN-γ could inhibit the expression of TGF-β1，CTGF，type Ⅰand type Ⅲ collagen (compared with model group，all Plt;0.05)，and the suppression of TGF-β1，type Ⅰand type Ⅲ collagen expression was more obvious in high-dose IFN-γ-treated group than those in low-dose group (Plt;0.05).Conclusions INF-γ possesses apparent anti-fibrosis effect that is similar to dexamethasone but with less side effect.Such effect may resulted from reduced production of type Ⅰand type Ⅲ collagen through expression inhibition of cytokines such as TGF-β1 and CTGF.
ObjectiveTo compare the expressive differences of plasma Kerbs von den lungen-6 (KL-6) in patients with idiopathic interstitial pneumonia (IIP) and connective tissue disease associated secondary interstitial pneumonia (CTD-SIP), and analyze the clinical significances.MethodsThe clinical data and peripheral blood of 399 inpatients with interstitial pneumonia and 50 healthy controls were collected from January 2011 to December 2014 in Nanjing Drum Tower Hospital. The level of plasma KL-6 was measured by chemiluminescence immunoassay method. The subjects were divided into IIP (n=233) group and CTD-SIP (n=166) group, usual interstitial pneumonia (UIP) pattern and non-UIP pattern, and stable (S) UIP group and acute exacerbation (AE) UIP group. Statistical analyses were performed by using IBM SPSS 19.0 (SPSS, Inc., Chicago IL, USA) to compare the differences of plasma KL-6 in groups.ResultsThere were more male subjects (61.8%) in the IIP group, and the average age of (62.3±12.5) years was significantly older (both P<0.01). Plasma KL-6 levels in the IIP ［(1 822.7±1 505.2) U/ml) and the CTD-SIP group ［(1 846.7±1 625.3) U/ml］ were significantly higher than the healthy control group ［(190.2±88.7) U/ml］ (both P<0.001). However, there was no any difference of KL-6, white blood cell count (WBC), lactate dehydrogenase (LDH), C-reactive protein (CRP) and erythrocyte sedimentation rate between the IIP and the CTD-SIP group. The level of plasma KL-6 was positively correlated to WBC, LDH and CRP in the IIP group (r=0.159, P=0.016; r=0.380, P<0.001; r=0.158, P=0.015, respectively); and it was positively correlated to LDH and CRP in the IIP group (r=0.187, P=0.016 and r=0.068, P=0.032) in the CTD-SIP group. There was no significant difference of plasma KL-6 between the UIP and non-UIP subgroups (P>0.05). The difference of plasma KL-6 between the S-UIP and AE-UIP subgroup was significant (P<0.001 and P=0.023). There was no any significant difference of plasma KL-6 among the subgroups with CTD patients (primary Sj?gren’s syndrome, n=90; rheumatoid arthritis, n=20; polymyositis/dermatomyositis, n=26; undifferentiated connective tissue disease, n=10; anti-neutrophil cytoplasmic antibody associated vasculitis, n=15 and systemic sclerosis, n=5) (P=0.785 2).ConclusionsPlasma KL-6 may be a useful biomarker for interstitial pneumonia. It can show the disease activities, but is not able to distinguish IIP from SIP.
Objective To explore the clinical effect and safety of sildenafil combined with bosentan in the treatment of connective tissue disease associated moderate-severe pulmonary arterial hypertension (CTD-MS-PAH ). Methods Seventy-six patients with CTD-MS-PAH during January 2013 to January 2017 were collected and divided into group A (41 cases) and group B (35 cases) using a stratified random sampling approach. The patients in group A received 25 mg sildenafil tablet therapy, three times a day. The patients group B received 25 mg sildenafil and 62.5 mg bosentan tablet therapy, twice a day. Both groups were treated for 12 weeks. Before and after the trial, all patients undertook six-minute walk test. Meanwhile the Borg dyspnea index score, the pulmonary artery systolic pressure (PASP), right ventricular diameter (RVD), B-type natriuretic peptide (BNP), the partial pressure of oxygen in artery (PaO2), blood pressure, heart rate, liver and kidney function were all measured. Results After the therapy, six-minute walking distance increased, Borg dyspnea index score decreased, PASP, RVD and plasma BNP decreased, and PaO2 increased in both groups (all P<0.05), but group B was superior to group A (allP<0.05). There were no significant differences in blood pressure, heart rate, liver or kidney function compared with those before the treatment in both groups (allP>0.05). Conclusion Sildenafil combined with bosentan can significantly decrease the level of pulmonary arterial pressure and effectively improve the cardiopulmonary function in CTD-MS-PAH patients with good safety.
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.