Objective To observe the influence of the transforming growth factor β1(TGF-β1) on the denervated mouse musclederived stem cells（MDSCs） producing the connective tissue growth factor（CTGF）at different time points in vitro. Methods MDSCs from the primarycultureof the denervated mouse skeletal muscle were isolated and purified by the preplate technique, and they were identified before the culture and after the culturein vitro with TGF-β1 (10 ng/ml) for 24 hours. Then, MDSCs were randomlydivided into 6 groups (Groups A, B, C, D, E and F) according to the different time points, and were cultured in vitro with TGF-β1 (10 ng/ml) for 0, 3, 6, 12, 24 and 48 hours, respectively. The levels of CTGF mRNA in MDSCs were measured by the real time RT-PCR and the expression of CTGF protein was detected by the CTGF Western blot. Results The immunohistochemistry revealed that before the adding of TGF-β1, MDSCs highly expressed Sca-1, with a positivityrate of 96%; however, after the adding of TGF-β1, the positive expression of Sca-1 decreased greatly, with a negativity rate gt;99%. The Western blot test showed that the ratios of CTGF to the average absorbance of βactin in Groups A-F were 0.788±0.123, 1.063±0.143, 2.154±0.153, 2.997±0.136, 3.796±0.153 and 3.802±0.175, respectively. In Groups AD，the absorbance increased gradually, with a significant difference between the abovementioned groups (Plt;0.05). However, in Groups D-F, there was no significant difference between the groups as the promotive tendency became less significant (P＞0.05). The RT-PCR test showed that the △Ct values in GroupsA-F were 1.659±0.215, 1.897±0.134, 2.188±0.259, 2.814±0.263,2.903±0.125 and 3.101±0.186, respectively. In Groups A-D, the increase in the △Ct value was gradual, but the differences were significant between the groups (Plt;0.05). But in Groups E and F, the promotive tendency became less significant（Pgt;0.05）. Conclusion TGF-β1 can promote the production of CTGF inthe mouse MDSCs cultured in vitro and the time-dependent relation exists for 3-12 hours.
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 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.
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.
Objective To explore the effect of connective tissue growth factor on the pathogenesis of hypertrophic scar and keloid tissue. Methods The content of hydroxyproline was determined and the expression of connective tissue growth factor gene was detected by the reverse transcription-polymerase chain reaction and image analysis technique in 5 normal skins, 15 hypertrophic scars and 7 keloid tissues. Results The contents of hydroxyproline in the hypertrophic scar(84.10±1.76) and keloid tissue (92.38±2.04) were significantly higher than that of normal skin tissue (26.52 ± 4.10) (P lt; 0.01). The index of connective tissue growth factor mRNA in the hypertrophic scar (0.78 ± 0.63) and keloid tissue (0.84 ± 0.04) were higher than that of normal skin tissue ( 0.09 ± 0.25) (P lt; 0.01). Conclusion Connective tissue growth factor may play an important role in promoting the fibrotic process of hypertrophic scar and keloid tissue.
Objective To improve the knowledge and diagnostic accuracy of combined pulmonary fibrosis and emphysema (CPFE) syndrome in connective tissue diseases (CTD) by summarizing the clinical characteristics of 20 CTD patients with CPFE and reviewing literatures. Methods The medical records of 20 CTD patients with CPFE from January 2011 to June 2015 were retrospectively analyzed. Results There were 11 males and 9 females. The average age was 47 years. Among them, 4 patients were smokers and 15 patients were nonsmokers. The average duration of CTD was 3.5 years with an average onset age of 41 years. Respiratory symptoms were reported in 17 patients and Velcro rale was found in 9 patients; The most common type of CTD disease in these 20 patients was inflammatory myopathy (9 patients, 45%) followed by systemic sclerosis (SSc) (4 patients, 20%). High resolution computerized tomography of lung showed typical radiological features of CPFE containing fibrosis lesions predominantly distributed in the subpleural (14 patients) and basal (18 patients) parts and emphysema mainly located in upper zones. Relatively normal results of lung volume and ventilation function, and markedly reduced carbon monoxide transfer capacity were observed. One patient was confirmed with pulmonary hypertension and 1 patient died from severe inflammation and acute respiratory distress syndrome. Conclusions The CPFE syndrome can be identified in CTD patients as an entity with male predominance, especially among patients with inflammatory myopathy and SSc. Higher risk of secondary pulmonary hypertension and acute lung injury in these patients may increase mortality. Early differentiation of CPFE from pure interstitial lung disease in CTD patients could be helpful in improving prognosis.
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)
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.
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.