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 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.
ObjectiveTo systematically review the efficacy of acellular dermal matrix (ADM) and subepithelial connective tissue flap (sCTG) on patients with gingival recession (GR).MethodsPubMed, EMbase, The Cochrane Library, CNKI, WanFang Data and VIP databases were electronically searched to collect randomized controlled trials (RCTs) about the efficacy of ADM and sCTG on patients with GR from inception to August 11st, 2019. Two reviewers indepeudently screened literature, extracted data and assessed the risk of bias of included studies, and then meta-analysis was performed by using RevMan 5.3 software and Stata 12.1 software.ResultsA total of 9 RCTs were included. The results of meta-analysis showed that: there were no significant differences in probing depth (PD) (MD3m=−0.04, 95%CI −0.18 to 0.11, P=0.63; MD6m=−0.01, 95%CI −0.13 to 0.12, P=0.90) and GR degree (MD3m=−0.10, 95%CI −0.37 to 0.18, P=0.48; MD6m=−0.02, 95%CI −0.33 to 0.29, P=0.89) in 3 and 6 months after operative between two groups. But the clinical attachment loss (CAL) in 3 months after operation (MD=0.33, 95%CI 0.00 to 0.66, P=0.05) and width of keratinized tissue (KTW) in 6 months after operation (MD=−0.48, 95%CI −0.76 to −0.20, P=0.000 7) of sCTG group were superior to ADM group, the differences were statistically significant.ConclusionCurrent evidence shows that there are no differences in PD and GR degree in 3 months and 6 months after operation between ADM and sCTG group. But the CAL in 3 months after operation and KTW in 6 months after operation of sCTG group is superior to ADM group. Due to limited quality and quantity of the included studies, more high-quality studies are needed to verify above conclusion.
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 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)
Objective To investigate the effects of matrine on cell proliferation and expression of connective tissue growth factor( CTGF) and hypoxia inducible factor-1α( HIF-1α) of human lung fibroblast ( WRC-5) in normoxia ( 21% O2, 74% N2 , 5% CO2 ) and hypoxia ( 1% O2, 94% N2 , 5% CO2 )conditions. Methods MRC-5 cells were cultured and divided into differrent groups interfered with different dose of Matrine ( final concentration of 0 ～3. 2 mmol / L) in normoxia or hypoxia for 24 h. Cells were dividedinto 8 groups according to culture conditions, ie. normoxiagroup( N0 group) , normoxia + matrine 0. 2 mmol / L group( N0. 2 group) , normoxia + matrine 0. 4 mmol / L group( N0. 4 group) , normoxia + matrine 0. 8 mmol / L group( N0. 8 group) , hypoxia group( H0 group) , hypoxia + matrine 0. 2 mmol /L group( H0. 2 group) , hypoxia +matrine 0. 4 mmol /L group( H0. 4 group) , and hypoxia + matrine 0. 8 mmol / L group( H0. 8 group) . The MTT assay was used to measure the cell proliferation activity. Western-blot assay was used to examine the expression of CTGF and HIF-1α. Results Hypoxia promoted the cell proliferation in all groups( P lt;0. 05) .Matrine inhibited the proliferation of WRC-5 cells in a concentration-dependent manner in hypoxia or normoxia conditions( P lt;0. 05) . The expression of CTGF andHIF-1αwas lower in normoxia and higher in hypoxia( P lt;0. 01) . Matrine inhibited the expression of CTGF and HIF-1αin a concentration-dependent manner in hypoxiaand normoxia( P lt;0. 05) . Conclusion Matrine can inhibit the cell proliferation and the expression of CTGF and HIF-1αof WRC-5 cells in normoxia and hypoxia in a concentration-dependent manner.
ObjectiveTo compare the half-year clinical efficacy of three different surgical root coverage methods including lateral sliding flap (LSF), subepithelial connective tissue graft (CTG) and acellular dermal matrix allograft (ADM). MethodsEighteen patients (24 teeth) with gingival recession treated in our hospital between December 2012 and July 2015 were selected and divided into three groups according to a certain sequence with 8 teeth in each group. The three groups of teeth were treated with LSF, CTG and ADM respectively. Gingival recession, probing depth and keratinized gingival width at both baseline and 6 months after surgery of all patients were recorded for inner- and inter-group comparison. ResultsAll three methods proved to be effective within 6 months with an awerage of 2.8-4.0 mm in decrease extent of gingival recession (P<0.01). LSF did not significantly change the probing depth (P>0.05) as the other two did (P<0.01). The differences among three surgical methods compared before and after surgery were all significant (P<0.05). ConclusionLSF, CTG and ADM are all effective surgical means for root coverage. Within 6 months, CTG presents better effects than ADM, and ADM better than LSF.
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 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.