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.
Objective To investigate the effect of cryopreservation (CP) on the expression of connective tissue growth factor (CTGF) in the renal tubular epithel ial cells. Methods A total of 40 male Wistar rats (weighing 230-250 g) were used in this study. En bloc removal with in situ cooling both kidneys and hypertonic citrate adenine preservation solution were adopted. The rat kidney was be preserved 0, 12, 24, 36 and 48 hours at 0-4℃ (n=8), respectively. The expression of CTGF of renal tubularepithel ial cells was detected by using immunohistochemistry and in situ hybridization analysis. Results The expression of CTGF was less in CP 0 hour group and CP 12 hours group, the positive unit (PU) values of CTGF protein were 5.91 ± 2.30 and 5.57 ± 2.40 (P gt; 0.05), respectively, and the PU values of CTGF mRNA were 6.24 ± 2.79 and 6.51 ± 2.43 (P gt; 0.05), respectively. The PU values of CTGF protein increased at CP 24 hours group (10.25 ± 2.92), CP 36 hours group (14.31 ± 2.83) and CP 48 hours group (18.11 ± 3.94, P lt; 0.05), respectively, and the PU values of CTGF mRNA increased at CP 24 hours group (15.24 ± 3.95), CP 36 hours group (19.20 ± 4.73) and CP 48 hours group (23.09 ± 4.40, P lt; 0.05), respectively; showing significant differences when compared with CP 0 hour group and CP 12 hours group (P lt; 0.05). Conclusion CTGF expression may increase with severe cold ischemia injury, and might play an important role in regeneration and repair of renal tubular epithel ial cell injury.
The aim of this research is to evaluate the effect of tetramethylpyrazine (TMP) and connective tissue growth factor (CTGF) miRNA plasmids on the expressive levels of CTGF, transforming growth factor-beta (TGF-beta) and type Ⅰ collagen of rat hepatic stellate cells (HSC) which are stimulated by high glucose. The rat HSCs which were successfully transfected rat CTGF miRNA plasmids and the rat HSCs which were successfully transfected negative plasmids were cultured in vitro. After stimulus of the TMP and the high glucose, the protein levels and gene expressive levels of CTGF, TGF-beta and type Ⅰ collagen were tested. The results indicated that high glucose increased the expression of CTGF mRNA, CTGF protein, TGF-beta mRNA,TGF-beta protein and type Ⅰ collagen (P<0.05). The expressive levels of CTGF mRNA, CTGF protein, TGF-beta mRNA, TGF-beta and type Ⅰ collagen in TMP group were lower than those in high glucose group and showed statistically significant differences (P0.05). Compared with high glucose group, the expressive levels of CTGF mRNA, CTGF protein, TGF-beta mRNA, TGF-beta and type Ⅰ collagen in rat CTGF miRNA plasmid interference group were significantly lower (P<0.05). However, no statistically significant difference was found in CTGF mRNA and CTGF protein levels between TMP group and CTGF miRNA group (P>0.05), while type Ⅰ collagen levels showed statistically significant differences (P<0.05). It is concluded that high glucose could promote the expressions of CTGF, TGF-beta and type Ⅰ collagen, and TMP and rat CTGF miRNA plasmids could reduce the expressions of CTGF, TGF-beta, type Ⅰ collagen.
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 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.
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 explore the clinical characteristics of patients with connective tissue disease with positive anti-small ubiquitin-like modifier activating enzyme (SAE) antibodies. MethodsRetrospectively select the patients who completed the screening of myositis autoantibodies in West China Hospital of Sichuan University between January 1, 2015 and May 30, 2021. Meanwhile, patients with positive anti-SAE antibodies were screened out. According to the clinical data of anti-SAE antibodies positive patients, they were divided into the following groups: tumor group and non-tumor group, ILD group and non-ILD group, inflammatory myopathy group and non-inflammatory myopathy group. Clinical symptoms, signs, laboratory examinations, imaging examinations and other clinical data of the above patients were collected. Results A total of 1 594 patients were screened for myositis autoantibodies, of which 56 were positive for anti-SAE antibodies, with a positive rate of 3.5%. In 56 patients, 32.1% in skin involvement, 35.7% in muscle involvement, 12.5% in joint involvement, 5.4% in dysphagia, 5.4% in weight loss, 58.9% in patients with interstitial lung disease (ILD) and 12.5% in patients with tumor history. There was no significant difference in age, sex, skin involvement, muscle involvement, joint involvement and respiratory system involvement between the tumor group and the non-tumor group (P>0.05). Except for age, the frequency of muscle involvement, and positive rate of anti-Ro-52 antibody, there was no significant difference in other indicators between the ILD group and the non-ILD group (P>0.05). Except for the positive rate of ILD, the frequency of skin involvement, the frequency of muscle involvement, the level of creatine kinase and hydroxybutyrate dehydrogenase (P<0.05), there was no significant difference in other indexes between the non-inflammatory myopathy group and the inflammatory myopathy group (P>0.05). Conclusions The patients with positive anti-SAE antibodies mainly present skin and muscle symptoms, and are prone to ILD, malignant tumor and dysphagia. Patients with positive anti-SAE antibodies and ILD were older, had less muscle damage, and had a higher positive rate of anti-Ro-52 antibody. Anti-SAE antibodies appear not only in patients with inflammatory myopathy, but also in non-inflammatory myopathy, often associated with a higher frequency of ILD and less muscle involvement.
ObjectiveTo explore the therapeutic effects of spleen aminopeptide on connective tissue disease-related interstitial lung disease (CTD-ILD) and its mechanism for anti-fibrosis. MethodsNinety patients with CTD-ILD admitted between February 2014 and May 2015 were recruited in the study. The CTD-ILD patients were randomly divided into group A (conventional therapy alone) and group B (conventional therapy plus spleen aminopeptide). Peripheral blood collected from CTD-ILD patients were subjected to performance of flow cytometric analysis and cytokine/chemokines profiling by liquid Chip and ELISA assay. Pulmonary function test and high resolution CT (HRCT) scan were performed before and after the treatments for 12 weeks. Human cytomegalovirus (HCMV) DNA in the patients' blood was tested by Q-PCR. ResultsSignificantly improved lung function and HRCT score were observed in group B, but not in group A. The levels of Treg and IFN-γ were significantly increased in group B, compared with those in group A where markedly increased IL-6, IL-10 and IL-17 were detected (P < 0.05). There was higher virus negative reversal rate in group B than that in group A (P < 0.05). ConclusionSpleen aminopeptid can effectively regulate deregulated immune microenvironment in CTD-ILD patients and inhibit HCMV replication, thereby block pulmonary fibrotic development.
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.
Objective To investigate the delay of the denervated skeletal muscle atrophy with the method of restraining the increment of the connective tissues by tetrandrine and hormone. Methods The left hind limbs of 42 male adult SD rats were made into models of the denervated gastrocnemius, and then the rats were randomly divided into 3 groups, with 14 rats in each. In Group A, tetrandrine (8 mg/L）was injected into the denervated gastrocnemius; in Group B, triamcinolone acetonide（1.6 g/L） was injected; in Group C (the control group),normal saline was injected. Enough samples were obtained according to the different observation indexes at 30 days after operation. Electromyography, muscle wet weight measurement, light microscopy,electron microscopy,and microimage analysis were performed. ResultsThe fibrillation potential amplitude was 0.195 8±0.041 9 μV in Group A and 0.185 2±0.050 3 μV in Group B, and there was no significant difference betweenthe two groups (Pgt;0.05). However,in Group C the fibrillation potential amplitude was 0.137 7±0.058 9μV. The fibrillation potential amplitude was significantly greater in Group A than in Group C（Plt;0.05）. The muscle wet weight was 1.740 0±0.415 9 g in Group A and 1.940 1±0.389 4 gin Group B, and there was no significant difference between the two groups(Pgt;0.05).However, in Group C the muscle wet weight was 0.800 0±0.100 0 g. The muscle wet weight was significantly greater in Group A than in Group C（Plt;0.05）.The microscopy showed that more remarkable atrophy occurred in the control group. The muscle fibers were more complete, thicker and larger, with more nuclei and clearer cross-lines. More connective tissue and flat cells could be observed in Groups A and B. The myogenic protein amount was 440.124 2±46.135 6 in Group A and 476.211 4±41.668 8in Group B, and there was no significant difference between the two groups(Pgt;0.05).However, in Group C the amount was 380.040 0±86.315 9.The myogenic protein amount was significantly greater in Group A thanin Group C（Plt;0.05）. The muscle fiber number, diameter, cross section, and connective tissue increment were all significantly greater in Group A than in Group C（Plt;0.05）; however, there wasno significant difference between Groups A and B (Pgt;0.05). The electron microscopy showed that there were more degeneration changes, such as muscle silk disorder, chondriosome disappearance, and hepatin reduction, could be observed inGroup C than in Groups A and B. Conclusion Tetrandrine and hormone can delay the denervated skeletal muscle atrophy by restraining the increment of the connective tissues.