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Objective:To evaluate the efficacy of recombinant humanized type Ⅲ collagen injected by microneedles in facial rejuvenation.Methods:Twenty-five cases recruited from July to December 2021 were included as the research objects in this study. Recombinant humanized type Ⅲ collagen was injected into the face skin using microneedles. Images were collected before the treatment and 1, 2, and 3 months after the end of the treatment. The VISIA image analysis system was used for comparative analysis. Patient′s satisfaction evaluation and the occurrence of complications after treatment were also recorded.Results:According to results of VISIA, the scores of spots were (32.06±6.92) and (27.59±7.69); red areas were (32.79±11.80) and (27.74±9.49); pores were (17.66±9.79) and (14.60±7.07) and brown spots were (43.61±11.93) and (37.59±16.22) after the treatment were lower than those before treatment, and the results were statistically different ( F=3.442, 4.209, 7.473, 7.113, P<0.05). One month after the end of the of treatment. Patient′s self-satisfaction reached 84% (21 cases); three months after the end of the treatment, the patient′s self-satisfaction reached 80% (20 cases). No serious adverse reactions were occurred. Conclusions:The recombinant humanized type Ⅲ collagen injected by microneedles can be effective and durable for facial rejuvenation and get high patient satisfaction. It has high safety and is worthy of clinical promotion.
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In this paper, 2 cases of collagen Type Ⅲ glomerulopathy were analyzed. The clinical manifestations mainly included nephrotic syndrome, proteinuria, hypertension and renal dysfunction. One patient showed that the complement factor H-related protein 5 (CFHR5) gene was likely a disease-causing mutation. The pathological examination of renal tissues showed hyperplasia of mesangial matrix, sub-endothelial insertion, and double-track formation. Immunohistochemistry of Type III collagen was positive. Electron microscopy revealed that massive collagen fibers (40-70 nm in diameter) deposited in the mesangial matrix and basement membrane. As for the follow-up results, the normal renal function had kept steady and the proteinuria was moderate in 1 case treated with angiotensin Ⅱ receptor blocker. Due to other system disease, another case developed into acute kidney injury and then received hemodialysis. The clinical manifestations of collagen Type Ⅲ glomerulopathy was atypical, the light microscope pathological features were various, and the disease was mainly diagnosed by electron microscopy and immunohistochemistry.
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Humans , Collagen Type III , Genetics , Glomerular Mesangium , Kidney Diseases , Kidney Glomerulus , ProteinuriaABSTRACT
Objective: To investigate the effect of echinococcus granulosus protoscolices on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into fibroblasts. Methods: Femur bone marrow of 4-week-old C57BL/6 mice was taken and BMSCs were isolated and cultured by adherent culture. Echinococcus granulosus protoscolices was extracted from the liver of sheep infected with echinococcus granulosus. The experiment was divided into two groups. The experimental group was co-cultured with the 3rd generation BMSCs and the echinococcus granulosus protoscolices, and the control group was the 3rd generation BMSCs. Before and after co-culture, the morphology of BMSCs and the activity of echinococcus granulosus protoscolices were observed by inverted microscope. After cultured for 1, 3, 5, and 7 days, the mRNA expressions of transforming growth factor β 1 (TGF-β 1), collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescent quantitative PCR, the protein expressions of TGF-β 1, collagen type Ⅰ, collagen type Ⅲ, Smad7, and phosphorylated Smad2/3 were detected by Western blot, and the contents of collagen type Ⅰ and collagen type Ⅲ in the supernatant of the two groups were detected by ELISA. Results: After 7 days of co-culture, the morphology of BMSCs changed into fusiform and irregular triangle, which was closer to the mouse fibroblasts. The relative mRNA expressions of TGF-β 1, collagen type Ⅰ, and collagen type Ⅲ in the experimental group were significantly higher than those in the control group; the relative protein expressions of TGF-β 1, collagen type Ⅰ, collagen type Ⅲ, and phosphorylated Smad2/3 in the experimental group were significantly higher than those in the control group, and the relative protein expression of Smad7 in the experimental group was significantly lower than that in the control group; the contents of collagen type Ⅰ and collagen type Ⅲ in the supernatant of the experimental group were significantly higher than those in the control group. The differences between the two groups were significant ( P<0.05). Conclusion: Echinococcus granulosus protoscolices may promote the secretion of collagen type Ⅰ, collagen type Ⅲ, and TGF-β 1 by TGF-β 1/Smad signal pathway, which can promote the fibrosis of BMSCs that related to the formation of fibrocystic wall by echinococcosis.
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Objective:To investigate the effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction (UUO) and to observe the effect of fluorofenidone on the expressions of collagen type Ⅰ (Col Ⅰ),collagen type Ⅲ (Col Ⅲ),α-smooth muscle actin (α-SMA),connective tissue growth factor (CTGF),platelet derived growth factor (PDGF) in the renal tissues of UUO rats.Methods:Male Sprague-Dawley (SD) rats were randomly divided into a sham-operated group,a UUO group,and a flurofenidone group (n=5).UUO model was induced by ligating the left ureter in rats.The rats were treated with 125 mg/(kg.d) fluorofenidone by gastric gavage in the fluorofenidone group at 24 h before the operation,and the rats were treated with the identical dose of 0.5％ sodium carboxyl methyl cellulose (CMC-Na) in the other 2 groups.The rats were sacrificed at 14 days after UUO.Pathological changes of the renal tissue were observed by HE and Masson staining,the mRNA expressions of Col Ⅰ,Col Ⅲ,α-SMA,PDGF and CTGF were detected by real-time PCR,and the protein expressions of Col Ⅰ,Col Ⅲ,PDGF and CTGF were detected by immunohistochemical staining.Results:The renal interstitial damage index,relative collagen area and mRNA and protein expressions of Col Ⅰ and Col Ⅲ in the renal tissues of the rats in the UUO group significantly increased (P＜0.05),and fluorofenidone could reduce these indexes (P＜0.05).Compared with the sham-operated group,the protein expressions ofα-SMA,PDGF,CTGF and the mRNA expressions of PDGF and CTGF in the renal tissues of the rats in the UUO group were increased,but fluorofenidone could decrease the protein expressions of α-SMA,PDGF,CTGF and the mRNA expressions of PDGF and CTGF (P＜0.05).Conclusion:Fluorofenidone (125 mg/kg·d) could attenuate renal interstitial fibrosis through inhibition offibroblast proliferation,myofibroblastic activation,PDGF and CTGF expression.
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Objective@#To investigate the effects of denatured collagen type Ⅰ on differentiation of human fibroblasts into myofibroblasts.@*Methods@#A small amount of normal skin donated by burn patients undergoing scar surgery was collected. Human fibroblasts were obtained by method of explant culture and then sub-cultured. The fourth passage of cells were used in the following experiments. (1) Fibroblasts were divided into normal collagen group and denatured collagen group according to the random number table, with 10 wells in each group. Fibroblasts in normal collagen group were cultured on normal collagen type Ⅰ coated coverslips. Fibroblasts in denatured collagen group were cultured on denatured type Ⅰ collagen coated coverslips. Expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical method, and the percentage of PCNA positive cells was calculated. (2) Another batch of fibroblasts were grouped and treated as in (1), with 12 wells in each group. Proliferation activity of cells was determined with methyl-thiazolyl-tetrazolium colorimetry method. (3) Another batch of fibroblasts were grouped and treated as in (1), and the microfilament morphology of cells was observed by rhodamine-phalloidin staining. (4) Another batch of fibroblasts were grouped and treated as in (1). Expression of α smooth muscle actin (α-SMA) of cells was detected by immunohistochemical method, and expression of OB-cadherin of cells was detected by immunofluorescence method. (5) Another batch of fibroblasts were divided into normal collagen, denatured collagen, and common coverslips groups according to the random number table, with 6 wells in each group. Fibroblasts in normal collagen and denatured collagen groups were treated as in (1), while fibroblasts in common coverslips group were cultured on coverslips without collagen coating. Expressions of α-SMA and OB-cadherin of cells were determined with Western blotting. (6) Another batch of fibroblasts were grouped and treated as in (5), and then the mRNA expressions of collagen type Ⅰ, collagen type Ⅲ, and α-SMA of cells were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with t test, one way analysis of variance, and least-significant difference test.@*Results@#(1) The percentage of PCNA positive cells in denatured collagen group was (83±9)%, significantly higher than (29±9)% in normal collagen group (t=13.53, P<0.01). (2) The proliferation activity of fibroblasts in denatured collagen group was 0.32±0.06, significantly higher than 0.25±0.05 in normal collagen group (t=3.06, P<0.01). (3) The microfilament of fibroblasts in normal collagen group was arranged vertically and in parallel way, paralleling the long axis of cells. The microfilament of fibroblasts in denatured collagen group was denser and thicker. (4) Most fibroblasts in normal collagen group showed long shuttle-like shape typically. Morphology of fibroblasts in denatured collagen group changed, and cells were obviously spreading. Expressions of α-SMA and OB-cadherin of fibroblasts in denatured collagen group were stronger than those in normal collagen group. (5) Expressions of α-SMA of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 1.69±0.41, 0.89±0.27, and 1.46±0.42. Expression of α-SMA of fibroblasts in denatured collagen group was significantly higher than that in normal collagen group (P<0.01). Expressions of OB-cadherin of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 5.17±0.28, 2.21±0.10, and 4.01±0.56. Expression of OB-cadherin of fibroblasts in denatured group was significantly higher than that in normal collagen group (P<0.01). (6) There was no significant difference in mRNA expression of collagen type Ⅰ of fibroblasts in denatured collagen, normal collagen, and common coverslips groups (F=2.71, P>0.05). The mRNA expressions of collagen type Ⅲ and α-SMA of fibroblasts in normal collagen group were significantly lower than those in denatured collagen group (P<0.01).@*Conclusions@#Denatured collagen type Ⅰ may influence the activity of fibroblasts, thus inducing fibroblasts differentiating into myofibroblasts.
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Objective:To investigate the mechanism of promoting wound healing of Shengji Huayu ointment. Methods: Db/db mice were divided into 5 groups according to the randomized block design: three Shengji Huayu ointment groups respectively at high (15 g·kg-1),middle (10 g·kg-1) and low(5 g·kg-1) dose,the positive control group(Beifuji) and the model control group, and balb/c mice were used in the normal control group,and each group was with 10 mice. The model of full-thickness skin defect in mouse skin was used,and after HE staining,IL-1β and collagen type I and type III in granulation tissue were observed by immunohis-tochemistry and image analysis. Results:HE staining showed that the wound healing was obvious in the high dose group and the mid-dle dose group. The expression of IL-1 β in each group was not obvious. The levels of collagen type I and type III in the three Shengji Huayu ointment groups were higher than those in the model control group (P<0.05).Conclusion: Shengji Huayu ointment can pro-mote wound healing and reduce scar formation in mice.
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Objective :To explore serum level of amino-terminal propepitide of type Ⅲ procollagen (PⅢNP) in pa-tients with arrhythmia and its correlation with other clinical indexes .Methods :A total of 135 patients with arrhyth-mia ,who were treated in our department from Apr 2014 to Apr 2017 ,were selected as arrhythmia group .Another 89 healthy subjects undergoing physical examination simultaneously were enrolled as healthy control group .Accord-ing to NYHA cardiac function classification ,arrhythmia group was further divided into class I group (n＝37) ,classⅡ group (n＝39) and class Ⅲ ～ Ⅳ group (n＝59).Serum levels of BNP and PⅢNP ,left ventricular end-diastolic di-mension (LVEDd) ,left ventricular end-systolic dimension (LVESd) and LVEF were measured and compared among all groups.Correlation among serum P Ⅲ NP ,BNP levels and cardiac function indexes were analyzed in these pa-tients .Results :Compared with healthy control group ,there were significant rise in serum levels of BNP [ (1132. 88 ± 32.84) μg/L vs .(1984.63 ± 84.61) μg/L] and PⅢ NP [ (26.44 ± 5.89) ng/ml vs.(52.51 ± 10.85) ng/ml] , LVEDd [ (53.38 ± 4.81) mm vs.(62.12 ± 5.35) mm] and LVESd [ (41.23 ± 5.93) mm vs.(53.19 ± 6.86) mm] , and significant reduction in LVEF [(49.85 ± 4.57)% vs.(34.83 ± 4.53)%] in arrhythmia group ,P＝0.001 all.A-long with cardiac function class rose ,there were significant rise in serum levels of BNP [(1242.68 ± 36.71) μg/Lvs. (1481.83 ± 46.09) μg/Lvs.(1938.39 ± 51.94) μg/L] and PⅢNP [ (34.36 ± 5.92) ng/ml vs .(47.81 ± 6.35) ng/ml vs .(60.94 ± 6.74) ng/ml] ,and class I group＜class Ⅱ group＜class Ⅲ ～ Ⅳ group ,P＝0.001 all.Pearson corre-lation analysis indicated that serum P ⅢNP level were significant positively correlated with LVEDd and LVESd ( r＝0.329 ,0.463 ,P＝0.043 ,0.029) ,and significant inversely correlated with LVEF (r＝ －0.351 ,P＝0.036).Con-clusion : The serum PⅢNP level rises in patients with arrhythmia ,it is significantly correlated with ventricular struc-ture and cardiac function indexes .
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Objective To evaluate the effect of miRNA-29 (miR-29) family on the synthesis of collagen Ⅰ and Ⅲ in chronically photodamaged (photoaged) skin.Methods Some cultured human dermal fibroblasts (HDFs) were divided into 2 groups:non-irradiated group receiving no treatment,and chronic photodamage group treated with repetitive ultraviolet A (UVA) radiation,which served as a chronically photodamaged cell model and was verified by flow cytometry and β-galactosidase staining.Western blot analysis was performed to determine the protein expression of collagen Ⅰ and Ⅲ,and real-time fluorescence-based quantitative PCR (qRT-PCR) to measure expression of 3 members of the miR-29 family (miR-29a-3p,miR-29b-3p and miR-29c-3p) in the above 2 groups.The differentially expressed miR-29c-3p between the above 2 groups was chosen for further functional tests.Some HDFs were divided into 4 groups to be transfected with fluorescein-labelled miR-29c-3p mimics (overexpression group),inhibitors (inhibition group),and their control RNA oligonucleotides (negative control group and inhibitor control group) respectively.The transfection efficiency was evaluated by the proportion of fluorescent cells,and the relative expression of miR-29c-3p in the above 4 groups was measured by qRT-PCR for evaluating the RNA interference efficiency,qRT-PCR was conducted to determine the mRNA expression of collagen type Ⅰ α1 (COL1A1) and collagen type Ⅲ α1 (COL3A1) genes,and Western blot analysis to measure the protein expression of collagen Ⅰ and Ⅲ.Results Compared with the non-irradiated group,the chronic photodamage group showed significantly increased proportion of senescent cells (36.47％ ± 3.20％ vs.12.56％ ± 1.46％,P ＜ 0.01) and G1-phase cells (71.70％ ± 2.43％ vs.41.89％ ± 1.86％,P ＜ 0.01),but significantly decreased proportion of S-phase cells (10.63％ ± 0.36％ vs.36.48％ ± 1.31％,P ＜ 0.01),which indicated that the chronically photodamaged cell model was established successfully.The protein expression of collagen Ⅰ and Ⅲ was significantly lower in the chronic photodamage group (0.40 ± 0.19 and 0.52 ± 0.10) than in the non-irradiated group (1.00 ± 0.12 and 1.00 ± 0.10,respectively,both P ＜ 0.01).The expression of miR-29c-3p was significantly higher in the chronic photodamage group than in the non-irradiated group (4.42 ± 2.05 vs.0.89± 0.10,P ＜ 0.05),while there were no significant differences in the expression of miR-29a-3p or miR-29b-3p between the 2 groups (both P ＞ 0.05).Twenty-four hours after transfection,the overexpression group and inhibition group both showed more than 90％ transfection efficiency which met the interference requirements.The expression of miR-29c-3p was significantly higher in the overexpression group than in the negative control group (224.17 ± 2.00 vs.2.45 ± 0.34,P ＜ 0.01),but significantly lower in the inhibition group than in the inhibitor control group (0.20 ± 0.08 vs.2.24± 0.14,P ＜ 0.01),suggesting that a RNA interference model was successfully established.The mRNA expression of COL1A1 and COL3A1 and the protein expression of collagen Ⅰ and Ⅲ were significantly lower in the overexpression group than in the negative control group and inhibition group (all P ＜ 0.05),and significantly higher in the inhibition group than in the inhibitor control group (all P ＜ 0.01).Conclusion The expression of miR-29c-3p is up-regulated in chronically photodamaged HDFs,likely by regulating the mRNA expression of COL1A1 and COL3A1 and the protein expression of collagen Ⅰ and Ⅲ.
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Objective To investigate the effects of different wavelength (595 nm,755 nm and 1064 nm) lasers on the mRNA expression of types Ⅰ and Ⅲ procollagen in cultured fibroblasts.Methods Fibroblasts from Kunming mice were cultured in vitro.They were divided into 595 nm laser group,755 nm laser group,1064 nm laser group and no laser irradiating group.The mRNA expression of the types Ⅰ and Ⅲ procollagen was detected by RT-PCR.Results The mRNA expression level of type Ⅰ procollagen in 1064 nm group was higher than that of 755 nm group,595 nm group and control group (P＜0.05).The expression levels of 755 nm group and 595 nm group were higher than that of control group (P＜0.05).But the difference between 755 nm group and 595 nm group was not statistically significant (P＞0.05).The mRNA expression level of type Ⅲ procollagen in 1064 nm group was higher than that in 755 nm group,595 nm group and control group (P＜0.05).755 nm group had higher expression than 595 nm group and control group (P＜0.05).But the difference between 595 nm group and the control group was not statistically significant (P ＞ 0.05).Conclusions Three wavelength lasers can directly promote mRNA expression of types Ⅰ and Ⅲ procollagen in fibroblasts.595 nm laser mainly promotes mRNA expression of type Ⅰ procollagen,and 755 nm laser promotes more mRNA expression of type Ⅲ procollagen than 595 nm laser.The most mRNA expression of types Ⅰ and Ⅲ procollagen is promoted by 1064 nm laser.
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Objective To investigate the effects and mechanisms of bone marrow mesenchymal stem cells (BMSCs) on pancreatic fibrosis in rats of chronic pancreatitis. Methods Thirty healthy male SD rats were randomly divided into three groups:control group, model group and transplanted group (n=10 for each group). Chronic pancratitis rat model was induced by retrograde injection of oleic acid into the biliopancreatic duct. The sham operation group was treated only with solvent. Transplanted group was given BMSCs through caudal vein injection at 1 week and 5 weeks after the model induction. All rats were weighed at 1 week, 4 weeks and 8 weeks in three groups. After 8-week feeding,pancreatic tissues were harvested for HE and picric-sirius staining. The contents of transforming growth factorβ1 (TGF-β1), typeⅠcollagen and typeⅢcollagen were detcted by using ELISA. Results Compared with the control group, the weights of rats were decreased at 4 weeks and 8 weeks in model group and transplantated group (P0.05). The pancreatic fibrosis score and pathological injury were ameliorated signicantly in transplanted group. The contents of TGF-β1, typeⅠcollagen and typeⅢcollagen in pancreas were increased in model group than those of control group and transplanted group (P<0.05). Conclusion BMSCs transplantation can reduce the collagen secretion and reduce the degree of pancreatic fibrosis in rats with chronic pancreatitis, which may be related to the inhibition of the release of TGF-β1.
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Objective To study the effect of paeonol (PAE) and panax notoginseny saponins (PNS) on the expressions of collagenⅠandⅢprotein and mRNA in rats with acute myocardial infarction (AMI), and to explore the molecular mecha?nism of improving myocardial fibrosis. Methods The rat model of AMI was made using the left anterior descending coro?nary branch ligation.According to the intervention rats were divided into model group, PAE (8 mg/kg) group, PNS (40 mg/kg) group, PAE (4 mg/kg)+PNS (20 mg/kg) group, PAE (8 mg/kg)+PNS (40 mg/kg) group and captopril positive control group (10 mg/kg). Sham operation group, only wear line without ligation. The left ventricular mass index (LVMI) was detected after treatment for 28 d. Masson staining was used to observe changes of myocardial fibrosis. Western blot assay and RT-PCR technique were used to detect protein and mRNA expression levels of collagenⅠandⅢ. Results The values of LVMI were increased in model group compared with those of sham operation group and treatment groups. Compared with PAE group and PNS group, values of LVMI were significantly decreased in PAE (4 mg/kg)+PNS (20 mg/kg) group and PAE (8 mg/kg)+PNS (40 mg/kg) group. There was a more significant decrease in formula high dose group (P < 0.01). The model group showed pathological change. There were different degrees of improvement in pathological structure in all treatment groups, more sig?nificant improvement was found in formula low dose group, formula high dose group and captopril positive control group. There were different degrees of increase in expressions of collagenⅠandⅢprotein and mRNA in model group compared with those of sham operation group and treatment groups. Compared with PAE group and PNS group, the protein and mRNA expression levels of collagenⅠandⅢwere significantly decreased in formula low dose group and formula high dose group,more significant decreased was found in formula high dose group (P<0.05). Conclusion Compound of paeonol and PNS can improve myocardial fibrosis in myocardial infarction rats, which may be related with reduced expressions of collagenⅠandⅢprotein and mRNA.
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Objective To observe the anti-fibrosis therapeutic effect and mechanism of Qingshen granule for treatment of patients with chronic renal failure (CRF) accompanied by damp-heat syndrome.Methods Sixty-eight patients with CRF accompanied by damp-heat syndrome were randomly divided into a control group and a observation group, and the study was completed only in 61 patients, 31 in the control group and 30 in the observation group. Thirty subjects having taken physical health examination were assigned in a healthy control group. All the patients in both treatment groups were treated with conventional western medical therapy and traditional Chinese medicine (TCM) retention enema, and for patients in observation group, Qingshen granule was given additionally, 1 bag (10 g) thrice a day taken orally. The therapeutic course was 8 weeks. The clinical therapeutic effect, the levels of serum creatinine (SCr), the glomerular filtration rate (eGFR), serum interleukin-17 (IL-17), collagen type Ⅲ (Col-Ⅲ) and nuclear factor-κB p65 (NF-κB p65) in peripheral blood mononuclear cells (PBMC) were measured before and after treatment in the two treatment groups, and the above results were compared with those in healthy control group.Results Clinically, the total effective rates of the disease and of the TCM syndrome in observation group were significantly higher than those in the control group (86.67% vs. 58.06%, 83.33% vs. 45.16%, bothP < 0.01). In the observation group, the level of SCr was obviously lower, and the level of eGFR was markedly higher after treatment, and compared with the control group, the changes in above data after treatment in observation group were more significant [SCr (μmol/L): 250.62±164.97 vs. 393.72±183.64, eGFR (mL·min-1·1.73 m-2): 33.42±17.24 vs. 39.72±23.85, bothP < 0.05]. After treatment, the levels of serum IL-17, Col-Ⅲ and NF-κB p65 in PBMC were obviously lowered in both treatment groups compared with those before treatment, the therapeutic effect in observation group being superior to that in the control group [IL-17 (ng/L): 17.47±8.87 vs. 25.51±16.69, Col-Ⅲ (μg/L): 17.06±8.76 vs. 23.77±10.44, NF-κB p65 (μg/L): 0.58±0.34 vs. 0.83±0.30, allP < 0.05].Conclusion The Qingshen granule can ameliorate the clinical symptoms, improve renal function, decrease the levels of serum IL-17, Col-Ⅲ and NF-κB p65 in PBMC, intervene renal fibrosis in patients with CRF and damp-heat syndrome, ultimately delaying the progress of CRF.
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Objective:To explore the effect of bone marrow mesenchymal stem cell transplantation on silicosis fibrosis in different time windows in rats.Methods:BMSCs were isolated and cultured from male 3-week-old SD rats in vitro.Fifty healthy female SD rats were randomly divided into 5 groups:control group,silicosis model group,early treatment group,middle treatment group,late treatment group(n=10).The silicosis model was made by one-time infusion of silica dust suspension using the non-exposed tracheal in-tubation(50 μg/ml),and 1 d,14 d,28 d of BMSCs were given for intervention therapy (1 ×106 ml-1 ).Rats in each group were sacrificed 14 days after treatment.The BMSCs identified by flow cytometry;the morphological changes of the lung tissues were observed by HE staining;the expression of MMP-9,collagen type Ⅰ and collagen type Ⅲ were detected by immunocytochemistry and Western blot analysis.Results: BMSCs in early silicosis ( 1 d ) and the middle silicosis ( 14 d ) compared to silicosis model group can significantly alleviate the pathological process of silicosis fibrosis (P0.05).Conclusion:Exogenous BMSCs transplantation on rat silicosis early pathological processes play a role in delaying , late treatment effect is not obvious.
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Background Extracellular matrix (ECM) fibrosis leads to corneal scaring during the process of cornea wound healing.Transforming growth factor-β1 (TGF-β1) is known to mediate overproduce of ECM components.Our previous study developed a three-dimensional model for corneal stromal cells culture in vitro.Objective The hypothesis of this study was to apply TGF-β1 in the three-dimensional culture system to establish a corneal stroma ECM fibrosis model.Methods Fresh bovine corneas were extracted for the culture of bovine keratocytes in constructed three-dimension culture system.The Pellets were cultured in the DMEM/F12+ 10％ fetal bovine serum (FBS) medium with 0.5 ng/ml or 1.0 ng/ml TGF-β1 or without TGF-β1,respectively.Calcein AM/(propidium iodide) PI staining was employed to assay the cell viability 2 weeks after culture.The expressions of α-smooth muscle actin (α-SMA),type Ⅰ collagen (Col Ⅰ) and Col Ⅲ mRNA and protein in the cells were detected by real-time PCR and Western blot respectively 48 hours,1 week and 2 weeks after cultured.The results were statistically analyzed.Results Cultured for 48 hours in the Pellet system,corneal stromal cells clustered and was identified alive by Calcein-AM/PI staining in 2 weeks.The relative expression levels of α-SMA,Col Ⅰ and Col m mRNA were elevated in both the 0.5 ng/ml and 1.0 ng/ml TGF-β1 supplement groups in comparison with the only DMEM/F12+10％ FBS group,with marked difference among the three groups (Fgroup =696.745,P＜0.001;Fgroup =35.166,P＜0.001;Fgroup =33.677,P＜0.001),and the expression levels increased with the lapse of culture time (Ftime =5.863,P＜0.05;Ftime =298.614,P＜0.001;Ftime =607.472,P＜0.001).The synthetic rate of Col Ⅲ mRNA was obviously faster than that of Col Ⅰ mRNA.Western blot showed that only a trace of α-SMA,Col Ⅰ and Col Ⅲ were detected 48 hours and 1 week after culture.The expression levels of α-SMA,Col Ⅰ and Col Ⅲ in Pellet system in 0.5 ng/ml TGF-β1 medium were 0.395±0.208,1.060±0.175 and 0.629±0.382,and in 1.0 ng/ml TGF-β1 medium were 0.758±0.228,1.201 ±0.187 and 0.753±0.468,respectively 2 weeks after culture,significant differences were shown among the three groups (α-SMA:F=10.691,P＜0.05;Col Ⅰ:F=14.094,P＜0.05;Col Ⅲ:F=10.995,P＜0.05).Conclusions Addition of TGF-β1 and serum enhance the assembly and fibrosis of ECM,showing the higher expressions of specific fibrotic markers in bovine keratocytes Pellet.This culture systerm can be used as a candidate three-dimensional model for corneal stroma ECM fibrosis.
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Objective To investigate the expressions of advanced glycosylation end products (AGE) in skin of mice with diabetes mellitus (DM) for different durations,and to evaluate their influence on collagen fibers.Methods Forty healthy 8-week-old male C57BL/6J mice were divided into DM group (n =20) and control group (n =20) to receive multiple intraperitoneal injections of low dose streptozotocin (50 mg/kg) and citric acid buffer (0.1 mol/L),respectively,for 5 consecutive days.Ten mice were sacrificed in each group on week 4 and 12 respectively after the last intraperitoneal injection,and full-thickness skin tissue samples were harvested from the middorsal region of each mouse.Then,hematoxylin-eosin (HE) staining was performed to observe histological changes,and total collagen content was estimated according to hydroxyproline content measured by an alkalinehydrolysis method.The cross-linking degree of collagen was determined by Edman degradation method using pepsin,the mRNA expression level of collagen type Ⅰ and Ⅲ by real-time quantitative PCR,the content of AGE by fluorospectrophotometry and Western blotting,and the level of malondialdehyde (MDA) by using a thiobarbituric acid method.Statistical analysis was carried out by t test.Results As light microscopy showed,the skin became obviously thinner in the diabetic mice with a progressive decrease in the number of collagen fibers in comparison with the control mice.On week 4 and 12 after the last injection,the diabetic mice exhibited a significant reduction in the content of hydroxyproline ((684.5 ± 76.7) vs.(787.7 ± 87.7) rg/g,(558.1 ± 73.1) vs.(757.8 ± 75.3) mg/g,both P ＜ 0.01) and in the levels of cross-linked collagen as well as mRNA expressions of collagen Ⅰ and Ⅲ (P ＜ 0.01 or 0.05),but a significant increase in the content of AGE ((37.47 ± 10.65) vs.(26.39 ± 3.74) AUF/mg hydroxyproline,(47.70 ± 5.66) vs.(29.91 ± 6.50) AUF/mg hydroxyproline,both P ＜ 0.01) and MDA ((6.62 ± 0.47) vs.(4.82 ± 0.56) μmol/L,(8.63 ± 0.36) vs.(5.15 ± 0.46) μmol/L,both P＜ 0.01) in skin tissue,compared with the control mice.The level of non-cross-linked collagen in skin tissue was also lower in the diabetic mice than in the control mice on week 12 (P ＜ 0.05).Moreover,the contents of hydroxyproline and the expression levels of collagen I in skin were significantly lower (P ＜ 0.05),but the levels of AGE and MDA were significantly higher (P ＜ 0.01) in the diabetic mice on week 12 than in those on week 4.Conclusions The characteristics of collagen fibers in skin are altered in diabetic mice when compared with normal control mice,which may be associated with increased AGE content and oxidative injury in skin.
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Objective To investigate the therapeutic effects and mechanism of anti-radiation pneumonia decoction(ARPD) on radiation induced lung fibrosis in rats.Methods One hundred and five male SD rats in a SPF grade were divided into Chinese medicine group,single radiation group and control group by random digits table method,with 35 in each group.After anesthetization,rats in Chinese medicine and single radiation groups were exposed to 6 MV X-rays at the dose of 15Gy.Rats in Chinese medicine group were treated with ARPD at the dosage of 10 ml·kg-1 ·d-1 once a day,but rats in single radiation group did not receive ARPD treatment.Rats in control group were treated with neither irradiation nor drugs.Five rats of each group were killed and the lung tissues and blood samples were collected at 15,30,60,75,90,105 and 140 d.The pathological changes of lung tissues were observed and the tissue protein and gene expressions of TGF-β1,PAI-1 and collagen type Ⅲ(C Ⅲ) were assayed by Western blot and RT-PCR.ELISA was used to detect serum TGF-β1 and plasma PAI-1.Tissue and serum HYP were determined by acid hydrolysis and alkaline hydrolysis methods respectively.Results Inflammation was found in the lung tissues of all the exposed rats.Obvious pathological lung fibrosis was found at 60 d,the inflammation and the fibrosis in treated group were slighter than those in single radiation group.In Chinese medicine group,the protein and gene expression levels of TGF-β1,PAI-1,C Ⅲ 30 d(Protein:t =2.49-3.74,t =2.63-4.57 and t =2.76-3.83;Gene:t =2.59-4.33,t =2.83-4.62 and t =2.83-3.96,P＜0.05),serum TGF-β1 and plasma PAI-1 15 dlater (t =2.85-6.27 and t =3.69-5.27,P＜0.05),and the levels of tissue and serum HYP60 dlater (t=3.65-4.40 and t =6.56-3.75,P＜0.05),all of them were lower than those in single radiation groups.There were significant positive correlations between tissue TGF-β1 and PAI-1 as well as C Ⅲ (Protein expression:r =0.604,0.759,P ＜0.05;Gene expression:r=0.519,0.816,P＜0.05).Conclusions ARPD may inhibit the pulmonary fibrosis by decreasing the levels of TGF-β1,PAI-1 and C Ⅲ.
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Objective To study the relationship between serum fibrosis parameters levels and chronic viral hepatitis B patients.Methods Radioimmunoassay and chemiluminescence were used to test the levels of hyaluronic acid(HA),collagen type Ⅳ(Ⅳ-C),laminin(LN) and procollagen type Ⅲ(PC Ⅲ) in 146 cases of chronic hepatitis B and 40 of normal controls,and the relationship with clinical parameters of liver function were analyzed.Results The levels of HA,Ⅳ-C,LN,PC Ⅲ in chronic hepatitis B patients were higher than those of normal control group (P ＜0.05 or P ＜ 0.01),there were statistical differen in different course of disease (P ＜ 0.05 or P ＜ 0.01) ; The levels of serum Ⅳ-C and PCⅢ had positive correlation with plasma PT,the same to HA with TBil and PCⅢ with ALT;The levels of HA,Ⅳ-C and LN had negative correlation with Alb,the same to LN with CHE.Conclusion The levels of HA,Ⅳ-C,LN and PC Ⅲ in chronic hepatitis B patients may reflect the situations of hepatic fibrosis and the degrees of liver function damage.
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ObjectiveTo study transcriptional characteristics of type Ⅲ procollagen gene in systemic scleroderma (SS)-derived fibroblast clones and their regulation by Radix Salviae miltiorrhizae(RSM).Methods Eight fibroblast clones with different collagen-producing capacity were previously obtained from patients with SS and normal human controls.Recombinant plasmids containing different deletions of the human alpha 1 chain of type 3 procollagen(COL3A1) gene promoter were constructed,and transiently transfected into the fibroblast clones.Dual-luciferase reporter assay system was used to evaluate the activities of these recombinants in the fibroblast clones and to select a proximal transcriptional regulatory sequence.Then,the fibroblast clones were transfected with the plasmid containing the selected regulatory sequence(phCOLH30.1) followed by the treatment with RSM injection(1 g/L) and active monomers of RSM,including salvianolic acid B(5 mg/L),tanshinone Ⅱ A (5 mg/L),danshensu(20 mg/L) and protocatechuic aldehyde(5 mg/L),for 48 hours.The transfected fibroblast clones receiving no drug treatment served as the water-soluble control,and those treated with only dimethyl sulfoxide as the lipid-soluble control.Subsequently,the fibroblasts were lysed and subjected to the quantification of cellular proteins and determination of luciferase activity.The activity of recombinant promoters was compared by t test for the selection of proximal transcriptional regulatory sequence,and the activity of phCOLH30.1 by two-way analysis of variance in the RSM-interfering test(if there was interaction,one-way analysis of variance was conducted; and if there was no interaction,the main effect was tested after the removal of interaction item).ResultsOf the 6 recombinants,the recombinant containing COL3A1 proximal promoter from -96 bp to +16 bp(phCOLH30.1) showed the highest transcriptional activity in nearly all of the fibroblast clones,and the activity was positively correlated with the collagen-producing capacity of fibroblast clones.Compared with the water-soluble control,RSM injection significantly downregulated the activity of phCOLH30.1 in fibroblast clones with high and low collagen-producing capacity from patients with SS (2.261 ± 0.619 vs.3.879 ± 0.309,1.462 ± 0.291 vs.2.150 ± 0.262,both P ＜ 0.01) and normal human controls (1.681 ± 0.263 vs.3.039 ± 0.271,1.121 ± 0.361 vs.2.223 ± 0.247,both P ＜ 0.01),salvianolic acid B decreased the phCOLH30.1 activity in SS-derived high collagen-producing fibroblast clones (2.309 ± 0.524,P ＜ 0.01 ) and in the normal control fibroblast clones with high and low collagen-producing capacity (2.126 ± 0.320 and 1.976 ± 0.362,both P ＜ 0.05).Tanshinone Ⅱ A only downregulated the phCOLH30.1 activity in SS-derived high collagen-producing fibroblast clones compared with the lipid-soluble control(2.975 ± 0.666 vs 5.379 ± 0.238,P ＜ 0.01 ).Neither danshensu nor protocatechuic aldehyde showed inhibitory effects on phCOLH30.1 activity in SS-derived or normal control fibroblast clones.ConclusionsThe type Ⅲ procollagen gene is activated at the transcriptional level in high collagen-producing fibroblast clones from patients with SS,and the activation could be suppressed by RSM injection,salvianolic acid B and tanshinone Ⅱ A.
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Objective To investigate the expression of connective tissue growth factor(CTGF),coll agen I and collagen Ⅲ in sacroiliac joint(SIJ)of patients with spondyloarthropathy(SpA).Methods Thirty patients with SpA,including 17 patients with grade Ⅱ saeroiliitis and 13 patients with grade Ⅰ sacroiliitis,were performed on CT guided needie biopsy of SIJ.After sacroiliitis were confirmed by staining with hematoxylin and eosin in sacroiliac joint tissue sample,immunohistochemical assay was performed to determine the expression of CTGF,collagen Ⅰ and collagen Ⅲ in sacroiliac ioint tissue.Univariate Chi-square test was used for data comparison between multiple groups and t-test was used for two group data comparison.Results Contrast to healthy controls,CTGF were found upexpressed on the cytoplasm of inflammatory cells in pannus and bone marrow of sacroiliac tissue samples of patients with SpA,while collagen I and collagen Ⅲ were found up-expressed in bone,cartilage and ligament tissue[(57.9±42.4)/HP vs(2.7±2.5)/HP P<0.05,0.298±0.080 vs 0.044±0.024 and 28.254±41.165 vs 0.105±0.054.P<0.05 respectively].Conclusion CTGF,collagen Ⅰ and collagen Ⅲ are up-expressed in SIJ of SpA patients.CTGF may play an important role in articular cartilage fibrosis and ossification of SpA.
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ObjectiveTo study the heat shock protein 47 ( HSP 47 ) andtype Ⅰ ,Ⅲ collagen relevance expression in keloid tissue. MethodsUSE immunohistochemistry to detect 21 patients( HS group) pathological keloid tissue HSP47, Ⅰ-type, Ⅲ collagen levels,and normal foreskin(control group) compared to the expression of HSP 47 and Ⅰ and Ⅲ collagen. ResultsHS group HSP 47 scar absorbance was( A value) ( 133.0 ± 58.7) ,it was significantly lower than the control group( 169.6 ± 53.2) ( t =2. 697 ,P =0.024) ;two groups of collagen type Ⅰ, Ⅲ collagen levels had statistically differentce(t =14.898,12.610,all P =0.000) ; HSP 47 level and type Ⅰ collagen were positively correlated(r =3.058,P =0. 034),and type Ⅲ collagen content was also positively correlated(r =4. 610, P =0. 029). ConclusionHSP 47 in keloid tissue could cause dermal layer assembly of collagen synthesis and significantly increased,and it was an important mechanism for the formation of keloids.