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Objective:To study the expression and clinical significance of collagen type Ⅰ alpha 2 chain (COL1A2) in glioma , and its effect on the migration and invasion of glioma cell lines.Methods:Through in-depth mining of the data related to COL1A2 in the Oncomine database, meta-analysis of its expression in different types of tumors, different grades and different molecular types of glioma, and then through the Chinese glioma genome map project (Chinese Glioma Genome Atlas, CGGA) database to explore the relationship between its expression level and the prognosis of glioma patients. The COL1A2 gene was functionally annotated by gene ontology (GO) and Pathway analysis. The annotation content includes cell components, biological processes, molecular functions and related signal pathways.Results:A total of 426 research results on COL1A2 in different types of tumors were collected in the Oncomine database, 114 of which were statistically different, 103 studies with increased COL1A2 expression, and 11 studies with decreased expression; the analysis shows there were 22 studies on high expression of COL1A2 in tumors, and no studies on low expression. Analysis of different grades of glioma and different molecular types of glioma Compared with the control group, COL1A2 was highly expressed in various types of glioma. Through the analysis of the gene chip data of the CGGA database, it was found that in glioblastoma, low expression levels of COL1A2 were significantly associated with an improved prognosis in patients with glioma ( P<0.05). Next, through GO and Pathway annotations, it was found that COL1A2 was involved in the biological processes of NAD metabolic salvage pathway, cell and cell signal transduction, circadian rhythm regulation and so on. Finally, through the construction of overexpression and knockdown cell lines in glioblastoma cell lines U87 and T98, scratch experiments and transwell cell function experiments confirmed that COL1A2 can significantly promote the migration and invasion of glioblastoma cell lines. Conclusions:Low expression levels of COL1A2 were significantly associated with improved prognosis in patients with glioma. Knockdown and overexpression of COL1A2 on glioblastoma cell lines U87 and T98 manifested that COL1A2 can promote glioblastoma cell lines migration and invasion ability. Based on the above results, COL1A2 may be used as an indicator for judging the prognosis of glioblastoma and as a potential biological target for therapy.
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Objective: To investigate the impact and related mechanisms of glucose fluctuations on aortic fibrosis in rats with type 1 diabetes mellitus. Methods: After injection of streptozotocin (STZ), male Sprague Dawley (SD) (8-12 weeks) rats (n=24) were randomly divided into three groups in accordance with the random number table: controlled STZ-induced diabetes (C-STZ) group (n=8); uncontrolled STZ-induced diabetes (U-STZ) group (n=8); STZ-induced diabetes with glucose fluctuations (STZ-GF) group (n=8). After three weeks, rats were sacrificed and aorta was obtained, aortic fibrosis was detected by Masson trichrome staining. The expression of collagen type 1 (collagen Ⅰ) was tested by immunofluorescence. The expression of runt-related transcription factor 2 (Runx2) was tested by immunohistochemistry. The mRNA levels of collagen Ⅰ and Runx2 were detected by quantitative real-time PCR (qRT-PCR). The protein expressions of collagen Ⅰ, Runx2 and nuclear factor (NF)-κB were determined by Western blot. Primary rat aortic smooth muscle cells (VSMCs) were cultured in three conditions: normal glucose (NG), high glucose (HG) and glucose fluctuations (GF). Cells in GF group were incubated for 72 hours with glucose alternating between 5.5 and 25 mmol/L every 12 hours. TPCA-1, the inhibitor of NF-κB, the expression of collagenⅠin different groups of cells was tested by immunofluorescence. The protein expressions of collagen Ⅰ, Runx2 and NF-κB were also determined by Western blot. Results: (1) The quantitative ratios of the area of fibrosis in the C-STZ group, U-STZ group, STZ-GF group were (8.42±0.10)%, (21.30±0.74)% and (44.39±1.09)% (P<0.05), respectively. The means of integral optical density (IOD) of collagenⅠ in the three groups were 11.92±0.88, 50.04±3.56 and 77.52±2.69, respectively (P<0.05). The mRNA levels of collagenⅠ in the three groups were 1.00±0.10, 2.02±0.28 and 2.83±0.33, respectively (P<0.05). The protein expressions of collagenⅠ in the three groups were 1.05±0.03, 2.06±0.32 and 4.93±0.25, respectively (P<0.05). (2) The average IOD of Runx2 in the three groups were 150.00±7.35, 204.84±2.32 and 391.48±7.13, respectively (P<0.05). The mRNA levels of Runx2 in the three groups were 1.02±0.02, 1.27±0.04 and 2.18±0.12, respectively (P<0.05). The protein expressions of Runx2 in the three groups were 1.03±0.01, 2.34±0.36 and 4.52±0.75, respectively (P<0.05). (3) The protein expressions of NF-κB in the three groups were 1.02±0.01, 1.96±0.13 and 2.64±0.21, respectively (P<0.05). (4) In vitro, application of inhibitor of NF-κB reversed glucose fluctuations-induced upregulation of protein levels of Col Ⅰ and Runx2 (P<0.05). Conclusion: Glucose fluctuations could aggravate aortic fibrosis through activating Runx2 via NF-κB signaling pathways.
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Animals , Male , Rats , Aorta , Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 1 , Fibrosis , Glucose , NF-kappa B , Rats, Sprague-DawleyABSTRACT
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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Establishing a stable resin-dentin hybrid layer is an effective method to improve the adhesion durability of the restoration. The biomodification of dentin by cross-linkers can enhance the mechanical properties of collagen and resistance to enzymatic hydrolysis while, inhibiting the process of demineralization and promoting the remineralization of dentin, which has the potential clinical applicability of preventing dental caries and improving adhesive property. This review summarizes the biomodification of dentin type Ⅰ collagen by different cross-linkers.
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OBJECTIVE: To observe the effects of nilotinib on silicon dioxide(SiO_2)-induced cell proliferation and collagen synthesis in human fetal lung fibroblast-1(HFL-1) cells and to explore the related mechanism. METHODS: ⅰ) HFL-1 cells were induced with different doses of SiO_2 suspension(0, 5,10, 25, 50 and 100 mg/L) for 24.0 hours. The expression of transforming growth factor-β1(TGF-β1), C-Abl, and platelet-derived growth factor receptor(PDGFR) was detected by Western blot, and the dose of SiO_2 in subsequent experiments was screened. ⅱ) HFL-1 cells were randomly divided into 6 groups: 1) the control group: no treatment; 2) the solvent control group: cells were treated with 0.10% dimethyl sulfoxide; 3) the SiO_2 stimulation group: cells were induced with SiO_2 suspension at a dose of 50 mg/L for 24.0 hours; 4)-6) the nilotinib groups: cells were induced with SiO_2 suspension at a dose of 50 mg/L for 24.0 hours and treated with nilotinib at the concentration of 5, 10, or 15 mmol/L for 24.0 hours. Cell proliferation was detected by MTS assay. The TGF-β1 protein secreted by cells was measured using enzyme linked immunosorbent assay. The expression of TGF-β1, C-Abl, platelet derived growth factor(PDGF), PDGFR and collagen typeⅠproteins was measured by Western blot. RESULTS: ⅰ) The dose of the SiO_2 in the experiments was set to 50 mg/L. ⅱ) The cell proliferation rate of HFL-1 cells in the SiO_2 stimulation group and the 3 nilotinib groups was higher than that in control group and solvent control group(P<0.05). The proliferation rates of HFL-1 cells in 10 and 15 mmol/L nilotinib groups were lower than that in SiO_2 stimulation group(P<0.05). The level of TGF-β1 and the protein relative expression levels of TGF-β1, collagen typeⅠ, C-Abl, PDGFR and PDGF in HFL-1 cells of SiO_2 stimulation group were higher than those in control group and solvent control group(P<0.05). The above indexes of HFL-1 cells in 15 mmol/L nilotinib group were lower than that in SiO_2 stimulation group(P<0.05); the above indexes of HFL-1 cells in 5 mmol/L nilotinib group were not significantly different from those in SiO_2 stimulation group(P>0.05). The level of TGF-β1 and the relative expression level of C-Abl protein in HFL-1 cells of 10 mmol/L nilotinib group were lower than those in SiO_2 stimulation group(P<0.05). CONCLUSION: Nilotinib can inhibit the proliferation of HFL-1 cells and reduce the expression of collagen typeⅠprotein induced by SiO_2. This process may be achieved by inhibiting tyrosine kinase-mediated signaling pathway.
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Objective To observe the expression of insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) in human pancreatic tumor tissues and investigate its significance and relationship with clinic pathological characteristics and tumor microenvironment of the pancreatic neoplasms.Methods A total of 236 patients with surgically resected pancreatic tissue from January 2007 to December 2017 were selected from the First Hospital of Shanxi Medical University.Totally 236 patients were divided into paracancer control group (normal pancreatic tissue adjacent to the tumor,n =111),benign group (benign or low-grade malignant tumor such as solid pseudopapillary tumor,n =37),and malignant group (malignant tumors such as pancreatic ductal adenocarcinoma,n =88).The histomorphology and collagen deposition were observed using hematoxylin-eosin (H-E) staining and Sirius red staining in the three groups.The expressions of IGFBPrP1,transforming growth factor β1 (TGFβ1),α-smooth muscle actin (α-SMA)and collagen type Ⅰ of pancreatic tissues in the three groups were detected by immunohistochemical staining.The relationship between IGFBPrP1 and TGFβ1,α-SMA or collagen type Ⅰ,and the relathionship between IGFBPrP1 and clinicopathological features of the pancreatic neoplasms were analyzed.T test and one-way analysis of variance were used for statistical analysis,and Spearman rank correlation test was used for correlation analysis.Results In benign group and malignant group,there were obvious cell atypia,and the cell atypia of malignant group was more significant than benign group.The contents of collagen fibers in benign group and malignant group were significantly higher than that in paracancer control group.IGFBPrP1,TGFβ1,α-SMA and collagen type Ⅰ were highly expressed in the endochylema of the tumor cells and (or) the myofibroblast.The expression level of IGFBPrP1 in highly differentiated ductal adenocarcinoma was significantly higher than that in moderately and poorly differentiated ductal adenocarcinoma ((9.46 ± 2.10) × 104 vs.(6.48 ± 1.38) × 104 and (6.07 ± 1.29) × 104);t =7.430 and 6.767,both P ＜ 0.05).The expression of IGFBPrP1 in human pancreatic neoplasms was positively correlated with TGFβ1,α-SMA and collagen type Ⅰ (r=0.530,0.619,0.625;all P ＜0.05).Conclusions IGFBPrP1 is highly expressed in pancreatic tumor tissue and its expression level may correlate with the histological grade of pancreatic neoplasms.The expression of IGFBPrP1 in human pancreatic tumor tissues may be accompanied by the activation of pancreatic stellate cells and the generation of cancer-related fibroblasts,and IGFBPrP1 may involve in the formation of tumor by changing the tumor microenvironment.
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Objective To detect the expression levels of collagen1 (colla-1),transforming growth factor-β1 (TGF-β1),a-smooth muscle actin (α-SMA) and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX-4) in mouse esophagus submitted to chronic restraint stress (CRS),in order to discuss stress-induced esophageal fibrosis and the role of oxidative stress.Methods 20 male Kunming mice were randomly divided into two groups,CRS and normal control (NC).The mice in CRS group were submitted to 2 h per day of restraint stress using home-made device for a period of 14 days,and the mice in both group were treated the same at rest of the time.Fibrotic changes of esophageal tissue were observed using Masson staining.The expression levels of NOX-4 and related fibrotic cytokines in esophageal tissues were detected by several methods such as immunohistochemistry,enzyme-linked immunosorbent assay (ELISA) and realtime polymerase chain reaction (qRT-PCR).Results Body weight in CRS group was significantly lower than NC group (8.75 ± 1.69 vs 12.69 ± 3.16),with statistically significant difference (t =3.11,P ＜ 0.05).Masson staining revealed that CRS mice showed distinct fibrosis of epithelial interstitium,while there was no distinct changes observed in NC mice.Immunohistochemical staining revealed intense staining for NOX-4 in epithelial,mucosal and submucosal layers of esophagi in CRS mice.ELISA showed that the serum level of NOX-4 in CRS mice was higher than NC mice (1.442 ± 0.05 vs 0.449 ± 0.08),with statistically significant difference (t =-27.32,P ＜ 0.01).Real-time PCR results showed that the expression of colla-1,TGF-β1,α-SMA and NOX-4 in CRS mice were as (2.443 ±0.36,2.78 ±0.13,2.244 ±0.18,2.448 ±0.440) times higher than NC mice,with statistically significant difference (t =-11.19,-38.86,-19.90,-10.37,P ＜ 0.01).Conclusions Fibrotic cytokines such as colla-1,TGF-β1 and α-SMA may participate in formation of stress induced esophageal fibrosis,and oxidative stress may play crucial role in the process of esophageal fibrosis.
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Objective@#To observe the effect of adenosine triphosphate (ATP) phosphorylation on type Ⅰ collagen mineralization and explore the role of small molecule compound ATP in biomimetic mineralization.@*Methods@#Fourier transform infrared spectroscopy (FT-IR) was used to analyze the phosphorylation of collagen molecules by different concentrations (0, 25, 50, 100 mmol/L) of ATP. The concentration of 50 mmol/L ATP was chosen to construct the phosphorylated collagen mineralization model. Transmission electron microscopy (TEM) was used to observed the ultrastructure of mineralized collagen and the collagen mineralization rate was further calculated by ImageJ software. The surface morphology of the collagen gel ATP group and the control group was observed by scanning electron microscopy (SEM) and the elemental analysis was performed by using an X-ray energy spectrometer. The artificial demineralized dentin samples were mineralized for 2 days and 4 days to compare the effect of ATP on dentin remineralization by SEM.@*Results@#FT-IR analysis showed that the formation of new peaks at wavenumbers of 642, 818, and 902 cm-1 indicated that ATP can phosphorylate type Ⅰ collagen. Through TEM and SEM observation, the mineralization degree of type Ⅰ collagen and demineralized dentin pretreated with 50 mmol/L ATP were significantly higher than that of the control group. Compared with the control group [(31.65±1.62)%], the mineralization rate of collagen in the ATP group [(100±0)%] was significantly increased after 2 days of mineralization (P<0.05).@*Conclusions@#ATP phosphorylation can effectively promote the mineralization process of type Ⅰ collagen.
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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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AIM:To investigate the regulatory effect of NADPH oxidase-4 (NOX-4) on PI3K signaling path-way in transforming growth factor-β1 (TGF-β1)-induced collagen type Ⅰ (collagen Ⅰ) synthesis from lung cancer cells and the mechanisms. METHODS:Human lung cancer A549 cells were cultured in vitro and stimulated with TGF-β1. The ex-pression of NOX family and collagen family at mRNA and protein levels as well as the PI3K class Ⅰ catalytic subunits and the activation of PI3K signaling pathway was measured. A549 cells were pre-treated with NOX-4 inhibitor diphenyleneiodo-nium (DPI), and the expression of collagen Ⅰ at mRNA level as well as the PI3K class Ⅰ catalytic subunits and the activa-tion of PI3K signaling pathway was measured upon TGF-β1 stimulation. RESULTS:TGF-β1 stimulated the expression of NOX-4 and collagen Ⅰ at mRNA and protein levels as well as the expression of PIK3CD and the activation of PI3K signaling pathway at a dose- and time-dependent manner. NOX-4 inhibitor DPI partly reversed TGF-β1-induced collagen Ⅰ expres-sion. Inhibition of NOX-4 down-regulated the degree of TGF-β1-stimulated activation of PI3K signaling pathway without effect on the expression of PIK3CD. CONCLUSION:NOX-4 participates in TGF-β1-induced collagen Ⅰ synthesis from lung cancer cells via regulating the activation of PI3K signaling pathway. TGF-β1/NOX-4/PI3K signaling pathway axis acts as a regulatory role in collagen Ⅰ synthesis from lung cancer cells.
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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 analyze gene mutations and the etiology of six fetuses with osteogenesis imperfecta detected by prenatal ultrasonography.Methods From March 2016 to May 2017,six gravidas of singleton pregnancy and their fetuses that were diagnosed with osteogenesis imperfecta by prenatal ultrasonography were enrolled in this study.Gravida 1 came to the Center of Prenatal Diagnosis of the First Affiliated Hospital of Zhengzhou University for prenatal diagnosis,while the other five were referred to that center after termination to identify genetic defects with their fetal tissues.Next-generation sequencing technology was carried out for exome sequencing in the genomes of six fetuses.Suspected mutations were confirmed by polymerase chain reaction and Sanger sequencing.Two hundred unrelated healthy individuals were analyzed with Sanger sequencing for validation of novel mutations.Results Fetus 1 carried a heterozygous mutation in collagen,type Ⅰ,alpha-1 (COL1A1) gene,c.724G＞C(p.Gly242Arg),which was found in the mother and brother but not in the father.Fetus 2 carried a known heterozygous mutation in COL1A 1 gene,c.2461G＞A(p.Gly821Ser),which was found in the mother but not in the father.Four heterozygous mutations,c.2282G＞A(p.Gly761Asp),c.1002+5G＞A in COL1A1 gene,c.1774G＞A(p.Gly592Ser) and c.3277G＞T(p.Gly1093Cys) in collagen,type Ⅰ,alpha-2 (COL1A2) gene,were respectively carried by fetuses 3 to 6,but not by their parents.Mutations of c.724G＞C(p.Gly242Arg),c.2282G＞A (p.Gly761Asp) and c.1002+5G＞A in COL1A1 gene and c.3277G＞T (p.Gly1093Cys) in COL1A2 gene were four novel mutations,which were not found in the 200 unrelated healthy individuals.The mother of fetus 1 who was highly suspected with osteogenesis imperfecta selected to continue the pregnancy because the family members had mild symptoms.After delivery,cord blood was collected for genetic test and the result was consistent with that of prenatal genetic diagnosis.Fetus 1 had no fractures during a six-month follow-up after birth.Conclusions Mutations in the COL1A1 and COL1A2 genes may be the etiology of osteogenesis imperfecta in these six fetuses.Results of this study could enrich the data on COL1A1 and COL1A2 mutations relating to osteogenesis imperfecta,and provide a basis for genetic counseling.
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Objective To study whether SGK1 is involved in the phenotypic transformation in adventitial fibroblasts (AF).Methods Vascular AF were separated from the thoracic aortas of SD rats.The AF not stimulated with TGF-β1 were divided into blank group,control group 1 and control group 2.The AF induced with 2.5,5.0,10.0 and 15.0 ng/ml TGF-β1 were divided into groups A-D.The AF pretreated with 50 μmol/1 EMD638683 and SB203580 were divided into inhibitor group 1 and inhibitor group 2.The AF stimulated with 5 ng/ml TGF-β1 were divided into stimulation group 1 and stimulation group 2.The expression of SGK1,α-SMA and collagen Ⅰ was detected by Western blot.Results The expression of SGK1 was significantly higher in groups AC than in blank group.The expression of α-SMA was significantly higher in groups B-D than in controlgroup (P＜0.05).The α-SMA and collage Ⅰ expression levels were significantly higher in stimulation group 1 than in control group 1,and were significantly lower in inhibitor group 1 than in stimulation group 1 (P＜0.05).The SGK1 expression level was significantly higher in stimulation group 2 than in control group 2 and was significantly lower in inhibitor group 2 than in stimulation group 2 (P＜0.05).Conclusion SGK1 participates in TGF-β1-induced phenotypic transformation via p38 MAPK and is thus involved in vascular remodeling.
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Objective To investigate degrees of fibrosis of adenomyosis(AM)myometrium and explore its relationship with dysmenorrhea. Methods Thirty AM patients who had hysterectomy from July, 2015 to December, 2016 in Beijing Obstetrics and Gynecology Hospital were selected as AM group; 28 cases of hysterectomy due to cervical lesions(none AM)were selected as control group. The area ratio of collagen fiber in the two groups was analysed by modified Masson stain, and the expression of collagen type Ⅰprotein in the two groups was analysed by immunohistochemical method. Results (1)The degree of fibrosis:the area ratio of collagen fiber and the expression of collagen type Ⅰof AM group [(34.5±5.1)％, 0.23±0.06] were significantly higher than those of control group [(26.7±10.1)％,0.18±0.08; all P<0.05].(2)The relationship between the degree of fibrosis and dysmenorrhea: the area ratio of collagen fiber and the expression of collagen type Ⅰ in severe dysmenorrhea, moderate dysmenorrhea, and none-mild dysmenorrhea were(35.3± 4.3)％,0.25±0.05;(35.7±3.2)％, 0.26±0.06;(25.0±2.9)％,0.15±0.03, there were significantly different among them(all P<0.01). And the area ratio of collagen fiber, the expression of collagen type Ⅰ were positively correlated with the degree of dysmenorrhea(r=0.50, 0.50; all P<0.05). Conclusions The area ratio of collagen fiber and the expression of collagen type Ⅰin AM are higher than in control group, and positively correlated with the severity of dysmenorrhea. These results suggest the degrees of fibrosis might be correlated with dysmenorrhea.
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Objective To investigate the important role of angiogenin-like protein 4 (Angptl4) in pulmonary fibrosis and to provide a new therapeutic targets for pulmonary fibrosis.Methods We established the pulmonary fibrosis animal models in rat by tracheal instillation of bleomycin.Then,we detected the expression of Angptl4 through real-time polymerase chain reaction (RT-PCR) and Western Blot.Rat lung fibroblast (RLF) was transfected into Angptl4-shRNA plasmid.Then we detected the changed collagen expression in RLF cells after transfection through RT-PCR and Western blot.Results The expression of Angptl4 was up-regulated in the bleomycin-induced rat pulmonary fibrosis model.The expression of both collagen Ⅰ and collagen Ⅳ in RLF cells transfected with Angptl4-shRNA plasmids were down-regulated compared with control after TGF-β treatment.Conclusions Inhibiting the expression of Angptl4 can reduce the expression of collagen fibers in lung tissue,then delaying the progression of pulmonary fibrosis.
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OBJECTIVE To investigate the effect of prenatal nicotine exposure on cardiac ejection function and myocardial fibrosis of the offspring of rats.METHODS Pregnant rats were sc given nicotine 6.0 mg· kg-1,once daily for 17 d.The body mass and heart mass of the offspring were detected at the 21th day of gestation,and 15 and 90 d after birth.Heart rate of 90 d offspring was recorded by ECG,and cardiac functions were detected by Doppler ultrasonography,including cardiac output (CO),stroke volume (SV),ejection fraction (EF),left ventricular long axis shortening fraction (FS),interventricular septum diastolic diameter (IVSd) and left ventricular posterior wall diastolic diameter (LVPWd).The myocardial ultrastructure was detected under an electron microscope.Masson staining was used to detect the myocardial collagen fiber deposition.The level of collagen protein type Ⅰ in heart tissue was detected by radioimmunoassay.RESULTS Compared with control group,prenatal nicotine exposure resulted in a decrease of heart mass and body mass in groups of 21 d fetal rats and 15 d offspring(P＜0.05,P＜0.01),but had no effect on the 90 d offspring.Compared with the normal control group,the heart rate of 90 d offspring increased [366+10 vs (418+10) min-1] (P＜0.05),CO,FS and EF decreased (P＜0.01),and IVSd and LVPWd increased (P＜0.05,P＜0.01).Electron microscopy revealed that in the heart of nicotine 90 d offspring,myocardial fiber arrangement was loosened and confused,while extracellular matrix increased.Masson staining showed collagen deposited in the myocardium.The level of collagen type Ⅰ in heart tissue increased [0.59±0.09 vs (0.40±0.05) tμg·g-1 tissue] (P＜0.01).CONCLUSION Prenatal nicotine exposure induces the increased level of cardiac collagen type Ⅰ,myocardial fibrosis and decrease of cardiac ejection function in adult offspring,which may lead to increased susceptibility to cardiovascular diseases.
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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 explore bone formation markers in dexamethasone intervention osteocalcin (OC),bone alkaline phosphatase (BAKP),and type Ⅰ original amino terminal propcptide (PINP) relationship with bone longitudinal growth.Methods The selected thirty-three 4-week-old male SpragueDawley (SD) rats were randomly divided into two groups:the dexamethasone group (n =18) and the control group (n =15).The rats in the dexamethasone group received dexamethasone (200 μg/100 g) by intraperitoneal injection for 10 days.The rats in the control group received matching volume sodium chloride solution.All rats were weighed everyday.The rats were killed by using 10％ chloral hydration at 8 AM of 11 th day.The length of tibiae was measured.The proximal tibiae were excised,fixed and decaleified.Mter paraffin embedded,sections in 5 μm thick were cut.The growth plate sections were stained by haematoxylin-eosin (HE) histochemistry method.Total height of growth plate was measured.The rats decaptitating and the blood were collected.Serum was separated and stored in-80 ℃ refrigerator for analysis.Enzyme -linked immuno sorbent assay (ELISA) method was used to detect the rat OC,BAKP and PINP values.Results The length of growth plate and tibiae of dexamethasone group were significantly decreased contrast the control group:the length of growth plate (P =0.001),and the length of tibiae (P =0.000).There were no significant differences between two groups of the value of OC,BAKP and PINP:OC (P =0.056),BAKP (P=0.122),and PINP (P =0.169).There was positive correlation between the serum OC and the length of tibiae (r =0.454,P =0.08) in control group,the PINP and OC (r =0.521,P =0.026) in dexamethasone group.Conclusions Glucocorticoid inhibit the longitudinal bone growth,to the osteoblasts (OC,BAKP and PINP) of growing rats is not obvious.
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Objective To explore the effects of different degrees of intermittent hypoxia (IH) on the activation and the secretion of extracellular matrix in MLg lung fibroblast cell line. Methods MLg lung fibroblast cells in logarithmic growth phase were exposed for 5%O2 for 100 seconds and 21%O2 for 120 seconds in 1 h, 4 h and 8 h groups (IH1, IH4 and IH8) and normoxia group (21%O2 for 8 h, N group). The cells in each group were collected at the end of experiment. Real-time PCR was used to measure the mRNA expression levels ofα-SMA and typeⅠcollagen (COL1) A1, and Western blot assay was used to detect the protein expression levels ofα-SMA and COL1. Results The mRNA and protein expression levels ofα-SMA and COL1 were significantly increased in IH1, IH4 and IH8 groups than those in N group (all P < 0.05). Furthermore, expression levels of α-SMA and COL1 showed a time-dependent increase with IH exposure time. Conclusion The intermittent hypoxia can promote the cell activation and the extracellular matrix secretion of mouse lung fibroblast cells, which may be related with the oxidative stress.
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Objective To explore the impact of different doses of butylphthalide on renal interstitial fibrosis in mice with obstructive nephropathy, and to discuss the correlation between Nrf-2 expression level and renal interstitial fibrosis. Methods Totally 72 male CD-1 mice of clean grade were selected and randomly assigned into 4 groups:sham operation group (Sham), model group (UUO), low dose of butylphthalide group (NBPL) and high dose of butylphthalide group (NBPH). In addition to the control group, UUO model was established in other three groups. NBPL group was given NBP 150 mg/(kg· d) by gavage since postoperative day 1,NBPH group was given NBP 220 mg/(kg·d) by gavage. Sham group and UUO group were given equal volume of saline by gavage. Six mice were sacrificed at the third, 7th, 14th day, respectively. The obstructive renal tissue was selected for immunohistochemical staining and Western blot assay. The expression levels of Nrf- 2 and type Ⅰ collagen were detected by Western blot assay. Results The IOD value of type Ⅰ collagen was increased with time in UUO group, and which showed a gradual decreasing trend in Sham group, NBPL group and NBPH group (P<0.05). The IOD values at different time points were significantly higher in UUO group, NBPL group and NBPH group compared to those of Sham group. The IOD values were significantly decreased in NBPL and NBPH groups than that of UUO group, and IOD value was lower in NBPH group than that of NBPL group (P<0.05). Western blot assay showed that IOD values of Nrf-2 and type Ⅰ collagen at 7 d and 14 d were increased in UUO group, NBPL group and NBPH group compared with those of Sham group. The IOD values of Nrf-2 protein were increased, and IOD values of type Ⅰcollagen were decreased, in NBPL group and NBPH group than those of UUO group. The IOD value of Nrf-2 protein was decreased in NBPL group than that of NBPH group, but the IOD value of type Ⅰcollagen was increased in NBPL group than that of NBPH group (P<0.05). Conclusion NBP can improve renal interstitial fibrosis in UUO mice, which may be related with the increased expression of Nrf-2 and the down-regulated expression of type I collagen.