Objective To explore the effects of DNA cross-linking repair 1B (DCLRE1B) gene on the migration and invasion ability of hepatocellular carcinoma cell. Methods Bioinformatics analysis was used to analyze the expression of DCLRE1B mRNA in hepatocellular carcinoma, and its relationship with the prognosis and related influencing factors of patients. Immunohistochemical staining was used to detect the expression of DCLRE1B protein in resected hepatocellular carcinoma tissues and their corresponding normal liver tissues. The DCLRE1B gene silenced Huh7 and HepG2 hepatocellular carcinoma cell lines were constructed by lentivirus, and the transfected effect was detected by Western blot. The migration and invasion of DCLRE1B silenced hepatocellular carcinoma cells were detected by scratch test and Transwell method. The changes of genes related to epithelial mesenchymal transformation (EMT) after DCLRE1B silencing were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results ① The biological information analysis results showed that: The mRNA expression of DCLRE1B was highly expressed in a variety of tumors including hepatocellular carcinoma (P<0.05). The mRNA expression of DCLRE1B was associated with the TNM staging of tumor (P<0.05). The relative expression level of DCLRE1B mRNA in hepatocellular carcinoma patients was related to their prognosis. The overall survival situation (P=0.038) and progression free survival situation (P=0.005) of hepatocellular carcinoma patients in the high expression group were worse than those in the low expression group. Univariate and multivariate Cox analysis showed that the expression of DCLRE1B gene was an independent factor affecting the prognosis of hepatocellular carcinoma (P<0.05). ② The positive rate of DCLRE1B protein expression in resected hepatocellular carcinoma tissues was higher than that in normal liver tissues (P<0.05). ③ Cell experiment results showed that: After stable silencing DCLRE1B gene of hepatocellular carcinoma cell (Huh7 and HepG2) constructed by lentivirus, the expression of DCLRE1B protein was significantly down regulated (P<0.05). After silencing DCLRE1B gene, the migration and invasion ability of hepatocellular carcinoma cells were significantly decreased (P<0.05). After silencing DCLRE1B, the mRNA expressions of E-cadherin, matrix metalloproteinase 9, and β-catenin were up regulated (P<0.05), and the mRNA expressions of N-cadherin and Vimentin were down regulated (P<0.05), but the mRNA expression of zinc finger transcription factor had no significant change, and the difference was not statistically significant (P＞0.05). Conclusion Silencing DCLRE1B gene can inhibit the migration and invasion ability of hepatocellular carcinoma cells, and its mechanism may be related to the process of EMT.
Abstract: Objective To observe the physical characteristics of decellularized porcine pulmonary valved conduits crosslinked by carbodiimide (EDC). Methods [WTBZ]Twenty porcine pulmonary valved arteries were mobilized on relative asepsis condition. They were cut longitudinally into three samples at the junction position of pulmonary valve (every sample was comprised of a part of the pulmonary conduit wall and the corresponding valve). The samples were randomly divided into three groups by lotdrawing method. Group A was the control group which was made up of the fresh porcine arterial valved conduit samples without any other treatments. Group B was comprised of porcine pulmonary samples decellularized by trypsindetergent digestion. Group Cincluded the decellularized porcine pulmonary samples crosslinked by EDC. We observed the water content, thickness, tensile strength, and shrinkage temperature of all the samples, based on which the physical characeteristics of these samples were analyzed. Results [WTBZ]Complete cellfree-pulmonary conduit matrix was achieved by trypsindetergent digestion. Compared with group A, in group B, the water content of pulmonary wall was significantly higher (P=0.000), while the water content of pulmonary valve was not significantly different; the thickness of pulmonary wall and valve (P=0.000，0.000) and tensile strength of pulmonary wall and valve (Plt;0.01） was significantly lower, while shrinkage temperature was not significantly different. Compared with group B, in group C, the water content of pulmonary wall was significantly lower (P=0.000), while the water content of pulmonary valve, and the thickness of pulmonary wall and valve were not significantly different; the tensile strength of pulmonary wall (Plt;0.01) and valve (P=0.000), and the shrinkage temperature of them (P=0.000, 0.000) were significantly higher. Compared with group A, in group C, the water content of pulmonary wall and valve, and the tensile strength of them were not statistically different, while the thickness of pulmonary wall and valve was significantly lower (P=0.000, 0.000), and the shrinkage temperature of them was significantly higher (P=0.000, 0.000). Conclusion [WTBZ]EDC crosslinking method is available for treating decellularized porcine pulmonary valved conduits in order to enhance its tensile strength, and decrease water content of pulmonary wall.
Riboflavin-ultraviolet A (UVA) collagen cross-linking has not only achieved good clinical efficacy in the treatment of corneal diseases such as dilatation keratopathy, bullae keratopathy, infectious keratopathy, and in the combined treatment of corneal refractive surgeries, but also its efficacy and safety in scleral collagen cross-linking have been initially confirmed. To better promote the application of cross-linking in the clinical treatment of corneal and scleral diseases, exploring controllability and predictability of the surgical efficacy are both important for evaluating the surgical efficacy and personalized precision treatment. In this paper, the progress on the cross-linking depth of riboflavin-UVA collagen cross-linking, and its relationship with the cross-linking effect will be reviewed. It will provide the reference for further application of this procedure in ophthalmology clinics.
Objective To manufacture fish swim bladder membrane material by crosslinking techniques, and to explore its physical and chemical properties and cytotoxicity. Methods After decellularization, the swim bladders were randomly divided into two groups. The swim bladders were treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) crosslinking method, surface hole making, and freeze-drying in crosslinking group, and only surface hole making and freeze-drying in non-crosslinking group. The physical and chemical properties of the materials were observed, including microstructure by scanning electron microscopy (SEM), mechanical properties (tensile strength and breaking elongation) by universal tensile machine, hydrophilicity by contact angle measuring instrument, porosity by ethanol infiltration method, degradation performance in vitro and thermal stability test, and the components of materials by infrared spectrum analysis. Mouse fibroblasts (L929) were cultured with the extracts of two groups of materials in order to determine the cytotoxicity of materials by using cell counting kit 8 (CCK-8) method. Results The porous structure and rough surface of materials were observed by SEM. Compared with the non-crosslinking group, the tensile stress of the crosslinking group was higher, the breaking elongation was lower, and the porosity increased, showing significant differences (P<0.05). There was no significant difference in contact angle between the two groups (P>0.05). The degradation was faster within the first 7 days and then tended to be smooth in the two groups. But the degradation rates of crosslinking group were significantly lower than those of non-crosslinking group (P<0.05). Differential scanning calorimeter showed that the denaturation temperature of the crosslinking group was (75.2±1.3)℃, which was significantly higher than that of the non-crosslinking group [(68.5±0.4)℃] (t=4.586, P=0.002). Compared with the non-crosslinking group, the crosslinking group produced new C=O bond and N-H bond, and no other new groups were introduced into the cross-linking group. CCK-8 method showed that the absorbance values of the crosslinking group and the non-crosslinking group were not significant when compared with the positive control group (P>0.05). Conclusion The fish swim bladder membrane obtained by crosslinking treatment with EDC/NHS method has good physical and chemical properties, no cytotoxicity, and is expected to be used as a dura mater repair material.
ObjectiveTo investigate the effects of modification of acellular bovine pericardium with 1-ethyl-3-(3-dinethylami-nopropyl) carbodimide (EDC)/N-hydroxysuccininide (NHS) or genipin and find out the best crosslinking reagent. MethodsThe cellular components of the bovine pericardiums were removed. The effects of decellularization were tested by HE staining. The acellular bovine pericardiums were crosslinked with EDC/NHS (EDC/NHS group) or genipin (genipin group). The properties of the crosslinked acellular matrix were evaluated by scanning electron microscope (SEM), matrix thickness, crosslinking index, mechanical property, denaturation temperature, enzymatic degradation, and cytotoxicity test before and after the crosslinking. Acellular bovine pericardium (ABP group) or normal bovine pericardium (control group) were harvested as controls. ResultsSEM showed that collagen fibers were reticulated in bovine pericardial tissues after crosslinked by EDC/NHS or genipin, and relative aperture of the collagen fiber was from 10 to 20 μm. The thickness and denaturation temperature of the scaffolds were increased significantly after crosslinking with EDC/NHS or genipin (P<0.05), while there was no significant difference between EDC/NHS group and genipin group (P>0.05). The difference had no statistical significance in crosslinking index between EDC/NHS group and genipin group (t=0.205, P=0.218). The degradation rate in EDC/NHS group and genipin group was significantly lower than that in ABP group and control group (P<0.05). Elastic modulus and fracture stress in EDC/NHS group and genipin group were significantly lower than those in ABP group (P<0.05), but there was no significant difference among EDC/NHS group, genipin group, and control group (P>0.05). The break elongation in EDC/NHS group and genipin group were significantly increased than those in ABP group and control group (P<0.05). The difference had no statistical significance in stability and mechanical properties between EDC/NHS group and genipin group (P>0.05). Cytotoxicity of genipin crosslinked tissue (grade 1) were much lower than that of EDC/NHS (grade 2) at 5 days. ConclusionAcellular bovine pericardium crosslinked with genipin has better biocompatibility than EDC/NHS.
ObjectiveTo investigate the changes of fibrinogen and classical markers of collagen metabolism [carboxy-terminal propeptide of type Ⅰ procollagen (PICP) and carboxy-terminal cross-linked peptide of type Ⅰ collagen (ICTP)] in peripheral blood and pericardial drainage after coronary artery bypass grafting (CABG) and/or heart valve replacement (VR), and to evaluate their relationship with postoperative atrial fibrillation (POAF) after cardiac surgery. MethodsPatients who underwent CABG and/or VR in the Heart Center of Beijing Chao-Yang Hospital from March to June 2021 were included. Peripheral blood and pericardial drainage fluid samples were collected before surgery and at 0 h, 6 h, 24 h and 48 h after surgery to detect PICP, ICTP and fibrinogen levels, and preoperative, intraoperative and postoperative confounding factors were also collected. PICP, ICTP and fibrinogen levels were measured by enzyme-linked immunosorbent assay (ELISA). ResultsA total of 26 patients with 125 blood samples and 78 drainage samples were collected. There were 18 males and 8 females with an average age of 64.04±7.27 years. The incidence rate of POAF was 34.6%. Among the factors, the fibrinogen level in pericardial drainage showed two peaks within 48 h after operation (0 hand 24 h after operation) in the POAF group, while it showed a continuous downward trend in the sinus rhythm (SR) group, and the change trend of fibrinogen in pericardial drainage was significantly different over time between the two groups (P=0.022). Fibrinogen in blood, PICP and ICTP in blood and drainage showed an overall decreasing trend, and their trends over time were not significantly different between the two groups of patients (P>0.05). Univariate analysis showed that fibrinogen at 24 h and 48 h after pericardial drainage, fibrinogen in preoperative blood, PICP immediately after surgery and right atrial long axis diameter were significantly higher or longer in the POAF group than those in the SR group. Multiple regression showed that fibrinogen≥11.47 ng/mL in pericardial drainage 24 h after surgery (OR=14.911, 95%CI 1.371-162.122, P=0.026), right atrial long axis diameter≥46 mm (OR=10.801, 95%CI 1.011-115.391, P=0.049) were independent predictors of POAF. ConclusionThis study finds the regularity of changes in fibrinogen and collagen metabolic markers after CABG and/or VR surgery, and to find that fibrinogen in pericardial drainage 24 h after surgery is a potential novel and predictive factor for POAF. The results provide a new idea for exploring the mechanism of POAF, and provide a research basis for the accurate prediction and prevention of clinical POAF.
ObjectiveTo establish a more accurately method to detect the residue of 1,4-butanediol diglycidyl-ether (BDDE) in the cross-linked sodium hyaluronic gel so as to provide a scientific testing method for the quality control. MethodsThe gas chromatography was used to explore the thermal stability of BDDE, and the residues of BDDE in sodium hyaluronic gel was detected by fluorescence spectrophotometry. The hyaluronidase was added to the BDDE standard solution and the improved fluorescence spectrophotometer was used to detect the BDDE residues in the cross-linked hyaluronic sodium gel. ResultsA good linearity was obtained as y=14.102x+1.103 (R2=0.999 8) for BDDE. BDDE was unstable under high temperature and long storage time. The relevant fluorescence intensity was detected with hyaluronidase solution. After adding hyaluronidase into the BDDE standard solutions, the advanced linearity was obtained as y=14.027x+7.062 (R2=0.999 9). ConclusionFluorescent spectrophotometry is a simple, rapid, and accurate method to analyze BDDE residue of cross-linked sodium hyaluronic gel. Due to the poor thermal stability, all the factors related to temperature must be excluded during the process, including the temperature control of every step. Furthermore, the adding of hyaluronidase in the pre-preparation of cross-linked sodium hyaluronic gel can bring interference. So when using fluorescent spectrophotometry, adjustment must be taken before the calibration curve is preparation.
Objective To study the release properties of basic fibroblast growth factor (bFGF) chitosan microspheres prepared by cross-linking-emulsion method using chitosan as a carrier material so as to lay a foundation for further study. Methods Using 0.6% sodium tripolyphosphate solution as a crosslinking agent and 1.5% solution of chitosan as a carrier material, bFGF chitosan microspheres were prepared by cross-linking-emulsion method. Laser particle size analyzer and Zeta electric potential analyzer were used to measure the particle diameter distribution, scanning electronic microscope to observe the morphology, and ELISA to determine the drug loading, the encapsulation rate, and the drug release properties. Results The particle size of bFGF chitosan microspheres ranged 20.312-24.152 μm. The microspheres were round with a smooth surface and uniform distribution, and it had no apparent porosity. The drug loading and encapsulation rate of microspheres were (7.57 ± 0.34) mg/g and 95.14% ± 1.58%, respectively. The bFGF chitosan microspheres could continuously release bFGF for 24 days; the bFGF level increased gradually with time and reached (820.45 ± 21.34) ng/mL at 24 days; and the microspheres had a burst effect, and the burst rate was 18.08%, and the accumulative release rate of the microspheres reached 82.05% during 24 days. Conclusion It is easy-to-operate to prepare the bFGF chitosan microspheres with the cross-linking-emulsion method. The bFGF chitosan microspheres have smooth surface, uniform distribution, and no apparent porosity.
Objective To compare the effectiveness and linear wear rates between high cross-linked polyethylene cup liner and traditional polyethylene cup liner. Methods A retrospective analysis was made on the clinical data of 61 patients (64 hips) who underwent primary total hip arthroplasty (THA) between January 2005 and March 2007. According to the type of cup liner, 61 patients (64 hips) were divided into 2 groups: high cross-linked polyethylene cup liner was used in 30 cases (31 hips, trial group), and traditional polyethylene cup liner in 31 cases (33 hips, control group). There was no significant difference in gender, age, body weight, etiological factor, and preoperative Harris score between 2 groups (P gt; 0.05). Complication was observed after operation; Harris score was used to assess hip function; X-ray film analysis was done to observe osteolysis and prosthesis loosening, and to measure cumulative penetration of the femoral head and linear wear rate. Results All incisions healed by first intention without complication. All patients were followed up. The mean follow-up was 6.3 years (range, 5-7 years) in trial group, and was 6.5 years (range, 4-7 years) in control group. There was no significant difference in Harris score between 2 groups at the 3 months after operation and last follow-up (P gt; 0.05). Osteolysis was observed in 3 cases (3 hips) of control group, but no osteolysis in trial group. X-ray films showed no aseptic loosening in the patients of 2 groups. The mean cumulative penetration of trial group were significantly lower than those of control group at 1-7 years after operation (P lt; 0.05). Linear wear rate of trial group [(0.025 ± 0.002) mm/year] was significantly lower than that of control group [(0.086 ± 0.005 ) mm/year] (Z=6.804, P=0.000). Conclusion High cross-linked polyethylene cup liner has the similar effectiveness as traditional polyethylene cup liner, but it has lower linear wear rate than the traditional polyethylene cup liner.
【Abstract】 Objective To compare the properties of collagen membranes before and after crossl inked and to establ ish the foundation of appl ication of collagen membranes. Methods Fresh bovine tendons were separated and collagen was extracted by washing, smashing and acetic acid dissolving. The collagen protein was determined by ultraviolet spectrophotometer and its characteristics were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), wavelength scanning and amino acids detecting. Collagen membranes were produced by lyophil ization. And then the biocharacteristics of the membranes before and after glutaraldehyde crossl inked were compared. BMSCs separated from volunteer’s bone marrow were seeded on collagen membranes before and after crossl inked by 2×103 in 100 μL medium, seven days after culture, the absorption spectrum of BMSCs was examined, and BMSCs were observed by scanning electron microscope (SEM). Results The contents of collagen protein were 2 mg/mL. The maximum absorption wave length appeared at about 230 nm. SDS-PAGE suggested that molecularweight of main bands was more than 66.2×103, the same as collagen marker from calf skin. There were 21.47% glycine, 12.04% pral ine and 10.18% hydroxyprol ine. No tryptophan was found. Before crossl inked, collagen membranes were in shape of white sponges and with big holes and the range of pH value was from 4.5 to 5.0. SEM showed reticular conformation and pore structure of collagen membranes, but the bore diameter was bigger. Their water-absorbing capacity was 61 times as much as their weight. The mechanical strength was 210 g/cm3. The dissolution time of collagenase was 90 minutes. After crossl inked, collagen membranes became thin, colorless, semi-transparent and compact with better tenacity. Under SEM, compact collagen fiber appeared reticular. There was lower water-absorbing capacity and pH value ranged from 6.5 to 7.0. The mechanical strength was 3 400 g/cm3 and the dissolution time of collagenase became longer. BMSCs could grow better either on before-crossl inked collagen membranes or on after-crossl inked ones. Conclusion As biomaterial scaffolds, after crossl inked collagen membranes were better than before-crossl inked ones.