Reactive oxygen species (ROS) are major contributors to radiation therapy-induced side effects in cancer patients. A fusion antioxidant enzyme comprising glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and a transmembrane peptide has been shown to effectively mitigate ROS-induced damage. To enhance its targeting capability, the fusion protein was further modified by incorporating a matrix metalloproteinase-2/9 substrate peptide (X) and the transmembrane peptide R9, yielding the antioxidant enzyme GST-SOD1-X-R9 (GS1XR). This modification reduced its transmembrane ability in tumor cells, thereby selectively protecting normal cells from oxidative stress. However, the use of non-human GST poses potential immunogenicity risks. In this study, we employed seamless cloning technology to construct an expression vector containing the human GST gene to replace the non-human GST gene, and then expressed and purified novel fusion antioxidant enzymes GS1R and GS1XR. The protective effects of newly constructed GS1R and GS1XR against hydrogen peroxide (H₂O₂)-induced oxidative damage in L-02 cells were then evaluated using GS1 as a control. Enzymatic activity assays revealed that the specific activity of GST in GS1XR remained unchanged compared to the unmodified protein, while SOD activity was enhanced. Exposure to 200 μmol/L H₂O₂ transiently activated the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway; however, this activation diminished after 24 h, reducing cell viability to 48.4%. Both GS1R and GS1XR effectively scavenged intracellular ROS, directly counteracting oxidative stress and promoting Nrf2 nuclear translocation, thereby activating antioxidant pathways and restoring cell viability to normal levels. The two enzymes showed comparable efficacy. In contrast, GS1, lacking transmembrane capability, was restricted to scavenging extracellular ROS and provided only limited protection. In conclusion, both novel fusion antioxidant enzymes demonstrated significant potential in safeguarding normal cells from ROS-mediated oxidative damage. The findings provide a foundation for further investigation in related field.
Humans ; Oxidative Stress/drug effects* ; Hydrogen Peroxide ; Antioxidants/metabolism* ; Glutathione Transferase/metabolism* ; Recombinant Fusion Proteins/pharmacology* ; Superoxide Dismutase-1 ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/biosynthesis*

Objective:To explore the clinical and imaging characteristics of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) caused by mitochondrial DNA 14453G>A (m.14453G>A) mutation.Methods:A case of MELAS caused by m.14453G>A mutation in the First Affiliated Hospital of Harbin Medical University on October 12, 2021 was reported. At the same time, the reported cases of MELAS and Leigh syndrome (LS) caused by the m.14453G>A mutation were reviewed. This enabled a comprehensive summarization, analysis, and comparison of these cases.Results:The patient was a female. She has suffered from the disease since 13-year old with seizures, accompanied by the disturbance of mood and the loss of memory. Brain magnetic resonance imaging findings consisted of lesions in frontal, parietal, occipital, temporal lobe and cerebellar. The patient was initially considered with autoimmune encephalitis and posterior reversible encephalopathy syndrome. Since direct sequencing of the complete mitochondrial genome from blood of the patient revealed m.14453G>A mutation in ND6 gene, and the mutation rate was 17.0%, the patient eventually diagnosed with MELAS based on clinical manifestations, imaging examinations, and genetic testing results. Using "m.14453G>A" as the search term, the relevant literature in China and abroad was retrieved and those with complete clinical data were identified. A total of 11 cases of m.14453G>A mutation including this case were reported, of whom 5 patients were diagnosed as MELAS, and 6 patients were diagnosed as LS. Among the 11 patients, those being adolescent or adult and with lesions in the cortex and subcortical white matter were probably be MELAS; those being infant or young child and with lesions in basal ganglia, thalamus and brainstem could be LS. Conclusions:Mitochondrial disease caused by m.14453G>A gene mutation shows a great heterogeneity, which can cause MELAS and LS. The clinical phenotype of the m.14453G>A mutation may be related to the age of onset and lesion′ s location.

Objective To explore the effects of secreted apoptosis-related protein 1 (SARP1) on apoptosis of the hyperstrophic scar fibroblasts (HSFB) and its regulating mechnisms.Methods The recombinant vector was identified by enzyme digestion analysis.And the virus supernatant of the recombinant vector was extracted from packaged 293 cells,then it infected the skin fibroblasts from hypertrophic scar patients,which aimed to promote its expression of SARP1 protein.After adenovirus infection,the expression of SARP1 in the fibroblasts was confirmed by RT-PCR and Western blot.The effect of SARP1 on proliferation of HSFB was detected by MTT assay,and the effect of SARP1 on apoptosis of HSFB was detected and change of the cells functions were analyzed by FACS.Results Recombinants were confirmed.After adenovirus infection,both protein and mRNA of SARP1 were detected in HSFB.And the mRNA value of SARPlwas detected to increase significantly by RT-PCR and the protein expression was detected to increase significantly by Western blot (P＜0.05).The proliferation in the groups of the adenovirus infection and HSFB was positively regulated by SARP1 (P＜0.01) and the apoptosis of them was inhibited by the expression of SARP1 as compared to the control groups of HSFB and Ad-EGFP.It showed that the apoptosis index decreased as compared the group of infected fibroblasts to the control group by flow cytometry.Conclusions SARP1 could be highly expressed in HSFB by adenovirus infection,exhibiting the proliferation-enhancing and apoptosis-inhibiting effects on HSFB.

Polyamidoamine-polyethylene glycol (PAMAM-PEG) copolymers were synthesized using IPDI as coupling reagent by two-step method. The copolymers were characterized by IR spectrum and 1H NMR spectrum, and the PEG conjugating ratios of the copolymers were calculated equal to 10% and 30% separately. MTT assay indicated that after PEGylation a lower cytotoxicity of the copolymers could be found, and with increasing PEG conjugating ratio the cytotoxicity decreased obviously. Agarose gel retardation assay demonstrated that PAMAM-PEG copolymers could be combined with DNA and PAMAM-PEG/DNA complexes were prepared by self-assembly. DLS measurement showed that when N/P > or = 50, the particle size of copolymer/ gene complexes was in a range of 150-200 nm, and the zeta potential was in a range of 10-25 mV. In vitro gene transfection illustrated that when N/P < or = 50, the gene transfection efficiency of PAMAM-PEG copolymers was a little less than that of PAMAM-G5, but the transfection efficiency can be raised by increasing N/P ratio or transfection time. Considering both cytotoxicity and transfection efficiency aspects PAMAM-PEG-13 was more effect than PAMAM-PEG-39 in PEGylation.

Objective To investigate the prevalence rate of metabolic syndrome (MS) and its related diseases in Bengbu community residents. Methods 3246 residents aged from 25 to 74 years were chosen through cluster random sampling method, including 1459 males and 1787 females. MS and its related diseases were examined. Results (1) The prevalence rate and standardized rate of MS was 20. 5% and 16.0%, respectively, which reached higher level all over China. The prevalence rate of male and female was 22. 9% and 18. 6%, respectively. The highest prevalence rate occurred in presenium patients. (2)The prevalences of various metabolic diseases were increased with aging except for overweigh/obesity and MS (P<0.01). The prevalence of various metabolic diseases was 1 times higher in elderly patients than that in young patients, and the prevalence rate of MS in male and female increased to 2.8 times and 2.7 times, respectively. The prevalence rate of type 2 diabetes mellitus increased biggest, male to 7.9 times and female to 12.8 times compared with the original level. (3) The multiple components clustering rate of MS was high, only 33.8% of individuals had no metabolic disorder. Conclusions The prevalence rates of MS and its related diseases are high in Bengbu community residents. It is necessary to take comprehensive measures to prevent and control MS in community.

O-Carboxymethylchitosan (O-CMC) in 1000 g batch was prepared from chitin as starting material and its chemical structure was confirmed by analysis of IR and NMR. O-CMC solution, sodium hyaluronate (HA) solution and physiological saline were used in Sprague-Dawley rat model for prevention of postsurgical adhesions; after 7 days of an abdominal operation, the 3 groups were evaluated according to Belluco standard, the mean scores of O-CMC group, HA group and physiological saline group were 2.5 +/- 3.1, 3.3 +/- 3.6 and 10.3 +/- 1.0, respectively. Histological inspection showed that in O-CMC group, mesothelial cells on peritonaeum or cecum surfaces were almost restored; in HA group the injured surface of peritonaeum was mostly repaired, but in physiological saline group the injured surface of cecum was just a little repaired; there were extensive adhesions between peritonaeum and cecum, and inflammatory response was quite serious. Experimental results indicated that O-CMC and HA had excellent efficiency and O-CMC was slightly better than HA for the prevention of postsurgical adhesions.
Abdomen ; surgery ; Animals ; Chitosan ; analogs & derivatives ; therapeutic use ; Female ; Intestinal Diseases ; prevention & control ; Male ; Postoperative Complications ; prevention & control ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tissue Adhesions ; prevention & control

This study was designed to investigate the changes of prostaglandin I2 (PGI2) and nitric oxide (NO) secreted by endothelialized polyurethane small diameter artificial blood vessel. The peripheral blood mononuclear cells of healthy adult were separated and induced into endothelial progenitor cells (EPCs), which were identified by the methods of discrepancy microphage and fluorescent immunology labeling. After the induced cells being seeded on the polyurethane small-diameter artificial vessels, the endothelialized polyurethane small diameter artificial blood vessels were divided into four different experimental groups, including stationary group, low-flow shear stress group (5 dynes/cm2), medium-flow shearstress group (15 dynes/cm2), and high-flow shear stress group (25 dynes/cm2). Then, the levels of 6-ketoprostaglandin F1alpha (6-keto-PGF1alpha) and NO of different time were measured by enzyme-linked immunosorbent assay and nitrate reductase methods. The peripheral blood mononuclear cells differentiated into EPCs. They presented typical "spindie-shaped" appearance, and they were positively labeled by fluorescent acetylated-LDL, lectin, FLK-1 and vWF. Shear stress enhanced the production of NO and 6-keto-PGF1alpha by EPCs in a dose-dependent manner. Therefore, shear stress increases the secretion of NO and PGI2 by EPC, which suggests that shear stress can improve the antithrombogenic potentials of endothelialized polyurethane small diameter artificial blood vessel.
Bioartificial Organs ; Biocompatible Materials ; chemistry ; Blood Vessel Prosthesis ; Cell Adhesion ; Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Epoprostenol ; metabolism ; Fibrinolytic Agents ; metabolism ; Humans ; In Vitro Techniques ; Leukocytes, Mononuclear ; cytology ; Nitric Oxide ; metabolism ; Polyurethanes ; chemistry ; Stress, Mechanical

In this study, the peripheral blood mononuclear cells of healthy adult were acquired and inducted by vascular endothelial growth factor, et cetera. The differentiated endothelial cells were observed and identified as EPCs by the double positive staining of fluorescent labeled acetylated-LDL and lectin, seeded on the polyurethane small-diameter artificial vessels, treated by shear stress of 15 dyn/cm2, and observed by scanning electronic microscopy. As a result, the peripheral blood mononuclear cells differentiated into EPCs. They were positively stained by labeled acetylated-LDL and lectin. Under observation of scanning electronic microscope, the unseeded polyurethane small-diameter artificial vessel being suited for the growth and spreading of the cells; the cell lineage on surface of artificial vessels of stationary group being arrayed in chaos, and that of shear stress group being arrayed in direction. Therefore, the peripheral cells can differentiate into EPCs, and EPCs can be used as novel source cells for the accelerated endothelialization of small diameter artificial vessel. Shear stress contributes to the mechanic moulding of cell lineage on the surface of artificial vessel.
Bioartificial Organs ; Biocompatible Materials ; Blood Vessel Prosthesis ; Cell Adhesion ; Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Polyurethanes ; chemistry ; Prosthesis Design ; Shear Strength ; Stem Cells ; cytology ; Stress, Mechanical

BACKGROUND: At present, after transplantation of small diameter artificial blood vessel, long-term patency rate is low due to being lacking of endothelial cells for lining and anti-thrombus characters. In some studies,mature endothelial cells were tried to be seeded in the artificial vessel to boost up its anti-thrombus capability so as to improve the long-term patency rate, but we got unsatisfied effect due to the defects of seed cells and scaffolds. Therefore, in clinic, proper seed cells and vascular scaffolds have been searched for improving the long-term low pateney rate in transplantation of small diameter artificial blood vessel.OBJECTIVE: To investigate the feasibility that differentiation of bone marrow mononuclear cells induced in vitro into endothelial-progenitor cells (EPCs) and seed polyurethane small diameter artificial blood vessel so as to provide proper seed cells for endotheliazation of polyurethane small diameter artificial blood vessel.DESIGN: Observation experiment SETTING: Cardivascular Medical Department and Staff Room of Immunology, First Hospital Affiliated to Sun Yat-sen University MATERILAS: This experiment was carried out at the First Hospital Affiliated to Sun Yat-sen University from September 2004 to May 2005. About 10 mL of bone marrow from healthy adult volunteers (n=7) was used in this experiment.METHODS: Bone marrow mononuclear cells of healthy adult were collected and put in the fibronectin pre-coated DMEM culture medium, then induced by vascular endothelial growth factor and basic fibroblast growth factor. Induced cells were observed under fluorescence microscope and identified with immunohistochemical staining. The induced and proliferated EPCs were seeded onto the surface of polyurethane small diameter artificial blood vessel. Morphological change was observed under scanning electron microscope.MAIN OUTCOME MEASURES: ① Cellular morphological change.② Staining results of immunohistochemical VWF and CD 34 antibody . ③ Adhesive growth status of EPCs on the polyurethane small diameter artificial blood vessel RESULTS: ① In the vascular endothelial growth factor and basic fibroblast growth factor and other inducers , bone marrow mononuclear cells differentiated into EPCs , presenting typical "spindle-shaped" appearance under an inverted fluorescence microscope and became to form a monolayer that arrayed in "cobblestone-like" ② Immunohistochemical staining showed von willebrand factor(VWF) and CD34 antigen stained positive. ③ Under the scanning electron microscope, surface of polyurethane small diameter artificial blood vessel without seeded cells presented typical polyporous honeycomb-like structure , and the size of hole suited the crawling of EPCs. After seeding the cells, we observed the adhesion, crawling and spreading of the EPCs on the surface of polyurethane small diameter artificial blood vessel. Some EPCs grew into the honeycomb-like holes were seen occasionally.CONCLUSION: Bone marrow mononuclear cells can be induced and differentiated into EPCs, while induced and differentiated EPCs well grow adhesively in the polyurethane small diameter artificial vessels, suggesting that differentiation of bone marrow mononuclear cells induced in vitro into EPCS, which can be used as seed cells for endothelialization of polyurethane small diameter artificial blood vessels.

In order to investigate the ability of PEG-PEI copolymers as gene carriers for delivery of VEGF165. A series of PEG-PEI copolymers with different PEG grafting was prepared and the cytotoxicity was evaluated. Simultaneously,the VEGF165 gene segment with HindⅢ and BamHⅠ site was obtained by PCR, which was cloned into pEGFP-C1. PEG-PEI/ pEGFP-VEGF165 complexes were formed by self-assembly and transfected HUVEc. Transfection efficiency was evaluated by measuring the percentage of cells expressing green fluorecensce protein. The VEGF expression was detected by ELISA, RT-PCR, and the effect of transfection on growth of endothelial cell was evaluated by MTT. The results suggested that the formation of PEG-PEI copolymers could help to reduce the cytotoxicity of PEI. After transfection, the strong expression of green fluorescence protein was observed by fluorescence microscopy. The transfection efficiency was influenced by the number of PEG side chains and N/P ratio. Of all copolymers tested, the transfection efficiency of PEG-PEI(5-25-1) at N/P = 30 reached a maximum, which was much higher than that of PEI. The expression of VEGF protein and mRNA increased significantly, and HUVEc proliferation was accelerated after transfection.These results indicates PEG-PEI copolymers can be used as effective gene carriers for delivery of pEGFP-VEGF165 gene.