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At present, acellular matrix is an effective replacement material for the treatment of skin damage, but there are few systematic evaluation studies on its performance. The experimental group of this study used two decellularization methods to prepare the matrix: one was the acellular matrix which sterilized with peracetic acid first (0.2% PAA/4% ethanol solution) and then treated with hypertonic saline (group A), the other was 0.05% trypsin/EDTA decellularization after γ irradiation (group B); and the control group was soaked in PBS (Group C). Then physical properties and chemical composition of the three groups were detected. Hematoxylin eosin (HE) staining showed that the acellular effect of group B was good. The porosity of group A and B were both above 84.9%. In group A, the compressive modulus of elasticity was (9.94 ± 3.81) MPa, and the compressive modulus of elasticity was (12.59 ± 5.50) MPa in group B. There was no significant difference between group A or B and group C. The total content of collagen in acellular matrix of group A and B was significantly lower than that of group C (1. 662 ± 0.229) mg/g, but there was no significant difference in the ratio of collagen Ⅰ/Ⅲ between group B and group C. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that there was no significant difference in microstructure. Qualitative detection of fibronectin and elastin in each group was basically consistent with that in group C. Therefore, acellular matrix of group B had better performance as scaffold material. The experimental results show that the acellular matrix prepared by γ-ray sterilization and decellularization of 0.05% Trypsin enzyme/EDTA could be used for the construction of tissue-engineered skin. It could also provide reference for the preparation and mounting of heterogeneous dermal acellular matrix. It was also could be used for electrostatic spinning or three-dimensional printed tissue engineered skin scaffold which could provide physical and chemical parameters for it.
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Dermis Acelular , Células Cultivadas , Matriz Extracelular , Porosidad , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Objective@#To explore the inhibitory effect of icotinib combined with cytokine induced killer (CIK) on various human lung adenocarcinoma cell lines in vitro.@*Methods@#The inhibitory effect of icotinib alone or icotinib combined with CIK on HCC827 and A549 cells was detected by cell counting kit-8(CCK-8). The apoptosis was detected by flow cytometry via Annexin V/PI staining. The effect of icotinib on CIK phenotype was detected by flow cytometry.@*Results@#The inhibitory rates of HCC827 cells treated with 1.5, 3, 6, 12 μmol/L icotinib were (5.64±0.05)%, (8.62±0.45)%, (14.57±0.65)% and (18.52±0.91)%, respectively. The inhibitory rates of A549 cells were (1.64±0.48)%, (2.09±0.28)%, (3.69±0.45)%, (4.41±0.58)%, respectively. At the same concentration, the inhibitory rate of HCC827 cells with icotinib treatment was significantly higher than that of A549 cells (P<0.05). When the effector/target ratio was 10∶1, 20∶1 or 40∶1, the inhibitory rates of HCC827 cells co-cultured with CIK were (15.17±2.33)%, (42.59±7.18)%, (62.59±8.95)%, respectively, and the inhibitory rates of A549 were(16.99±2.81)%, (46.31±1.89)%, (58.24±4.23)%, respectively. The inhibitory rate of HCC827 cells co-cultured with CIK was not significantly different from that of A549 cells at the same effector/target ratio (P10∶1=0.299, P20∶1=0.318, P40∶1=0.366). When the effector/target ratio of CIK combined with 6 μmol/L icotinib was 10∶1, 20∶1 or 40∶1, the inhibitory rates of HCC827 cells were (37.07±3.50)%, (76.03±6.55)%, (80.34±10.69)%, respectively, and the inhibitory rates of A549 cells were(25.72±1.41)%, (52.76±3.82)%, (62.26±1.94)%, respectively. The inhibitory rates of 6 μmol/L icotinib combined with CIK were significantly higher than those of icotinib group and CIK group alone at the same effector/target ratio (P<0.05), except for the effector/target ratio at 40︰1 on A549 cells (P=0.089). Moreover, all of the combination index (CI) of combined group were <1 (P<0.05). The apoptotic rates of HCC827 and A549 cells induced by icotinib combined with CIK were significantly higher than those of icotinib group and blank control group (P<0.05), especially the proportion of late apoptotic or necrotic cells.Increasing effector/target ratio of CIK contributed to stronger inhibition(P<0.05). The expressional rate of CIK phenotype with or without icotinib treatment was not significantly different from each other(P>0.05).@*Conclusions@#EGFR mutant lung adenocarcinoma cells are more sensitive to icotinib, while the EGFR mutation status has no effect on the killing effect of CIK cells. icotinib combined with CIK has a synergistic effect on the inhibition of tumor growth, and icotinib has no any impact on the phenotype of CIK cells.
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Objective:To discuss the effect and mechanism of autophagy inhibitor 3-MA on arsenic trioxide inducing apoptosis of acute T-cell leukemia cell line Jurkat cells.Methods:Proliferation inhibition of Jurkat cells treated with arsenic trioxide was detected by XTT.Morphological characteristics of Jurkat cells treated with different concentrations arsenic trioxide were observed by electron mi-croscope.Microtubule-associated protein 1 light chain 3B (LC-3B) protein expression was detected by Western blot and flow cytome-try.Apoptosis rates of Jurkat cells treated with 3-MA combining arsenic trioxide were detected by flow cytometry using AnnexinV-FITC/PI double staining.Results:Arsenic trioxide inhibited the growth of Jurkat cells in a dose and time dependence.We observed different morphological characteristics of autophagy , apoptosis and necrosis accompanying more autophagosomes in Jurkat cells which were treated with arsenic trioxide 2.5,5,10 μmol/L after 24 h.LC3B mean fluorescence intensity (MFI)relative multiples were(3.1±0.2) fold,(4.6±0.31)fold,(34.2±4.5)fold with 5 μmol/L arsenic trioxide treated Jurkat cells 0,24,48 h,and the P values between each of the two groups were less than 0.05,which increased depending time consistently with the growth inhibition rates.LC-3B protein expression gradually increased treated Jurkat cells with arsenic trioxide after 24 h,48 h.The growth inhibition rate (60.6±8.3)%was significantly different treated with arsenic trioxide combining 3-methyl adenine ( 3-MA ) while it was ( 33.4 ±9.1 )% treated with arsenic trioxide alone, however, LC-3B protein expression gradually decreased.Jurkat cell apoptosis rate ( 44.96 ±3.60 )% was significantly increased treated with arsenic trioxide combining autophagy inhibitor(3-MA) while it was (2.94±0.26)% treated with arsenic trioxide alone, and this difference was statistically significant.Conclusion: 3-MA increased apoptosis rates of Jurkat cells inducing by Arsenic trioxide and it may be related with inhibition of autophagy and induction of apoptosis.
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BACKGROUND:The treatment of diabetic foot has become a“bottleneck”. The transplantation of autologous bone marrow or peripheral blood endothelial progenitor cells is a new way for the treatment of diabetic foot in recent years. OBJECTIVE:To review the effects of vascular endothelial growth factor receptor 2 and the integrin in treatment of diabetes foot by transplantation of endothelial progenitor cells. METHODS:The first author searched PubMed database and Chinese periodical ful-text database for related articles published between January 1998 and December 2012, withdiabetic foot, endothelial progenitor cells, VEGFR-2, integrin, synergistic effectin English and Chinese, respectively. A total of 98 articles were searched, and 60 were included after excluding repetitive articles. RESULTS AND CONCLUSION:In the diabetic foot treatment, autologous bone marrow or peripheral blood endothelial progenitor cells has around increasing attention, and gradual y become the focus of this field. Therapy with endothelial progenitor cells has gradual y developed into a new method for diabetic foot. Vascular endothelial growth factor receptor 2 and the integrin may exhibit synergistic effects in the treatment of diabetes foot. However, the mechanism remains poorly understood. Further studies are needed to wel understand their mechanism of action.
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10.3969/j.issn.2095-4344.2013.23.002
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Objective To investigate the effects of mannan-binding lectin(MBL) on TNF-α production induced by peptidoglycan (PGN) and its mechanism in human THP-1/CD14 monocytes.Methods The THP-1/CD14 cells were stimulated for 24 h with PGN at the indicated ratios after pretreated with human natural MBL at concentrations ranging from 1 to 20 mg/L for 2 h.The content of TNF-α and IL-6 in culture supernatants were detected by ELISA,and the levels of TNF-α and IL-6 mRNA expressions in these cells were determined by RT-PCR.FACS was used to investigate the interaction of MBL with THP-1/CD14 cells and the impact of MBL on PGN binding to THP-1/CD14 cells.Western blot was used to detect PGN-induced NF-κB translocation in THP-1/CD14 cells.Results ELISA showed that secretion of TNF-α and IL-6 from THP-1/CD14 cells could be induced by PGN ;The productions of TNF-α and IL-6 by THP-1/CD14 cells induced with PGN were profoundly inhibited by MBL at higher concentrations (10-20 mg/L) but not MBL at lower concentrations (1 mg/L).RT-PCR analysis also indicated that the mRNA expressions of TNF-α and IL-6 in THP-1/CD14 cells were decreased by MBL at higher concentration,compared to the corresponding THP-1/CD14 cells stimulated with PGN only.FACS showed that the binding of MBL to THP-1/CD14 cells was evident in a Ca2+-dependent manner.PGN could competitively inhibit the binding of MBL to THP-1/CD14 cells.MBL could competitively inhibit the binding of PGN to THP-1/CD14 cells by binding to THP-1/CD14 cells directly.Similarly,MBL at higher concentration (20 mg/L) decreased the NF-κB translocation in THP-1/CD14 cells.Conclusion MBL may inhibit TNF-α and IL-6 production induced by PGN in THP-1/CD14 cells through NF-κB signaling pathways,suggesting that MBL can play some roles in the regulation of PGN-induced inflammatory response.