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Objective:To observe the expression of deleted in malignant brain tumor protein 1 (DMBT1) in rat acute respiratory distress syndrome (ARDS) model induced by sepsis and its relationship with ARDS related biomarkers.Methods:Forty-eight healthy male rats were randomly divided into sham operation group (Sham group) and ARDS model group, and the rats in each group were further divided into three subgroups at 6, 12 and 24 hours after operation, with 8 rats in each subgroup. The rats in the Sham group were exposed to the cecum only, and sepsis induced ARDS model was reproduced by cecal ligation and puncture (CLP) in the ARDS model group. The general performance was observed at 6, 12, 24 hours after operation. Abdominal aortic blood of rats was collected, and the levels of DMBT1, surfactant-associated protein D (SP-D), vascular endothelial growth factor (VEGF), interleukins (IL-6, IL-10) in serum were determined by enzyme-linked immunosorbent assay (ELISA). The lung tissues were collected, and the lung wet/dry weight (W/D) ratio was determined. The lung tissue pathological changes were observed under light microscope after hematoxylin-eosin (HE) staining, and the lung tissue injury score was evaluated. The expression of DMBT1 protein in lung tissue was determined by Western blotting. The relationship between the serum DMBT1 and SP-D, VEGF, IL-6, IL-10, lung tissue injury score were analyzed by Pearson correlation analysis.Results:Rats in the ARDS model group showed obvious pathological manifestations after operation. The alveolar structure destruction, inflammatory cell infiltration, and alveolar hemorrhage were observed under microscope. Compared with the Sham group, the lung tissue injury score and the lung W/D ratio at 12 hours after operation in the ARDS model group were significantly increased (lung tissue injury score: 3.35±0.13 vs. 1.16±0.07, lung W/D ratio: 5.36±0.44 vs. 4.38±0.35, both P < 0.05), and pulmonary edema was present, which suggested that the ARDS model caused by CLP was successfully reproduced. The results of ELISA and Western blotting showed that the levels of serum DMBT1, SP-D, VEGF and IL-6 in the ARDS model group increased gradually with time, while the level of IL-10 increased first and then decreased. Compared with the Sham group, the levels of DMBT1 in serum and the expressions of DMBT1 protein in lung tissue in the ARDS model group were significantly increased from 6 hours after operation [serum (ng/L) : 231.96±19.17 vs. 187.44±10.19, lung tissue (DMBT1/β-actin): 2.05±0.19 vs. 0.93±0.25, both P < 0.05], and the levels of SP-D, VEGF, IL-6 and IL-10 in serum were significantly increased from 12 hours after operation [SP-D (ng/L): 73.35±8.05 vs. 43.28±5.77, VEGF (ng/L): 89.85±8.47 vs. 43.19±5.11, IL-6 (ng/L): 36.01±2.48 vs. 17.49±1.77, IL-10 (ng/L): 84.55±8.41 vs. 39.83±5.02, all P < 0.05]. Pearson correlation analysis showed that serum DMBT1 was positively correlated with serum SP-D, VEGF, IL-6, IL-10 and lung injury score at 12 hours and 24 hours in the ARDS model group (12 hours: r values were 0.946, 0.942, 0.931, 0.936, 0.748, respectively; 24 hours: r values were 0.892, 0.945, 0.951, 0.918, 0.973, respectively; all P < 0.05). Conclusion:DMBT1 is a novel early biomarker of ARDS by affecting alveolar epithelial cell, alveolar capillary permeability and inflammatory response.
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Objective: To establish a method for the induction of peripheral blood mononuclear cells to hepatocyte-like cells, and preliminarily investigate cell response to injury under the effect of acetaminophen (APAP). Methods: The surface marker CD45 of peripheral blood mononuclear cells wase detected cells by using flow cytometry and immunofluorescence methods. The cellular morphology of induced hepatocyte-like cells was observed under an inverted microscope. Real-time fluorescent quantitative PCR (RT-PCR) was used to detect the expression level of hepatocyte-specific genes, such as cytochrome (CY) P1A2, CYP3A4, CYP2C9, albumin (ALB), alpha-fetoprotein (AFP), and hepatocyte nuclear factor (HNF)4α mRNA. Immunofluorescence method was used to detect intracellular hepatocyte markers AFP, HNF4α, and ALB expression at the protein level. Biochemical analyzer was used to detect hepatocyte-specific secretory functions of AFP, ALB, and urea. Luciferase chemiluminescence method was used to detect the activity of key drug metabolizing enzyme CYP3A4. Colorimetric assay was used to detect the effect of the drug acetaminophen on hepatocyte-like cells, and alanine aminotransferase (ALT) was used as an indicator of liver cell injury. The statistical differences between the data were compared with t-test and rank-sum test. Results: The positive expression rate of CD45 cell surface markers isolated from peripheral blood mononuclear cells was about 98%, and hepatocyte-like cell morphology changes appeared on 15th day of induction. Compared with isolated mononuclear cells, CYP1A2, CYP3A4, CYP2C9, ALB, AFP and HNF4α mRNA was markedly elevated. The expression level of AFP, ALB and HNF4α protein were equally increased, and the secretory function of AFP, ALB and urea were enhanced. Compared with primary hepatocytes, CYP1A2, CYP2C9, AFP, HNF4α mRNA, and CYP3A4 mRNA did not decrease. The expression levels of AFP, ALB, and HNF4α proteins in the cells did not decrease, and the secretory function of AFP, ALB, and urea did not decrease. In addition, the CYP3A4 enzyme activity produced by hepatocyte-like cells was similar to that of primary hepatocytes. Compared with hepatocyte-like cells incubated without APAP, hepatocyte-like cells incubated with APAP had higher ALT level. Under the effect of APAP, the ALT level of hepatocyte-like cells was higher than isolated mononuclear cells. Conclusion: Peripheral blood mononuclear cells can be induced into hepatocyte-like cells with partial characteristics of hepatocytes, including the activity of CYP3A4, a key enzyme of hepatocyte drug metabolism. Additionally, preliminarily ALT secretory features reflect the hepatocytes injury under the effect of acetaminophen.
Subject(s)
Acetaminophen/pharmacology , Cell Differentiation , Cells, Cultured , Hepatocytes , Leukocytes, Mononuclear , RNA, MessengerABSTRACT
Objective To analyze the relationship of closed staple height with tissue damage and compression pressure, so as to provide theoretical references and guidance for the surgeon to choose the appropriate staple cartridge and height, as well as improve the safety of operation. Methods The finite element model of stapled colorectal end-to-end anastomosis was established based on analysis of staple-tissue interaction. Large intestine tissues with different wall thicknesses (1.0-1.5 mm) were compressed by closed staples with 4 different height to compare changes in stress distributions and average radial pressure. Results When the tissues were compressed by closed staple with height of 1.0, 1.1, 1.2 and 1.5 mm, respectively, the average radial stress of compressed tissues with wall thicknesses of 1.2, 1.3, 1.4, and 1.5 mm were 56.0, 58.6, 59.7 and 57.3 kPa, respectively, which was close to the optimal compression pressure. Stress concentrations were found in contact area of the staple and tissues，with the maximum stress being 2 783, 1 750, 1940 and 2 030 kPa, respectively. Conclusions Tissue damage cannot be completely avoided in anastomotic surgery, and stress concentration is generally located near contact region of the staple and tissues. The optimal closed staple height ranges in 50%-60% of the uncompressed tissue height.
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By developing a novel endoscopic succession closing device to overcome the shortcomings of existing devices that cannot deploy several clips at a time, to perform structural analysis on different clamp structures and to validate their performances in tissue closure through finite element analysis. Methods Comparative analyses of three clamp structures, namely, the aligning tooth structure (original, clamp A), the staggered tooth structure (clamp B), a combination structure with page break angle and staggered tooth (clamp C), were performed to analyze pressure and its distribution on tissues when clamping the stomach wall. Displacement of 7.5 mm was then applied on the clamps to simulate the effect of the operating procedures of the device and tissue kick-back. Results The maximum stresses of the clamp A and B were located on the first pair of teeth which was closest to the rotating shaft, with the stress being 10.39 kPa and 10.11 kPa, respectively. The maximum stress (11.35 kPa) of the clamp C was located on the second pair of teeth. For clamp A and B, the longer the distance to shaft, the larger pressure on stomach tissues. While for clamp C, the pressure on device-tissue interface showed little change along the path. Under tensile displacement, clamp A and B slipped off from the tissue when displacements reached to 5 mm and 6.5 mm, respectively, while clamp C did not. Conclusions Clamp with page break angle and staggered tooth can exert the uniform max pressure to tissues and provide a larger contact area away from the rotating shaft, thus improving anti-slippage and performance of the novel endoscopic closing device.
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Objective By developing a novel endoscopic succession closing device to overcome the shortcomings of existing devices that cannot deploy several clips at one time,to perform structural analysis on different clamp structures and to validate their performances in tissue closure through finite element analysis.Metbods Comparative analyses of 3 clamp structures,namely,the aligning tooth structure (original,clamp A),the staggered tooth structure (clamp B),a combination structure with page break angle and staggered tooth (clamp C),were performed to analyze pressure and its distribution on tissues when clamping the stomach wall.Displacement of 7.5 mm was then applied on the clamps to simulate the effect from operating procedures of the device and tissue kick-back.Results The maximum stresses of the clamp A and B were located on the first pair of teeth which was closest to the rotating shaft,with the stress of 10.39 kPa and 10.11 kPa,respectively.The maximum stress (11.35 kPa) of the clamp C was located on the second pair of teeth.For clamp A and B,the longer the distance to shaft,the larger pressure on stomach tissues.While for clamp C,the pressure on device-tissue interface showed little change along the path.Under tensile displacement,clamp A and B slipped off from the tissue when displacements reached to 5.0 mm and 6.5 mm,respectively,while clamp C did not slip off.Conclusions Clamp with page break angle and staggered tooth can exert the uniform maximum pressure to tissues and provide a larger contact area away from the rotating shaft,thus improving the anti-slippage and performance of the novel endoscopic closing device.
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Objective By developing a novel endoscopic succession closing device to overcome the shortcomings of existing devices that cannot deploy several clips at one time,to perform structural analysis on different clamp structures and to validate their performances in tissue closure through finite element analysis.Metbods Comparative analyses of 3 clamp structures,namely,the aligning tooth structure (original,clamp A),the staggered tooth structure (clamp B),a combination structure with page break angle and staggered tooth (clamp C),were performed to analyze pressure and its distribution on tissues when clamping the stomach wall.Displacement of 7.5 mm was then applied on the clamps to simulate the effect from operating procedures of the device and tissue kick-back.Results The maximum stresses of the clamp A and B were located on the first pair of teeth which was closest to the rotating shaft,with the stress of 10.39 kPa and 10.11 kPa,respectively.The maximum stress (11.35 kPa) of the clamp C was located on the second pair of teeth.For clamp A and B,the longer the distance to shaft,the larger pressure on stomach tissues.While for clamp C,the pressure on device-tissue interface showed little change along the path.Under tensile displacement,clamp A and B slipped off from the tissue when displacements reached to 5.0 mm and 6.5 mm,respectively,while clamp C did not slip off.Conclusions Clamp with page break angle and staggered tooth can exert the uniform maximum pressure to tissues and provide a larger contact area away from the rotating shaft,thus improving the anti-slippage and performance of the novel endoscopic closing device.
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OBJECTIVE: To explore the toxicity of 1,2-dichloroethane( 1,2-DCE) and its metabolites on human astrocytes( HAs). METHODS: Different doses of 1,2-DCE( 5. 00,10. 00,25. 00,50. 00 and 100. 00 mmol/L),2-chlorohydrins( 5. 00,25. 00,50. 00,100. 00 and 200. 00 mmol/L),2-chloroacetaldehyde( 1. 00,5. 00,10. 00,20. 00 and 50. 00 mmol / L) and chloroacetic acid( 0. 01,0. 05,0. 10,0. 50 and 1. 00 mmol / L) were used for treating HAs in vitro during their logarithmic phase. After 24 hours of culture,the morphology of HAs was observed by fluorescent inverted phase contrast microscope. The survival rate and the inhibition ratio of HAs were detected by CCK-8 colorimetry to estimate the50% inhibiting concentration in 24 hours( 24 h-IC50). The apoptosis of HAs was tested by double-labeling and flow cytometry using Annexin Ⅴ-fluorescein isothiocyanate and propidium iodide. RESULTS: The morphology of HAs changed in varying degrees after 24 hours exposure to 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid. The changes included smaller size of cells,pseudopodia tapering,increased intracellular particles and suspension of circular cells and decreased transparency of cells. With the increasing does of 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid exposure,the survival rates of HAs decreased( P < 0. 01),while its inhibition ratios increased( P <0. 01). They all showed dose-effect relationship. 24 h-IC50 of the above 4 chemicals were 56. 25,235. 00,26. 43 and1. 38 mmol / L,respectively. The 1,2-DCE,2-chlorohydrins and chloroacetic acid could induce the apoptosis of HAs and the apoptosis rate of HAs was positively correlated with the 3 kinds of chemicals( P < 0. 01). CONCLUSION: 1,2-DCE and its metabolites 2-chloroacetaldehyde,2-chlorohydrins and chloroacetic acid can lead to toxic damage and induce the apoptosis of HAs. Chloroacetic acid has the strongest toxicity among the metabolites.
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Objective To design a novel endoscopic successive hemostasis and closing device, and to validate whether the device can meet the needs of tissue closure by finite element analysis. Methods By using the novel device, the target tissue was clamped and the clip was then pushed to pierce the tissue. Under the compression between the clip and the inner side of the grasper, the thinner arms of the clip were forced to bend and close to stay in the tissue, and then the inverse displacement of 2 mm was applied on the clip. The elastic limit and tensile strength of the clip were set as 239.0 and 901.0 MPa, respectively. Results Deformation did not occur in the piercing process of the clip, with the maximum stress of 212.6 MPa. The deformed shape of the clip in the bending process matched its design expectation, with the maximum stress of 727.7 MPa. The maximum stress of the clip was 75.8 MPa under 2-mm inverse displacement. Material failure was not found in the bending process or with 2-mm inverse displacement, and the maximum stress in the whole process was 741.0 MPa. Conclusions The novel endoscopic successive hemostasis and closing device proposed in this study can deploy 4 clips at one time, together with an independent grasper for gathering tissues, which can shorten the reloading time and improve the accuracy of clip deployment. The effectiveness and safety of the device is also proved by using finite element method.
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Objective To evaluate the role of miRNA-214 in myocardial injury in septic mice.Methods Sixty-six healthy male Kunming mice,weighing 25-30 g,were randomLy (random number) divided into4 groups:sham operation group (Sham group,n =18),cecal ligation and puncture group (CLP group,n =18),CLP + miRNA-214 precursor treated group (pre-miR-214 group,n =12),CLP + miRNA-214 inhibitor treated (anti-miR-214 group,n =12),and the rest six mice were treated with miRNA-214 precursor or inhibitor intravenously.Sepsis was induced by CLP,pre-miR-214 group and anti-miR-214 group were respectively treated with miRNA-214 precursor or miRNA-214 inhibitor before the CLP,Sham group were exposed to the cecum only.Mice were sacrificed and hearts were removed for determination of miRNA-214 expression by RT-PCR in Sham and CLP group at 6 h,12 h,and 24 h.Blood samples were collected from inferior vena cava at 12 h and 24 h after CLP for determination of the B-type natriuretic peptide (BNP) and cardiac troponin-I (cTnI) concentration by ELISA,then the mice were sacrificed and hearts were removed for determination of the inflammatory factor concentration by ELISA,cardiac myocyte apoptosis rate by flow cytometry and histopathological changes by microscopic examination.Statistical analysis was carried out with SPSS 13.0 software for One-way ANOVA.Results (1) The miRNA-214 expression was higher in CLP group than that in Sham group at 6 h,12 h and 24 h;(2) Compared with CLP group,the miRNA-214 expression in pre-miR-214 group was increased,but decreased in anti-miR-214 group at 12 h;(3) Compared with CLP group,the concentrations of the serum BNP,cTNI,tumor necrosis factor-alpha (TNF-α) and the interleukin-6 (IL-6) were lower than those in pre-miR-214 group,whereas the interleukin-10 (IL-10) was increased.However,the levels of these variables changed just in opposite direction in anti-miR-214 group at 12 h,24 h;(4) Compared with CLP group,the myocardial cell apoptosis rate was decreased in pre-miR-214 group,but increased in anti-miR-214 group at 24 h;(5) The microscopic examination showed that myocardial injury was attenuated in pre-miR-214 group compared with CLP group.Conclusions The miRNA-214 expression was increased in myocardial injury in CLP mice,suggesting miRNA-214 expression relating to myocardial protection in sepsis.
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Objective: To investigate the molecular mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) into myocardial cells induced by 5-azacytidine (5-aza), and to explore the expression and significance of DLL4-Notch signaling in this process. Materials and Methods: hUCMSCs were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. After treatment with 5-aza for 24 h, hUCMSCs was continued to culture, the expression of GATA4 and NKx2.5 at 4 weeks after induction, DLL4 and Notch1 mRNA at 1d, 3d, 5d, 7d after induction were detected. The expression of cardiac troponin I (cTnI) after 4 weeks was determined by immunocytochemistry. Results: hUCMSCs treated with 5-aza were stained positively for cTnI 4 weeks after induction. The expression of Notch1 and DLL4 mRNA in the 5-aza-induced group was stable and significantly higher than that in the control group (mean Ct value for the Notch1 gene: 0.51 ± 0.21 in the 5-aza-induced group vs. 7.85 ± 0.35 in the control group; mean Ct value for the DLL4 gene: 1.60 ± 0.49 in the 5-aza-induced group vs. 12.42 ± 0.73 in the control group). Similar results were observed for Nkx2.5 and GATA4 genes. The expressions of Nkx2.5 and GATA4 mRNA in the 5-aza group were 4.72 ± 0.58 and 3.76 ± 0.06 times higher than that in the control group, respectively, with statistical significance. Conclusions: hUCMSCs can be differentiated into myocardial cells by 5-aza induction in vitro. 5-Aza may affect this process by regulating the expression of GATA4 and Nkx2.5 genes. The DLL4-Notch signal pathway may be involved in this process.