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Objective@#To investigate whether microRNA(miR)-214 can improve hyperglycemia induced pyroptosis in H9c2 cells through targeting caspase-1.@*Methods@#H9c2 cells of rats those in good growth condition were selected and incubated into the T25 culture bottle after digestion and passage. Cells were cultured in an incubator at 37 ℃ with 5%CO2, repeat passage was made after cell density reached about 80%, The 5th to 8th generations of cells were selected for the subsequent experiments. To observe the effect of overexpression of miR-214 on pyroptosis and caspase-1 expression in H9c2 cells induced by hyperglycemia, the cells were divided into 4 groups: Control group(H9c2 cells cultured normally), Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimic-negative control+hyperglycaemic group(MNC+HG group, H9c2 cells were transfected with miR-214 mimic-negative control for 24 hours and then treated with 50 mmol/L hyperglycaemic for 24 hours). In order to further verify the anti-pyroptosis effect of miR-214 was mediated by targeted inhibition on caspase-1, cells overexpressing caspase-1 were used in the rescue experiment. The cells overexpressing caspase-1 were divided into 4 groups: Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimics+hyperglycosis+recombinant adenovirus (Ad-caspase-1-EGFP) group with caspase-1 gene and EGFP green fluorescent protein expression (mimics+HG+Ad-caspase-1-EGFP group, H9c2 cells were transfected with caspase-1-green fluorescent protein-carrying adenovirus for 48 hours, followed by transfection of miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycaemia for 24 hours), miR-214 mimics+HG+Ad-EGFP empty virus group (mimics+HG+Ad-EGFP group, H9c2 cells were transfected with empty adenovirus containing green fluorescent protein for 48 hours, followed by transfection with miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycosis for 24 hours). The mRNA expression levels of miRNA-214 and caspase-1 in cells were detected by real-time quantitative PCR. The expression and localization of caspase-1 protein were detected by immunofluorescence assay. Western blot was used to detect protein expression levels of procaspase-1, cleaved caspase-1, NLRP3 and ACS with β-actin as internal reference. The secretion of IL-1β and IL-18 in cell culture medium was detected by ELISA. The correlation between miR-214 and caspase-1 was detected by double luciferase reporter gene.@*Results@#(1) The mRNA expression levels of miR-214 and caspase-1 in each group: the mRNA expressions of miR-214 in HG group and MNC+HG group were significantly lower than that in control group(P<0.05). The mRNA expression of miR-214 in mimics+HG group was significantly higher than that in control group (P<0.05). The mRNA expression levels of caspase-1 in HG group and MNC+HG group were significantly higher than that in control group(P<0.05). The mRNA expression level of caspase-1 in mimics+HG group was lower than that in control group(P<0.05). (2) The expression of caspase-1 in each group: the green fluorescence intensity in the control group was weak, which was strong in the HG group and MNC+HG group. The green fluorescence expression was weaker in mimics+HG group than in HG group. (3) ASC and NLRP3 protein expression levels in each group: ASC and NLRP3 protein expression levels in HG group and MNC+HG group were higher than those in control group(P<0.05). ASC and NLRP3 protein expression levels were significantly lower in mimics+HG group than in mimics+HG group (P<0.05). (4) The secretion of IL-1β and IL-18 in the cell culture medium of each group: the content of IL-1β and IL-18 in the cell culture medium of HG group and MNC+HG group was significantly higher than that of control group (P<0.05). The content of IL-1β and IL-18 in the cell culture medium of mimics+HG group was significantly lower than that of the HG group (P<0.05). (5) Correlation between miR-214 and caspase-1: miR-214 specifically binds to caspase-1 3 ′UTR. Meanwhile, Western blot results showed that cleaved caspase-1 protein expression levels were significantly higher in both HG group and MNC+HG group than in control group (P<0.05). The levels of cleaved caspase-1 were significantly lower in mimics+HG group than in HG group (P<0.05). There was no significant difference in procaspase-1 expression among groups (P>0.05). (6) The expression levels of procaspase-1, cleaved caspase-1, ASC and NLRP3 in each group in rescue experiment: there was no significant difference in the expression of procaspase-1 in each group (P>0.05). Cleaved caspase-1, ASC and NLRP3 protein expressions were significantly lower in mimics+HG group than in HG group (P<0.05). However, cleaved caspase-1, ASC and NLRP3 protein expressions were significantly higher in mimics+HG+ Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). (7) The expression of IL-1β and IL-18 in rescue experiment: the secretions of IL-1β and IL-18 in the cell culture medium of the mimics+HG group were significantly lower than that of HG group (P<0.05), which were significantly higher in mimics+HG+Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05).@*Conclusion@#miR-214 can improve the hyperglycemia induced pyroptosis in H9c2 cells by targeted inhibition of the caspase-1.
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Objective@#To explore the imaging characteristics and related influencing factors of in-stent neoatherosclerosis (ISNA) in patients with restenosis after drug-eluting stent(DES) implantation with optical coherence tomography(OCT).@*Methods@#A total of 25 cases of coronary heart disease patients(DES placement time ≥8 months) with coronary artery angiography showing DES in-stent restenosis (ISR) in Zunyi medical college affiliated hospital from July 2013 to December 2015 were included in this study and patient's data were retrospectively analyzed.In these patients with ISR, OCT images were acquired before percutaneous coronary intervention. Patients were divided into the ISNA group (12 patients and 12 lesions) and non-ISNA group(13 patients and 13 lesions) according to the result of OCT. ISNA on OCT was defined as neointima formation with the presence of lipids or calcification.@*Results@#(1) The incidence of chronic kidney disease and increased low-density lipoprotein cholesterol level in ISNA group were significant higher than that in non-ISNA group(all P<0.05). The stent implantation time in ISNA group was longer than that in the non-ISNA group(53.0(14.0, 81.0) months vs. 15.0(8.5, 32.5) months, P<0.01). In addition, clinical manifestation of acute coronary syndrome was present in 8 out of 12 patientsin ISNA group, and stable angina pectoris was found in 10 out of 13 casesin non-ISNA group(P<0.01). (2) Quantitative analysis of OCT showed that the lumen area was less in ISNA group than in non-ISNA group((3.45±1.82)mm2 vs. (4.17±1.68)mm2, P<0.01), and neointimal area(3.89(2.26, 5.52)mm2 vs. 2.96(1.99, 4.22)mm2, P<0.01), neointimal load (53.15(40.18, 67.30)% vs. 41.54(32.08, 56.91)%, P<0.01), neointimal thickness(0.98(0.63, 1.36)μm vs. 0.72(0.51, 1.03)μm, P<0.01) were higher in ISNA group than in non-ISNA group.(3)Qualitative analysis of OCT showed that the prevalence of homogeneous intima was less in the ISNA group than in the non-ISNA group ((41.42±22.56)% vs.(72.06±18.68)%, P<0.05), on the contrary, the heterogeneous intima was more common in the ISNA group ((58.57±22.56)% vs. (27.94±18.68)%, P<0.05). There was no significant difference between two groups in the peri-stentmicrovessels (9/12 vs. 5/13,P>0.05), and prevalence of intraintimalmicrovessels was higher in the ISNA group than in non-ISNA group (7/12 vs. 2/13, P<0.05). In addition, thin cap fibrous plaque(7/12 vs. 0, P<0.01), disrupted intima with visible cavity (7/12 vs. 1/13, P<0.05),andintraluminal red thrombus(7/12 vs. 1/13, P<0.05) were significantly higher in ISNA group than in non-ISNA group.@*Conclusions@#Results of OCT show that ISNA occurs frequently in patients with ISR after DES implantation. The stent implantation time, incidence of chronic kidney disease and higher low-density lipoprotein cholesterol level are associated with the formation of ISNA in these patients.
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OBJECTIVE:To investigate the effects of intensive atorvastatin therapy on postoperative blood lipid,inflammation reaction and major adverse cardiac events (MACE) in non-ST segment elevation myocardial infarction (NSTEMI) patients before PCI.METHODS:A total of 120 NSTEMI patients underwent selective PCI were randomly divided into control group (60 cases) and observation group (60 cases).Both groups were given Aspirin enteric-coated tablet 0.3 g orally,once a day+Clopidogrel sulfate tablet 300 mg orally,once a day,immediately after admission.After operation,they were given medicine continuously for consecutive 12 weeks.Control group was given Atorvastatin calcium tablet 80 mg orally,immediately after operation,and then was given 40 mg,once a day,for consecutive 12 weeks.Observation group was additionally given Atorvastatin calcium tablet 40 mg orally 6 h before operation on the basis of control group.The levels of TG,TC,HDL-C,LDL-C,hs-CRP,TNF-αt and IL-10 before and after PCI,the incidence of postoperative MACE,postoperative re-hospitalization rate and the occurrence of ADR were observed in 2 groups.RESULTS:There was no statistical significance in the levels of TG,TC,HDL-C or LDL-C between 2 groups before and after operation (P＜0.05).After operation,the levels of hs-CRP,TNF-α and IL-10 in 2 groups were significantly higher than before operation,and the observation group was significantly lower than the control group,with statistical significance (P＜ 0.05).There was no statistical significance in the incidence of postoperative MACE,postoperative re-hospitalization rate or the incidence of ADR between 2 groups (P＞0.05).CONCLUSIONS:Intensive atorvastatin therapy before PCI can effectively reduce the levels of inflammatory response in NSTEMI patients,but have no significant changes in blood lipid levels and MACE risk,without increasing the incidence of ADR.
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Forensic entom otoxicology is a branch of forensic m edicine, w hich applies entom ology, toxicology and other related studies to solve the poisoning cases. It has an obvious advantage in the investigation on poisoning death. B ased on the expounding definition and research of entom otoxicology, this paper review s research progress and application value in som e aspects of forensic m edicine, such as the effects of drugs/toxins on the growth and developm ent of sarcosaphagous insects and the qualitative and quantitative analysis of the drugs/toxins in the poisoned body tissue.
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Objective To explore in vitro mesenchymal stem cell (MSC) transfection of lentiviral vector mediated calcitonin gene‐related peptide(CGRP) gene and its effects on biological properties of MSC .Methods MSC were isolated ,cultured and identi‐fied .MSC were infected by lentivirus encoding recombinant enhanced green fluorescent protein (EGFP) gene and CGRP (Lv‐EG‐FP‐CGRP) .The transfection efficiency was determined by the inverted fluorescence microscope and flow cytometry .The expression levels of CGRP were detected in CGRP‐modified MSC by using real‐time PCR ,immunocytochemistry and enzyme‐linked immu‐nosorbent assay (ELISA) .The proliferation ,aging and differentiation ability of MSC were evaluated by MTT ,β‐galactosidase stai‐ning and inducing differentiation respectively .Results After 48 h of MSC transfection by Lv‐EGFP‐CGRP ,EGFP/CGRP could be expressed stably .When multiplicity of infection (MOI) was 30 ,the transfection efficiency reached more than 80% .Compared with the MSC group and the MSC‐EGFP group ,the mRNA and protein expression levels of CGRP in CGRP‐modified MSC(MSC‐CGRP group) were markedly increased(all P<0 .01) .The results of MTT ,β‐galactosidase staining and inducing differentiation assay dem‐onstrated that the transfected CGRP basically had no effect on the proliferation ,aging and endotheliocyte differerntiation of MSC . Conclusion MSC is a kind of ideal genetic vector cell ,which can serve as the target cell of CGRP gene transduction for the applica‐tion of gene therapy and lays the foundation for follow‐up in vitro and vivo experiments .
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Objective To explore the effect of transplantation of mesenchymal stem cells (MSCs) transfected with the human receptor activity modifying protein 1 (hRAMP1) gene on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) after carotid balloon angioplasty was performed in rabbits with carotid atherosclerosis.Methods Density gradient centrifugation and adherent culture were carried out to obtain MSCs,which were then transinfected with an adenovirus vector carrying the hRAMP1 gene or an empty adenovirus vector.A rabbit model of atherosclerotic stenosis and balloon angioplasty was successfully established.Results were randomly divided into three groups:the hRAMP1-MSCs group,theadipose-derived MSCs (Ad-MSCs) group and the control group.MSCs were transinfected with Ad-EGFP-hRAMP1,Ad-EGFP or PBS by transplantation into the injured carotid arteries.Homing and differentiation were assessed with MSCs harvested at 7 d.With MSCs collected at 28 d,Western blotting was used to measure the expression of the hRAMP1 target gene in the carotid artery; the neointima and media area in the injured carotid arteries were estimated; carotid artery morphology was examined with H&E staining; and the proliferation and apoptosis of VSMCs were determined by immunohistochemistry and TUNEL.Results The expression of CD31 and EGFP was found in proliferating neointima lesions at 7d in the hRAMP1-MSCs group and the Ad-MSCs group.At 28d of MSC transplantation,the level of RAMP1 significantly increased in the hRAMP1-MSCs group,compared with the Ad-MSCs and control groups [(63.0±4.9) vs.(28.3±2.5) and (27.2±7.2),all P＜0.05],but there was no differencein the RAMP1 level between the Ad MSCs group and the control group (P＞0.05).Positive expression of the α-smooth muscle antibody (α-SMA) was found in all three groups at 28 d of MSC transplantation.The thickness of the hyperplastic neointima significantly decreased in the hRAMP1-MSCs group,compared with the other two groups (P＜0.05),and was lower in the Ad-MSCs group than in the control group (P＜0.05).The expression of proliferating cell nuclear antigen (PCNA) was lower in the hRAMP1-MSCs group than in the Ad-MSCs and control groups at 28d of MSC transplantation (P ＜0.05),while the PCNA level was lower in the Ad-MSCs group than in the control group (P＜ 0.05).The VSMC apoptosis rate significantly increased in the hRAMP1-MSCs group,compared with the Ad MSCs and control groups (P＜0.05),and was the lowest in the control group (P＜0.05).Conclusions Gene-modified stem cell therapy can effectively inhibit vascular intimal hyperplasia,thereby reducing restenosis after angioplasty.
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Objective To investigate the effect of transplantation of CXC receptor 4 (CXCR4)gene-modified bone marrow mesenchymal stem cells (BMSCs) on repairment of carotid injure in rats.Methods BMSCs were cultured and transfected with lentivirus vector carrying CXCR4 gene to generate CXCR4 gene-modified BMSCs (CXCR4-BMSCs).CXCR4 expression was detected by Western blot.Rat model of carotid artery balloon injury was established.Rats were randomly divided into the PBS control group (n=12),CXCR4-BMSCs group (n=12) and BMSCs group (n=12).Two weeks after transplantation,the injured arteries were obtained.The homing of BMSCs was detected by immunofluorescence with green fluorescent protein (GFP).Platelet endothelial cell adhesion molecule (CD31) expression was detected by immunofluorescence staining.At 4 weeks after transplantation,proliferating cell nuclear antigen (PCNA) expression was determined by immunohistochemical staining,and the vascular morphological changes were observed by hematoxylineosin staining (HE).Results Compared with the control and BMSCs groups,the protein level of CXCR4 was increased in CXCR4-BMSCs group (both P＜0.05).The percentage of GFP-positive cells homing were much more in CXCR4-BMSCs group than in BMSCs group [(58.8±4.4)％ vs.(36.2±5.0) ％,P＜0.05].The CD31 expression were higher in CXCR4-BMSCs group than in BMSCs group [(58.8±4.3)％ vs.(28.8±4.2)％,P＜0.05].Compared to the control group,the PCNA expression was decreased in CXCR4-BMSCs and BMSCs groups [(21.0±4.2) ％,(36.5±4.9) ％vs.(78.3±3.5) ％,both P＜0.05].There was a significant difference in PCNA expression between the CXCR4-BMSCsgroupandBMSCs group [(21.0±4.2)％vs.(36.5±4.9)％,P＜0.05].The neointimal area and the ratio of neointimal/medial area were decreased in CXCR4 BMSCs and BMSCs group as compared with the control group [(0.205±0.018) mm2,(0.323±0.071) mm2 vs.(0.536 ± ±0.054) mm2; (1.039±0.123),(1.660±0.404) vs.(2.460±0.328); all P＜0.05],and there were significant differences in neointimal area and the ratio of neointimal/medial area in CXCR4-BMSCs group and BMSCs group [[(0.205±0.018) mm2 vs.(0.323±0.071) mm2,(1.039±0.123)vs.(1.660±0.404),both P＜0.05].Conclusions CXCR4 gene-modified BMSCs may increase the CXCR4 expression in BMSCs.CXCR4-BMSCs transplantation is more effective than BMSCs transplantation in increasing BMSCs homing capacity,reducing the reendothelialization and vascular restenosis.
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Objective To construct lentiviral vector carrying rat′s calcitonin gene-related peptide(CGRP) gene for the following-up study on the function of CGRP .Methods CGRP gene segment was subcloned into shuttle plasmid ,become Puc57-CGRP .The pLenO-DCE-CGRP expression vector was be constructed by double digests .The pLenO-DCE-CGRP and 4 auxiliary packaging plas-mids were co-transfected into 293T cells .Cells were cultured for 48 hours .The supernant was collected and concentrated ,and then the viral titers were tested by multiple proportions dilution method and flow cytometer .The expression levels of CGRP were detec-ted in CGRP-modified 293T cells by Real-time PCR .Results The results of digestion and sequencing show that the pLenO-DCE-CGRP vector was constructed successfully .The titer of the lentiviral particles was 5 .1 × 108 TU /mL .Conclusion The high-titer lentvirus vector containing CGRP gene is constructed successfully ,which lay a foundation for transfecting mesenchymal stem cell (MSC) and studying the function of CGRP .
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Objective To observe the effect of adenovirus- receptor activity modifying protein-1 (RAMP1) on nuclear factor(NF κB) and myocardial fibrosis in heart of rabbit with myocardial infarction. Methods Myocardial infarction (MI) models were developed in 54 rabbits and they were randomly divided into RAMP1 group,EGFP group and control group according to whether pAd2-EGFP-RAMP1,pAd2-EGFP or PBS was injected into infarction region and its border.At 7 d,14 d and 28 d after injection,Left ventricular function indices such as LVEF,LVSd,LVDd and LVFS were estimated by echocardiogram,the expression of NF-κB in myocardium was detected by Western blot,the plasma level of TNF-α was measured by ELISA,and fibrosis and collagen content was measured by Masson stain. Results At 7 d after adenovirus injection,the expression of RAMP1was significantly increased in RAMP1 group (67.33 ± 3.97)％ as compared to EGFP group(20.59 ±3.26) ％ and PBS group ( 23.80 ± 5.08) ％ ( P ＜ 0.05 ).The expression of NF κB was decreased in RAMP1 group ( 26.54 ± 5.13 ) ％ versus EGFP group (62.60 ± 6.18) ％ and PBS group (62.95 ±5.17)％ (P＜0.05).The plasma level of TNF-α was lower in RAMP1 group than in EGFP group and in PBS group at different time[7 d:( 136.74 ± 5.42) μg/L vs.( 196.97 ± 14.17) μg/L,(203.67 ±13.90)μg/L; 14 d:( 154.51 ± 13.61 )]μg/L vs.( 112.22±6.74 )μg/L,(160.46± 14.27)μg/L ;28 d;(51.10± 5.62)μg/L vs.( 95.55 ± 9.94 )μg/L,( 98.96 ± 12.68) μg/L,all P＜ 0.05].The collagen content was reduced in RAMP1 group as compared with EGFP group and PBS group at 14 d and 28 d [14 d:(7.10±0.98)％ vs.(19.52±2.32)％,(17.91±0.96)％ ;28 d:(17.04±2.44)vs,(34.10±5.59) ％,(33.98±4.33)％,all P＜0.01].At 28 d after infarction,the infarct size was decreased in RAMP1 group (26.54 ± 5.13) ％ compared with EGFP group (32.20 ± 3.73) ％ and control group (35.58±2.65) ％ (P＜0.01),and better heart function appeared in RAMP1 group. Conclusions The high-expression of RAMP1 could decrease the collagen deposition and fibrosis in the border of infarction and improve heart function through lower expression of NF-κB and decreasing the plasma level of TNF-α.