ABSTRACT
Objective To construct a human renal epithelial cell line HEK293T by CRISPR-Cas9-based site-directed knock-in of vascular endothelial growth factor 165 (VEGF165) gene, and avoid the off-target effect caused by lentivirus infection. Methods The VEGF165 expression vector with homologous arm (pUCm-T-VEGF165 plasmid) and the sgRNA expression vector [pSpCas9(BB)-2A-Puro-sgRNA plasmid] were designed and constructed based on the DNA sequence of the EZH2 gene, and then co-transfected into HEK293T cells. The expression of VEGF165 mRNA was detected by qPCR and the expressions of VEGF165 proteins were detected by Western Blot. Results The qPCR and Western Blot results showed that, comparing with the control, the pUCm-T-VEGF165 plasmid and pSpCas9(BB)-2A-Puro-sgRNA plasmid, the expression of the co-transfection plasmid were significantly increased, i.e. 3.42±0.30 vs. 1.02±0.21, 1.13±0.16 and 0.98±0.18 for the VEGF165 mRNA level (all P<0.01), and 1.13±0.16 vs. 1.02±0.06, 0.88±0.03 and 0.80±0.05 for the VEGF165 protein level (all P<0.01), respectively. Besides, the expression of EZH2 was significantly down-regulated, i.e. 0.14±0.06 vs. 1.08±0.11, 1.02±0.12 and 1.13±0.16 for the EZH2 mRNA level (all P<0.01), and 0.23±0.03 vs. 1.05±0.13, 0.91±0.04 and 0.81±0.06 for the EZH2 protein level (all P<0.01), respectively. This result showed that the VEGF165 was successfully inserted into the EZH2 genome, interfering the EZH2 expression. Conclusions VEGF165 gene can be successfully knocked into HEK293T cells by CRISPR/Cas9 system.
ABSTRACT
Objective To analyze the independent risk factors for hemorrhagic transformation (HT)after endovascular mechanical thrombectomy in patients with acute ischemic stroke (AIS).Methods From March 2015 to February 2018,patients with AIS treated with mechanical thrombectomy at the Department of Neurology,TEDA Hospital were selected.The patients with hemorrhagic infarction (HI) or parenchymal hematoma (PH) were used as the case group,and those without HT were used as the control group.The independent risk factors for HI or PH after mechanical thrombectomy in patients with AIS were determined by multivariate logistic regression analysis.Results A total of 132 patients with AIS were enrolled in the study,and 60 (45.4％) developed HT,of which 37 were HI (28.03％) and 23 were PH (17.42％).Multivariate logistic regression analysis showed that after adjusting for gender,alcohol consumption,fasting blood glucose and glycated hemoglobin,diabetes (odds ratio [OR] 3.485,95％ confidence interval[CI]l.121-6.928;P=0.019),atrial fibrillation (OR 3.962,95％ CI 1.143-7.514;P =0.007) and high fasting blood glucose (OR 3.254,95％ CI 1.107-6.549;P =0.036) were the independent risk factors for HI after mechanical thrombectomy in patients with AIS;after adjusting for gender,hyperlipidemia and glycosylated hemoglobin,diabetes (OR 3.348,95％ CI 1.120-6.709;P =0.025) and high fasting blood glucose (OR 3.172,95％ CI 1.129-7.023;P =0.014) were the independent risk factors for PH after mechanical thrombectomy in patients with AIS.Conclusion Diabetes,atrial fibrillation and high fasting blood glucose were the independent risk factors for HT after mechanical thrombectomy in patients with AIS.
ABSTRACT
Objective To investigate the association between matrix metalloproteinase (MMP)-9 gene rs20544 polymorphism and hemorrhagic transformation (HT) in patients with acute ischemic stroke (AIS). Methods Patients with AIS admitted to the Department of Neurology, TEDA Hospital from March 2016 to September 2018 were enrolled. They were divided into HT group and non-HT group depending on whether HT occurred. HT was defined as no bleeding found in the first imaging examination, and the head CT rescaning indicated a high-density lesion in the low-density area. MMP-9 gene rs20544 single nucleotide polymorphism was determined by TaqMan ? SNP genotype analysis kit. Multivariate logistic regression analysis was used to determine the independent association between rs20544 polymorphism and HT. Results A total of 204 patients with AIS were enrolled, aged 66.91 ± 9.07 years, 89 males (43.63% ), and 45 (22.06% ) developed HT. There were significant differences in atrial fibrillation, diabetes, fasting blood glucose, and triglyceride between the HT group and the non-HT group (all P<0.05). There was also a significant difference in rs2054 genotype distribution between the HT group and the non-HT group (χ2 =7.067; P=0.029 ). Multivariate logistic regression analysis showed that after adjusting atrial fibrillation, diabetes, fasting blood glucose, triglyceride, and hyperlipidemia, rs20544 CC genotype (odds ratio 2.074, 95% confidence interval 1.368-4.041) and CT genotype (odds ratio 1.571, 95% confidence interval 1.109-2.544) were the independent risk factors for HT. Conclusion MRP-9 gene rs20544 single nucleotide polymorphism is associated with increased susceptibility to HT in patients with AIS.
ABSTRACT
Objective To study on the role of histone methylation enzyme enhancer of zeste homolog 2 (EHZ2) and vascular endothelial growth factor 165 (VEGF165) in momymoya disease. Methods The animal model of moyamoya disease was established by ear vein injection of horse serum in New Zealand rabbits. VEGF165 was over-expressed in situ by packaging lentivirus. Real-time quantitative PCR and Western Blot were used to detect the expression of VEGF165, EZH2 and H3K27me3 in the brain tissues of the animal models. Results Compared with the normal control group, the expression levels of mRNA and protein of EZH2 in the moyamoya disease model group were increased (EZH2 mRNA:P<0.01), and the level of histone H3K27me3 was increased. After overexpression of VEGF165 in the moyamoya disease model group, the expression levels of mRNA and protein of EZH2 was further increased (EZH2 mRNA: P<0.01), and the level of histone H3K27me3 was also increased. Conclusions EZH2 plays a certain role in the pathogenesis of moyamoya disease, and the expression of EZH2 is regulated by VEGF 165, which provides a theoretical basis for the study of the pathogenesis of moyamoya disease.
ABSTRACT
Objective To investigate the feasibility,effectiveness,and technical superiority of Solumbra thrombectomy for treatment of acute large cerebral artery occlusion stroke.Methods 32 patients who had acute large cerebral artery occlusion stroke and received mechanical thrombectomy in TEDA Hospital of Tianjin between January 2013 and August 2016,were divided into two groups:stent group(with conventional stent-retrievers,n=21) and Solumbra group (with Solumbra thrombectomy,n =11).Clinical characteristics,variables correlated with operation,and clinical outcomes were compared and analyzed retrospectively.Results There were no differences in basic clinical and radiographic parameters between stent group versus Solumbra group (all P＞0.05).Moreover,there were no differences between Solumbra group versus Stent group in rates of embolus to new territory(18.2 ％ vs.28.6％,P=0.425),in times of thrombectomy(2.2± 1.0 vs.2.4± 1.3,P=0.657),in nonsymptomatic intracranial hemorrhage (18.2％ vs.14.3％,P =0.572),in symptomatic intracranial hemorrhage(18.2％ vs.9.5％,P =0.427),in TICI 2b/3 revascularization(81.8％ vs.81.0％,P =0.670),in puncture-to-reperfusion time[(66.4±39.0)min vs.(51.6±34.5)min,P=0.279],and in NIHSS at 7 days(11.6 ± 7.7 vs.11.3 ± 7.2,P =0.925).A modified Rankin Scale(mRS ≤2) is a variable of recovery of function and good clinical outcome at 90 days.The levels of mRS were similar(P =0.490)between Solumbra and stent groups,but there was a tendency to higher rate of good clinical outcome at 90 days in Solumbra group (63.6％,7/11)than in stent group (47.6％,10/21).Conclusions Solumbra thrombectomy as intravascular revascularization technique is an effective and safe strategy for endovascular recanalization of acute large cerebral artery occlusion stroke.Compared with conventional Stent-Retriever thrombectomy,Solumbra thrombectomy has a good clinical outcome tendency at 90 days after operation.
ABSTRACT
Objective To investigate the feasibility, safety and technical superiority of mechanical thrombectomy using a direct aspiration first-pass thrombectomy (ADAPT) in treatment of patients with acute cerebral artery occlusion. Methods A retrospective study was conducted on all patients with acute ischemic stroke treated with mechanical thrombectomy in our institution from January 2013 to August 2016.Patients using ADAPT or stent retriever as a first-line endovascular procedure were compared for clinical characteristics, procedural variables and clinical outcomes. The technical superiority of ADAPT was analyzed in depth. Results During observation period, a total of 91 cases were performed endovascular treatment with mechanical thrombectomy. ADAPT was designed in 46 cases as a first-line endovascular procedure and was utilized in 38 cases (82.6%;ADAPT group), while primary stent retriever thrombectomy was performed in 21 patients(stent group). There was no significant difference in baseline clinical or radiographic factors between ADAPT and stent groups. Although rates of good neurological outcome (modified Rankin Scale(mRS) score≤2) at 90 days were similar between the ADAPT and stent groups (61%(23/38) vs 48%(10/21), P=0.247), National Institute of Health Stroke Scale (NIHSS) score at seven days (6.0(2.0, 9.3) vs 9.0(5.5, 18.5),Z=-2.031,P=0.021) and full recovery rate of neurological outcome (mRS score=0, 37%(14/38) vs 10%(2/21), P=0.022) were significantly better in the ADAPT group than in the stent group. There were no significant differences in rates of embolus to new territory (21%(8/38) vs 29%(6/21), P=0.365), Thrombolysis In Cerebral Infarction (TICI) 2b/3 grade revascularization (84%(32/38) vs 81%(17/21), P=0.507) and symptomatic intracerebral hemorrhage (0%(0/38) vs 10%(2/21), P=0.123) between the ADAPT and the stent groups, but the figures were better in the ADAPT group. Conclusions Mechanical thrombectomy using ADAPT is feasible and safe compared with stent retriever, with higher full recovery rate of neurological outcome and better NIHSS score.It is a method worthy of further exploration for endovascular mechanical recanalization.
ABSTRACT
Objective To detect the transferred vascular endothelial growth factor (VEGF)165 gene expression in rhesus autologous bone marrow mesenchymal stem cells (MSCs), and to explore the functional viability of transgenic MSCs. Methods MSCs from rhesus bone were isolated by Ficoll, which were used to detect the phenotype. After the culturing, the expression vector pcDNA-eGFP-VEGF165 was transfected into bone marrow MSCs. Fluorescence microscope and flow cytometry were used to detect the enhanced green fluorescent protein (eGFP) expression. At the same time, the phenotype in transfected MSCs was also indentified. The VEGF165 expression level was detected by RT-PCR. Results The highly purified MSCs were collected successfully. The transfected MSCs and daughter cells showed expressions of eGFP and VEGF165, which also remained the characteristics of MSCs. Conclusion The VEGF165 gene that is transfected into MSCs can maintain characteristics of MSCs, and stably express foreign genes.
ABSTRACT
Objective To investigate the effect of cerebral ischemia on functional parameters of affected arteries and probe into the possible pathogenesis of ischemia-reperfusion injury.Methods Intraluminal suture ischemic model was used by occlusion of left middle cerebral artery in rats.Two hours later,the middle cerebral artery segments were isolated from both ischemia and control groups for measurement of changes in vessel diameter induced by increasing pressure and vasoactive compounds.And then,distensibility,myogenic tone,reactivity to 5-HT and ACh were calculated and compared between groups.Results In lower pressure range,ischemic vessels showed an increased myogenic tone(at 40 mm Hg,1 mm Hg=0.133 kPa,19.3%±0.4% vs 10.0%±0.2%,t=20.568,P=0.000)and decreased diameter.In higher pressure range,ischemic vessels showed an increased diameter.distensibility and decreased myogenic tone(at 120 mm Hg,12.0%±0.2% vs 21.8%±0.4%,t=-23.575,P=0.000).In normal pressure range,myogenic tone was not altered after ischemia. Both groups constricted to 5-HT and dilated to ACh,however,the response was significantly diminished after ischemla.Conclusion These findings demonstrate that contractile and diastolic function of affected artery was impaired after ischemia,a result that may contribute to ischemia-reperfusion injury by losing upstream cerebrovascular resistance and increasing perfusion on the microcirculation.
ABSTRACT
Objective:To investigate the effect and mechanism of hypoxia-inducible factor-1α (HIF-1α)in perihematomal brain tissue on the early formation of edema in patients with hyper-tensive intracerebral hemorrhage.Methods:The perihematomal brain tissue in 32 patients with hypertensive intracerebral hemorrhage were treated with hematoma evacuation.The expressions of HIF-1α and vascular endothelial growth factor(VEGF)were measured by immunohistochem- istry staining technique.The volume of perihematomal edema was determined on the basis of reoperative head CT scan.The results of staining and the volume of edema were analyzed by using double-blind method.Resets:Neuron and vascular endothelial cell swelling were observed 4 hours after cerebral hemorrhage,and the scattered HIF-1α positive neurons were visible.The expressions of HIF-1α reached the peak(at 24 to 48 hours,and went on high expressions at 49 to 72 hours.There was significant difference as compared with the normal brain tissue(P＜0.01). There were positive correlations between the expressions of HIF-1α and VEGF (r=0.72,t=6.37,P＜0.01)and the volume of brain edema(r=0.64,t=4.56,P＜0.01).Conclusions: The expression of HIF-1α in perihematomal brain tissue of hypertensive cerebral hemorrhage iS associated with the early formation of cerebral edema.HIF-1α may accelerate the formation of brain edema by inducing and regulating the expression of VEGF.
ABSTRACT
Objective To investigate the expressions of hypoxia-inducible factor-1 alpha (HIF-1?) and its relation with secondary brain damage in perihematomal issue in human intracerebral hemorrhage. Methods Perihematomal brain issue were collected in the course of evacuation of hematoma in 32 patients with hypertensive intracerebral hemorrhage. Expressions of HIF-1? were observed by immunohistochemistry and the neuronal apoptosis were observed by terminal deoxynucleotidyl transterrase mediate dUIP nick end labeling (TUNEL) staining and HE staining. Results HIF-1? protein immunohistochemical staining positive cells ((2.8?0.8)/HP) were identified dispersedly from 4 h after acute hemorrhagic stroke in perihematomal brain issue, and reached the peak at 24—48 h ((12.5?3.9)/HP). The expressions of HIF-1? kept high at 49—72 h ((12.2?1.8)/HP) after acute hemorrhagic stroke. Nervous cells and vascular endothelium cells had been swelled at 4 h after acute hemorrhagic stroke. TUNEL positive cells appeared from 12 h ((11.2?4.1)/HP), increased markedly at 24—48 h ((29.7?8.4)/HP), and reached the peak ((33.2?4.3)/HP) at 49—72 h after acute hemorrhagic stroke. There was a statistically significant correlation between HIF-1? expressions and TUNEL positive cells (r=0.788, t=7.02, P