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OBJECTIVE@#To explore the effects of Ena/VASP gene family on the expression of glycoprotein (GP) Ib-IX complex in human megakaryoblastic leukemia Dami cells.@*METHODS@#SiRNAs targeting Ena/VASP gene family were designed and synthesized to interfere Enah, EVL and VASP gene expression. When the siRNAs were transfected into Dami cells by using LipofectamineTM 2000 for 48 h, the expression of GPIb-IX complex was detected by quantitative real-time PCR, Western blot and flow cytometry.@*RESULTS@#We successfully established siVASP , siEVL and si Enah Dami cell lines. And it was found that the expression of GPIb-IX complex had no evident reduction in siEVL or siVASP Dami cells at both mRNA and protein level, while the total protein and membrane protein of GPIb-IX complex were obviously reduced when Enah was knocked down.@*CONCLUSION@#Enah could affect the expression of GPIb-IX complex in human megakaryoblastic leukemia Dami cells, but the underlying mechanism still needs to be further explored.
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Humanos , Linhagem Celular , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Leucemia/metabolismo , Plaquetas/metabolismoRESUMO
OBJECTIVE@#To explore the regulation of mitochondria on platelet apoptosis and activation, and the relationship between platelet apoptosis and activation.@*METHODS@#Platelets were isolated from peripheral venous blood of healthy volunteers. Cyclosporin A (CsA), which has a protective effect on the function of platelet mitochondria, BAPTA, which can chelate calcium ions across membranes in platelets, and NAC, an antioxidant that reduces the level of intracellular reactive oxygen species, were selected for coincubation with washed platelets, respectively. By flow cytometry, platelet aggregator was used to detect the changes of platelet mitochondrial function and platelet activation indexes after different interventions.@*RESULTS@#H89, staurosporine, and A23187 led to platelet mitochondrial abnormalities, while CsA could effectively reverse the decline of platelet mitochondrial membrane potential caused by them. Antioxidant NAC could reverse platelet mitochondrial damage correspondingly, and completely reverse platelet shrinkage and phosphatidylserine eversion induced by H89. BAPTA, prostaglandin E1, acetylsalicylic acid and other inhibitors could not reverse the decline of platelet mitochondrial membrane potential.@*CONCLUSION@#Mitochondrial function plays an important role in platelet apoptosis and activation. Abnormal mitochondrial function causes the imbalance of reduction/oxidation state in platelets, which leads to platelet apoptosis. Platelet apoptosis and activation are independent signal processes.
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Humanos , Plaquetas/metabolismo , Antioxidantes/farmacologia , Mitocôndrias/fisiologia , Ativação Plaquetária , Apoptose , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/farmacologiaRESUMO
OBJECTIVE@#To explore the main factors of platelet spreading and provide the foundation for related research.@*METHODS@#Platelets (2×107/ml) were draw from C57BL/6J mouse and kept at 22 ℃ for 1-2 hours. Platelets (2×107/ml) were were allowed to adhere and spread on the fibrinogen-coated slides, after staining F-actin in platelets, the platelets were observed with the confocal microscopy. The effects of different concentrations of fibrinogen (10 μg/ml, 30 μg/ml, 100 μg/ml) and kinds of agonists [thrombin(0.01,0.05,0.1 U/ml), ADP(5,10,20 μmol/L), U46619(0.125,0.25,0.5 μmol/L)] on platelets were analyzed. The platelet spreading was successful if the spreading rate was higher after treated with agonists.@*RESULTS@#Compared to the group which coated with 10 μg/ml and 100 μg/ml fibrinogen, the platelet density is optimal when coated with 30 μg/ml fibrinogen. In addition, under the stimulation of thrombin, ADP and U46619, the spreading rate of platelets showed a certain concentration-dependent increasing.@*CONCLUSION@#The platelet spreading is easily influenced by various factors, the platelet spreading can be induced successfully at 0.1 U/ml thrombin, 20 μmol/L ADP and 0.5 μmol/L U46619 on the slide coated with 30 μg/ml fibrinogen.
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Animais , Humanos , Camundongos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Difosfato de Adenosina , Plaquetas/fisiologia , Fibrinogênio , Camundongos Endogâmicos C57BL , Adesividade Plaquetária/fisiologia , Trombina/farmacologiaRESUMO
Under the situation of regular epidemic prevention and control of COVID-19, the hospitals need to establish an emergency personnel pool and update it regularly. The author introduced the experience of establishing and managing the emergency personnel pool in a hospital, including the use of information technology to achieve scientific grouping and dynamic management, strengthening the emergency response ability, epidemic prevention and control training and assessment of all staff, ensuring their mental health and logistics support, and improving the performance assessment, salary and welfare system of emergency personnel.From December 2021 to May 2022, the members of the emergency personnel pool completed a total of 62 emergency support tasks. The response time and team gathering time of all emergency tasks were reduced to less than 1 hour. A total of 4 421 medical personnel were sent. The nucleic acid test results of COVID-19 during the tasks were negative, the infection rate was zero, and no adverse events occurred.
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OBJECTIVE@#To investigate the regulatory effect of zyxin on the distribution of platelet cytoskeleton.@*METHODS@#Platelets were isolated from zyxin-knockout (Zyx@*RESULTS@#After zyxin gene was knockout, the expressions of cytoskeleton proteins β-actin, α-actinin, filamin A, and myosin Ⅱ A in resting and Jas-induced platelets were significantly increased. In the platelet spreading on fibrinogen surface, F-actin was increased in Zyx@*CONCLUSION@#Zyxin significantly regulates the distribution of platelet cytoskeleton, which plays an important role in maintaining platelet cytoskeleton homeostasis.
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Animais , Camundongos , Actinina , Actinas , Plaquetas , Citoesqueleto , ZixinaRESUMO
PURPOSE@#Treatment of irreducible femoral intertrochanteric fractures often requires open reduction. However, the technique unavoidably causes patients to suffer greater trauma. As such, minimally invasive techniques should be employed to reduce the surgical-related trauma on these patients and maintain a stable reduction of the fractures. Herein, a minimally invasive wire introducer was designed and used for the treatment of femoral intertrochanteric fractures. The effectiveness of using a wire-guided device to treat irreducible femoral intertrochanteric fractures was evaluated.@*METHODS@#Between 2013 and 2018, patients with femoral intertrochanteric fractures who were initially treated by intramedullary nail fixation but had difficult reduction using the traction beds were retrospectively reviewed. Decision for an additional surgery was based on the displacement of the fracture. The patients were then divided into two groups: those in the control group received an open reduction surgery while those in the observation group received a closed reduction surgery using a minimally invasive wire introducer to guide the wire that could assist in fracture reduction. The operation time, blood loss, visual analogue scale scores, angulation, reduction, neck-shaft angle, re-displacement, limb length discrepancy, and union time were then recorded and analyzed to determine the efficiency of the wire introducer technique. Categorical variables were analyzed by using Chi-square test, while continuous variables by independent t-test and the Mann-Whitney test accordingly.@*RESULTS@#There were 92 patients included in this study: 61 in the control group and 31 in the observation group. There were no significant differences in baseline demographic factors between the two groups. All surgeries were successful with no deaths within the perioperative period. The average follow-up time for the patients was 23.8 months. However, the observation group had a significantly shorter operation time, lower visual analogue scale score, less intraoperative bleeding, and shorter fracture healing time. There were no significant differences in the angulation, reduction, neck-shaft angle, and limb length discrepancy between the two groups.@*CONCLUSION@#The minimally invasive wire guide achieved a similar effect to that of open reduction in the treatment of intertrochanteric fractures with difficult reduction. Moreover, the minimally invasive wire introducer is a good technology that accurately guides the wire during reduction. Indeed, it is an effective technique and achieves good clinical outcomes in restoration of irreducible femoral intertrochanteric fractures.
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Objective: Securidaca inappendiculata is a medicinal plant frequently used in the treatment of inflammatory diseases in south China. In this study, we aimed to explore its bioactive constituent which contributes to the anti-inflammatory activity. Methods: Polyphenol-enriched and polyphenol-deprived fractions (PRF and PDF, respectively) were separated from the ethanolic extract by HPD300 macroporous resin-based method, and their anti-inflammatory activities were investigated on a lipopolysaccharide (LPS)-induced acute lung injury (ALI) model in rats. The possible mechanism of action in alleviating acute inflammation was studied using RAW264.7 cells. Results: Both Folin-Ciocalteu and
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Objective: To analyze the clinical features of patients with odontogenic sinusitis (OS) treated by endoscopic sinus surgery (ESS). Methods: A retrospective investigation was carried out in our 27 (16 males and 11 females) cases with OS aged (49.74±14.42) years old. Subjects were hospitalized between January 2018 and November 2020 from Department of Otorhinolaryngology Head and Neck Surgery, Beijing Chaoyang Hospital. The medical history, symptoms, result of nasal endoscopy and paranasal sinus computed tomography (CT) were analyzed statistically by SPSS 19.0. Results: OS mainly occured on unilateral sinuses, with a duration of (8.56±11.79) months. Seventy point four percent (19/27) of the patients had a course within six-month, only 11% was over 12 months (3/27). Symptoms mostly showed as nasal obstruction (88.9%; 24/27), runny nose (81.5%; 22/27), nasal stinks (16/19) and postnasal drip (10/10). Sixty-three percent (17/27) of the OS patients had a dental history. Nasal endoscopic examination revealed a swelling of the ostiomeatal complex (77.8%; 21/27), medial wall interhal displacement of maxillary sinus (55.6%; 15/27), white emulsion-like purulent secretion in the middle meatus (70.4%; 19/27) and nasal polyps (59.3%; 16/27). Etiology of OS included implant-related problems (14.8%; 4/27) and periodontal disease (85.2%; 23/27). Conclusions: OS is usually unilateral sinusitis with a short history. Its clinical features show nasal stinks, white emulsion-like purulent secretion in the middle meatus and imaging findings of unilateral maxillary sinusitis with tooth-related lesions.
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Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença Crônica , Endoscopia , Seio Maxilar/cirurgia , Sinusite Maxilar/cirurgia , Estudos Retrospectivos , SinusiteRESUMO
OBJECTIVE@#To investigate the effect of protein kinase A (PKA) activation on aggregation funetion of platelets in vitro.@*METHODS@#The peripheral blood of healthy adults were collected, and the washed platelets were gained from collected peripheral blood. The washed platelets were treated with PKA activator Forskolin, then the platelet aggregation was induced by using Ristocetin, Thrombin, Collagen and ADP respectively, the platelet aggregation level was detected by the platelet aggregator.@*RESULTS@#Compared with the controls, 5 μmol/L forskolin significantly inhibited ADP and collagen-induced platelet aggregation (P<0.001), and showed mild inhibiting effect on Thrombin-induced platelet aggregation (P<0.05). 2.5-10 μmol/L forskolin significantly inhibited ADP and Collagen -induced platelet aggregation (P<0.001); but not showed significantly inhibitory effects on Ristocetin-induced platelet aggregation (P>0.05).@*CONCLUSION@#PKA activation inhibits agonists-induced platelet aggregation.
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Humanos , Plaquetas , Proteínas Quinases Dependentes de AMP Cíclico , Agregação Plaquetária , Inibidores da Agregação Plaquetária , Ristocetina , TrombinaRESUMO
<p><b>OBJECTIVE</b>To investigate the factors that influence FeCl-induced mouse mesenteric arteriole thrombosis model.</p><p><b>METHODS</b>Platelets were isolated from donor mice and labeled with Calcein-AM. Mice were transfused intravenously with Calcein-AM labeled platelets. The influence of mouse ages (3-6 weeks, 6-10 weeks and >10 weeks), transfused platelets counts (1×10, 1×10and 2×10platelets) and concentrations of FeCl(6%, 12%, 24% and 48%) on FeCl-induced thrombosis model were compared.</p><p><b>RESULTS</b>The occlusion time was 16 min for mice aged 3-6 weeks, which was shorter than that for 6 mice aged 6-10 weeks(25 min)(P<0.05) and that for mice aged >10 weeks(38 min)(P<0.01). The occlusion time resulting from transfusion of 1×10and 2×10of pletclets was 15-18 min, which was shorter than that of transfusion 1×10platelets (30 mins). The occlusion time resulting from transfusion of 6% and 12% FeClwas from 15 to 20 min, however the transfusion of 24% and 48% FeClall in all leads to vessel occlusion within 10 min.</p><p><b>CONCLUSION</b>The factors influencing the success of FeCl-induced mouse thrombosis model are more. Transfusion of 1×10to 2×10labeled platelets to 3-6 week-old mice, and 6% to 12% of FeClshould be used to induce thrombosis, and the experimental conditions should be optimized for this animal model, therefore, it is easier for us to set up a mouse mesenteric arteriole thrombosis model.</p>
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@#Objective To explore the development and implementation of International Classification of Functioning, Disability and Health (ICF) (International Chinese Version) in China. Methods A total of 332 research papers in ICF had been selected from CNKI from 2001 to 2017 and analyzed using Citespace III. The time, authors and institutions distributions had been explored. Results There was a sus-tainable increase of ICF publications in Chinese from 2001. The core authors of ICF are QIU Zhuo-ying and his research team. Chinese Journal of Rehabilitation Theory and Practice has published most researches in ICF in China. ICF Chinese version has been widely imple-mented in the areas of disability statistics at population level, clinical and services, management and service information, and social policy. With the guidance and coordination of WHO Family of International Classifications Collaborating Center (WHO-FIC CC) and cooperation with WHO-FIC CCs Germany, Australia, and Italy, and cooperation with WHO CC Rehabilitation in China, remarkable achievements has been made in the fields of ICF research and implementation. Conclusion It is a promising new research area to research and implement ICF in China. New ideologies and tools has been developed and significant influences have been made from ICF research and implementation in the fields of health care, rehabilitation, education, social security and employment, social services, disability studies, and informatics.
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@#Objective To explore the development and implementation of International Classification of Functioning, Disability and Health (ICF) (International Chinese Version) in China. Methods A total of 332 research papers in ICF had been selected from CNKI from 2001 to 2017 and analyzed using Citespace III. The time, authors and institutions distributions had been explored. Results There was a sus-tainable increase of ICF publications in Chinese from 2001. The core authors of ICF are QIU Zhuo-ying and his research team. Chinese Journal of Rehabilitation Theory and Practice has published most researches in ICF in China. ICF Chinese version has been widely imple-mented in the areas of disability statistics at population level, clinical and services, management and service information, and social policy. With the guidance and coordination of WHO Family of International Classifications Collaborating Center (WHO-FIC CC) and cooperation with WHO-FIC CCs Germany, Australia, and Italy, and cooperation with WHO CC Rehabilitation in China, remarkable achievements has been made in the fields of ICF research and implementation. Conclusion It is a promising new research area to research and implement ICF in China. New ideologies and tools has been developed and significant influences have been made from ICF research and implementation in the fields of health care, rehabilitation, education, social security and employment, social services, disability studies, and informatics.
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<p><b>OBJECTIVE</b>This study was aimed to investigate the regulatory effect of protein disulfide isomerase (PDI) on platelet GPIbα ectodomain shedding.</p><p><b>METHODS</b>The washed platelets were obtained from healthy volunteers. Platelets were incubated with PDI inhibitor bacitracin before stimulation with PMA (Phorbol-12-myristate-13-acetate), dibucaine and collagen. The N-terminal domain of GPIbα in supernatant was detected by Western blot, the GPIbα expression and the intraplatelet ROS levels were measured by flow cytometry.</p><p><b>RESULTS</b>neither GC content nor GPIbα expression was changed after the washed platelets from the healthy donors were incubated only with PDI inhibitor. The washed platelets were incubated with PDI inhibitor before stimulation with different stimulin, PMA, dibucaine or collagen, and then GPIbα was cleaved and ROS levels were elevated more than that in the controls.</p><p><b>CONCLUSION</b>PDI participates in the induced GPIbα ectodomein shedding, and the effect of PDI in this process maybe depend on the change of ROS level inside platelets. These results might provide a new point of view for the platelet drug development.</p>
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Humanos , Plaquetas , Colágeno , Citometria de Fluxo , Complexo Glicoproteico GPIb-IX de Plaquetas , Isomerases de Dissulfetos de ProteínasRESUMO
<p><b>OBJECTIVE</b>To explore clinical effects of suturing-assisted locking plate in treating elderly proximal humeral fractures.</p><p><b>METHODS</b>From January 2005 to January 2013, 55 elderly patients with three- and four-part fractures of proximal humeral fractures were divided into treatment group and control group. In treatment group, there were 31 patients including 12 males, and 19 females aged from 65 to 85 with an average of (74.00±5.42) years old, and treated with suturing-assisted locking plates; 19 patients were Neer 3-part fractures, and 12 patients were Neer 4-part fractures of proximal humerus; 23 patients were suffered from low-energy injuries and 8 patients were caused by high-energy injuries. In control group, there were 24 patients including 7 males, and 17 females aged from 65 to 85 with an average of (72.79±5.34) years old, and treated with locking plates; 16 patients were Neer 3-part fractures, and 8 patients were Neer 4-part fractures of proximal humerus; 17 patients were suffered from low-energy injuries and 7 patients were caused by high-energy injuries. Operative time, blood loss during operation, and bone healing time between two groups were observed and compared. Postoperative Neer scoring were used to evaluate recovery of shoulder joint function.</p><p><b>RESULTS</b>All patients were followed up from 6 to 24 months with an average of 16.1 months. In treatment group, blood loss was (495.806±143.150) ml, function of Neer scoring was 22.645±2.443, range of action was 18.194±2.613, anatomy was 7.935±1.504 and total score of Neer scoring was 77.161±8.335; while in control group, blood loss was (641.667±169.851) ml, function of Neer scoring was 13.958±1.989, range of action was 13.083±2.165, anatomy was 5.500±1.978 and total score of Neer scoring was 58.792±7.313. There were sigificant difference between two groups in these indexes.</p><p><b>CONCLUSION</b>Suturing-assisted locking plate for the treatment of proximal humerus fractures in elderly, has advantages of less blood loss, simple fracture reduction and rapid recovery of shoulder joint, and is a effective method.</p>
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Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Placas Ósseas , Estudos de Casos e Controles , Recuperação de Função Fisiológica , Fraturas do Ombro , Cirurgia Geral , Articulação do Ombro , SuturasRESUMO
Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.
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Humanos , Apoptose , Plaquetas , Biologia Celular , Metabolismo , Caspase 3 , Metabolismo , Potencial da Membrana Mitocondrial , Fosfatidilserinas , Metabolismo , Proteína Quinase C , MetabolismoRESUMO
This study is aimed to establish a high-performance liquid chromatography (HPLC) method for simultaneous determination of chlorogenic acid, caffeic acid, 1,3-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, luteolin-7-O-beta-D-glucoside, 3,4-dicaffeoylquinic acid, linarin and luteolin in Chrysanthemum indicum. The separation was carried out on a Shim pack VP-ODS (4.6 mm x 250 mm, 5 microm) column eluting with mobile phases of methanol (A) and water containing 0.3% phosphoric acid (B) in gradient mode (0-9 min, 85% -80% B; 9-12 min, 80% -70% B; 12-15 min, 70% -65% B; 15-20 min, 65% -60% B; 20-23 min, 60% -55% B; 23-29 min, 55% -54.4% B; 29-32 min, 54.4% -45% B; 32-37 min, 45% -5% B; 37-45 min, 5% -85% B) at the flow rate of 1.0 mL x min(-1). The column temperature was 35 degrees C and the detection wavelength was set at 326 nm. The good separation of chlorogenic acid, caffeic acid, 1,3-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, luteolin-7-O-beta-D-glucoside, 3,4-dicaffeoylquinic acid, linarin and luteolin was achieved within 40 min. Calibration curves of the eight effective components showed good linear relationship (r > 0.999 5, n = 7). The average recoveries were within 97.03%-102.3% (RSD < 2.0%, n = 6). The method is simple, accurate and repeatable and can be used for the quality control of Ch. indicum.
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Calibragem , Cromatografia Líquida de Alta Pressão , Métodos , Chrysanthemum , Química , Flavonoides , Controle de QualidadeRESUMO
<p><b>OBJECTIVE</b>To explore the role of the amino acids between 551 and 565 in the cytoplasmic domain of glycoprotein (GP) I b alpha in the VWF binding to GP I b alpha.</p><p><b>METHODS</b>The VWF binding to GP I b alpha induced by ristocetin was analyzed by flow cytometry, in three GP I b-IX-expressing Chinese hamster ovary (CHO) cell lines 1b9, delta 565 and delta 551, adhesion of above cells on VWF by flow chamber analysis at shear rate of 200 s(-1). The spread of GP I b-IX-expressing cells were stimulated with botrocetin on VWF-coated coverslips by confocal microscope.</p><p><b>RESULTS</b>The VWF binding to GP I b alpha was higher in delta 565 cells stimulated by ristocetin than in delta 551 or 1b9 cells. The number of delta 565 cells adhered on the VWF-coated-chamber was more than that of controls at shear rate of 200 s(-1). Moreover, the surface spreading areas of delta 565 cells were greater than that of the controls on VWF-coated coverslips.</p><p><b>CONCLUSIONS</b>The amino acids between 551 and 565 in the cytoplasmic domain of GP I b alpha regulates the VWF binding to GP I b alpha.</p>
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Animais , Cricetinae , Feminino , Sequência de Aminoácidos , Células CHO , Cricetulus , Adesividade Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas , Genética , Metabolismo , Fator de von Willebrand , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To explore the choice of operative approach for old inferior-cervical fracture-dislocation and analyze the clinical effects of anterior operative approach.</p><p><b>METHODS</b>From January 2003 to May 2010, 17 patients with inferior-cervical fracture-dislocation delayed for more than 4 weeks were treated with continued closed skull traction and anterior decompression, bone graft and internal fixation with steel plate. Among the patients, 11 patients were male and 6 patients were female with an average age of 41 years (ranged from 24 to 56 years). The time between injury and operation was from 4 weeks to 3 months. According to Frankel grade, grade A was in 7 cases, B in 4, C in 2, D in 2, E in 2. Neurological function, bone fusion height of vertebral body and cervical sequence and curvature were observed.</p><p><b>RESULTS</b>The incision of 17 cases obtained primary healing. There was 1 case with hoarseness, and symptoms disappeared after 1 month. The mean time of follow-up was 23 months (ranged from 4 to 47 months). The X-ray films showed satisfactory reduction and good alignment and lordosis. The Frankel grade improved obviously at final follow-up, grade A was in 5 cases, B in 5, C in 1 , D in 3, E in 3.</p><p><b>CONCLUSION</b>Single anterior operative approach can successfully reduce old inferior-cervical fracture-dislocation of DF stage I , II and some stage III; anterior decompression, bone graft and internal fixation with steel plate is a safe, effective method for old inferior-cervical fracture-dislocation.</p>
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Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Vértebras Cervicais , Cirurgia Geral , Fixação Interna de Fraturas , Métodos , Luxações Articulares , Cirurgia Geral , Fraturas da Coluna Vertebral , Cirurgia GeralRESUMO
The aim of this study was to construct Chinese Hamster Ovary (CHO) cell models expressing recombinant wild-type GPIb-IX and mutant GPIb-IX complex, so as to provide the platform to study the related physiologic functions of GPIb-IX. The plasmids were extracted from E.coli expressing wild-type or deletion mutant GPIbalpha and were identified by digestion with EcoR I. Three plasmids containing GPIbalpha, GPIbbeta, and GPIX genes were co-transfected into CHO cells, and then the expression of GPIb-IX complex was detected by immune coprecipitation, Western blot and flow cytometry. The results showed that the expression of GPIb-IX complex could be detected in the lysate and on the surface of CHO cells at 48 hours after transfection. In conclusion, CHO cell models expressing recombinant wild-type or mutation GPIb-IX complex has been successfully constructed.
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Animais , Cricetinae , Células CHO , Cricetulus , Mutação , Plasmídeos , Complexo Glicoproteico GPIb-IX de Plaquetas , Genética , Proteínas Recombinantes , GenéticaRESUMO
<p><b>OBJECTIVE</b>To explore the regulatory role of protein kinase A (PKA) in platelet surface glycoprotein (GP) I balpha expression.</p><p><b>METHODS</b>Washed platelets from healthy volunteers were incubated with PKA inhibitor. The N-terminal fragment of GP I balpha (glycocalicin, GC) in the supernatant of platelet suspensions was detected by Western blot and GP I balpha surface expression by flow cytometry. Calpain activity was determined by cytoskeletal proteins proteolysis and calpain surface expression by flow cytometry. The effect of PKA inhibitor on ristocetin-induced platelet aggregation was measured by platelet aggregometer.</p><p><b>RESULTS</b>After PKA was inhibited in washed platelets, GP I balpha was cleaved and released to the supernatant, which significantly decreased the surface expression of GP I balpha (P < 0.05). The event was suppressed by pre-treatment with various calpain inhibitors, indicating that PKA inhibitor-mediated shedding was calpain dependent. The actin-binding protein (ABP) and talin proteolysis demonstrated that calpain was activated by PKA inhibitor and expressed on the platelet membrane. Ristocetin-induced aggregation was inhibited by PKA inhibitor.</p><p><b>CONCLUSION</b>PKA inhibition results in calpain-dependent GP I balpha shedding, which thus reduces GP I balpha surface expression and GP I balpha-dependent platelet aggregation. These results might provide a view to develop new drugs for thrombotic diseases.</p>