CD62P在人血白细胞、血小板上调表达促进DVT形成的临床研究

发布时间：2018-03-18 17:17

  本文选题：P-选择素　切入点：深静脉血栓　出处：《昆明医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：[目的] 在课题组前期利用Affymetrix Rat230A cDNA基因芯片技术筛选TDVT(Traumatic Deep Venous Thrombosis,TDVT)大鼠静脉内皮组织中上调差异性表达基因基础上,查阅Ncibi Gene和Gene Card数据库(2012)确定与血小板活化及内皮细胞粘附密切相关的选择素家族成员CD62P (P-selectin,p-选择素)作为进一步研究对象。结合CD62P主要定位于血小板的a-颗粒和内皮细胞的Weibel-Palade小体内的机制,利用RT-PCR技术检测各实验组血样白细胞、血小板中CD62P的转录表达,初步探讨CD62P基因在深静脉血栓形成过程中转录表达情况。为进一步研究CD62P参与血小板活化及内皮细胞粘附的分子机制,以及CD62P是否有望成为临床中预测和早期诊断DVT的特异性分子标志物提供理论参考。 [材料和方法] 一、确定实验研究对象,收集各实验组血液样本,通过RT—PCR技术检测CD62P基因在血液白细胞、血小板转录表达情况 1、参考2012年第9版《美国胸科医师协会抗栓与血栓预防临床实践指南—深静脉血栓形成的诊断》制定本研究的DVT诊断标准,按下列危险因素评估高危人群：血栓病史或家族史、长期卧床或制动、高龄、肥胖；内科急性因素：呼吸衰竭、急性脑卒中、急性心力衰竭、急性心肌梗死、急性感染性疾病、急性风湿性疾病等；内科基础及慢性因素：慢性心力衰竭、恶性肿瘤、偏瘫、慢性肺部疾病、糖尿病、肥胖、胶原血管病及易栓症、炎症性肠病、肾病综合征、代谢综合征等疾病。此外,中心静脉置管、永久性起搏器置入、口服避孕药,激素替代治疗,妊娠及产后6周等；外科危险因素包括主要为普通外科手术(结、直肠肿瘤)、骨科大手术、血管外科手术(涉及下肢静脉手术)、妇科手术(分娩、盆腔肿瘤)、神经外科手术(肢体运动障碍及颅高压脱水治疗)；其中骨科大手术包括人工全髋、膝关节关节置换术,股骨颈骨折和股骨转子间骨折,股骨干骨折,骨盆骨折,脊柱骨折合并脊髓损伤,手术时间45min,术中应用止血带。 评估危险因素后,结合临床表现(下肢疼痛、肿胀等、Homans征、Neuhof征)和彩色多普勒超声检查综合诊断。纳入研究对象排除其它高危因素(肥胖、怀孕、糖尿病、肿瘤、高血压、高脂血症、免疫系统疾病、全身炎性反应综合征等因素)(详见附录2),于2010年3月-2012年3月共纳入58例病例,正常对照组20例(健康人群)、血栓未形成组20例(创伤行手术后及骨科大手术后1天无形成血栓的患者)、血栓形成组18例(为DVT形成患者),三组间的年龄、性别构成、体重指数等差异无统计意义。三组均采集清晨空腹静脉血液样本,血栓未形成组于手术后第1d采集,血栓形成组于确诊后第1d采集。提取各组样本总RNA并进行质量检测； 2、应用Oligo6.69引物设计软件,设计CD62P基因反转录引物,将各组人血液总RNA反转录为cDNA; 3、采用PCR检测CD62P基因在各个实验组中的转录表达情况,以GAPDH为内参,利用光密度进行半定量分析； 二、结果数据处理和分析 将所有试验记录数据分类并统计,统计数据采用x±s表示,组间比较采用单因素方差分析,由SPSS13.0统计软件进行数据处理,p0.05表示差异有显著性意义。 [结果] 与正常对照组(A组)相比,血栓未形成组(B组)有表达,血栓形成组(C组)呈现高表达。A组与B组相比,P0.05；A组与C组相比,P0.05；C组与B组相比,P0.05。 [结论] 通过各实验组人血白细胞、血小板中CD62P基因转录表达量的RT-PCR检测结果表明：血栓形成组及创伤后血栓未形成组与健康人群相比CD62P在基因转录层面呈现明显上调表达趋势,深静脉血栓形成组表达量大于创伤术后及骨科大手术后血栓未形成组。据此,初步推测该基因可能在DVT形成和机体应激反应中发挥着重要作用,与血栓形成密切相关。
[Abstract]:[Objective]
TDVT Affymetrix Rat230A cDNA in the screening of gene chip technology using ourprevious (Traumatic Deep Venous Thrombosis, TDVT) of rat vein endothelial tissue increase the differential gene expression on the basis of Ncibi Gene and Gene Card access database (2012) to determine the associated with platelet activation and endothelial cell adhesion of the selectin family member CD62P (P-selectin p- selectin), as a further study. The mechanism of a- particles with CD62P mainly localized in platelets and endothelial cells in Weibel-Palade body, the detection of each experimental group blood cells using RT-PCR technology, transcriptional expression of platelet CD62P, preliminary study of CD62P gene expression in the transcription process for molecules of deep vein thrombosis. Further study of the mechanism of CD62P involved in platelet activation and endothelial cell adhesion, and whether the CD62P is expected to become a clinical prediction and early It provides a theoretical reference for the diagnosis of specific molecular markers of DVT.
[materials and methods]
First, to determine the subjects of the experimental study, collect the blood samples from the experimental groups and detect the transcription and expression of the CD62P gene in the blood white blood cells and platelets by RT - PCR technique.
1, reference ninth edition in 2012. American College of chest physicians antithrombotic and prevention of thrombosis in DVT diagnosis standard in this study, clinical practice guidelines > diagnosis of deep venous thrombosis, according to the risk assessment of high-risk groups: history or family history of thrombosis, immobilization, high age, obesity; acute respiratory factors: failure, acute heart failure, acute stroke, acute myocardial infarction, acute infectious diseases, acute rheumatic diseases; medical foundation and chronic factors: chronic heart failure, malignant tumor, hemiplegia, chronic lung disease, diabetes, obesity, collagen vascular disease and thrombophilia, inflammatory bowel disease, nephrotic syndrome, metabolic syndrome disease. In addition, Central venous catheter, permanent pacemaker, oral contraceptives, hormone replacement therapy, pregnancy and postpartum 6 weeks; surgical risk factors include general surgery ( Node, rectal cancer), Department of orthopedics, surgery, vascular surgery (involving venous surgery), gynecological surgery (delivery, pelvic tumor surgery (Department of neurosurgery), limb movement disorder and intracranial pressure dehydration therapy); the Department of orthopedics operation including hip, knee arthroplasty, femoral neck fracture and femoral intertrochanteric fracture, femoral shaft fracture, pelvic fracture, spine fracture combined with spinal cord injury, the operative time was 45min, the application of tourniquet during operation.
Assessment of risk factors, clinical manifestations (lower limb pain, swelling, Homans syndrome, Neuhof syndrome) and color Doppler ultrasonography diagnosis. Included in the study to exclude other risk factors (obesity, pregnancy, diabetes, cancer, hypertension, hyperlipidemia, immune system disease, systemic inflammatory response syndrome and other factors) (see Appendix 2), in March 2010 -2012 year in March a total of 58 cases, 20 cases of normal control group (healthy people), non thrombosis group (20 cases of trauma surgery and Department of orthopedics 1 days after surgery without thrombosis patients), 18 cases of thrombosis group (DVT patients) between the three groups, age, gender, body mass index had no significant difference. The three groups were collected fasting venous blood samples, non thrombosis group in operation after the 1D acquisition, thrombosis group confirmed by 1D after acquisition. The extraction of total RNA of each sample and quality Testing锛，

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