人血白细胞EPCR基因表达、产物干扰PC活化与DVT形成的相关性研究

发布时间：2018-03-12 08:35

  本文选题：深静脉血栓　切入点：内皮细胞　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] 依据PubMand和Gene Card数据库分类查询深静脉血栓形成时期具有明显差异性表达基因的相关文献报道,表明：在动物实验及体外细胞实验中发现可溶性EPCR (sEPCR)呈抑制PC/APC活性的作用,它抑制PC的活化,使凝血酶的生成增多而加大血栓形成风险。结合前期对形成DVT大鼠股静脉动物模型Affymetrix Rat230AcDNA基因芯片的统计研究,筛查锁定深静脉血栓形成上调内皮细胞蛋白C受体(EPCR)基因；本实验针对深静脉血栓形成患者及创伤手术后患者的白细胞,采用RT-PCR技术检测白细胞中EPCR基因的表达变化,分析在内皮细胞损伤时期EPCR基因的差异表达水平,及其表达产物通过凝血酶-血栓调节素干扰PC活化过程,结合血液样本实验室凝血功能检查进一步分析其在参与DVT形成中所发挥的作用关系。 [材料和方法] 查询Ncbi和GeneCard数据库(2012)中人类的基因序列,锁定血栓形成期具有显著上调的内皮细胞蛋白C受体基因,采用RT-PCR检测人血白细胞中EPCR基因的表达变化水平 对前期形成DVT大鼠股静脉动物模型Affymetrix Rat230A cDNA基因芯片统计研究,查询Ncbi和Gene Card数据库中人类的基因序列,锁定在创伤前后、血栓形成期具有明显差异表达变化的内皮细胞蛋白C受体基因。依据《静脉血栓栓塞症预防的NICE指南》(2012)制定本实验研究临床志愿者及研究对象的观察、诊断和纳入标准(排除危险因素如：吸烟、家族史及近期接受各类外科手术、创伤或长期住院治疗史。判断高危人群：ICU病房的危重患者,急性心肌梗死、心肺衰竭、缺血性脑卒中以及严重的肺部疾病患者,恶性肿瘤等。此外,口服避孕药,激素替代治疗,妊娠及产后6周,肿瘤化疗,糖尿病、急性风湿性疾病、炎症性肠病、肾病综合征、代谢综合征等疾病),收集实验对照组资料、患者入院病史、临床症状体征及实验室检查资料。依据研究对象纳入标准选取深静脉血栓形成的患者18例为血栓组,选取骨科创伤术后20例病人为血栓未形成组、选取体检结果提示健康的志愿者20人作为对照组。各组研究对象在年龄、体重及性别方面无明显统计学差异,实验对照组采集晨起空腹外周血5m1,血栓未形成组和血栓形成组均于入院后、术后和确诊后1天经肘正中静脉抽取晨起空腹外周血5m1。血液标本经3.8%枸橼酸钠抗凝,3000r/min离心10分钟,取离心后沉淀成分分装置于-20℃冻存待测；采用RT-PCR检测各组人血白细胞中内皮细胞蛋白C受体基因的表达。PCR反应扩增(预变性、变性、退火、延伸、终末延伸),对PCR产物进行RT-PCR分析,识别特异性扩增产物,经1.5%琼脂糖凝胶电泳检测评估各组总RNA纯度及质量。操作步骤均严格按照说明书执行；采用SPSS13.0统计软件进行统计分析,单因素方差分析。以P0.05为显著性检验水准。 结果： 1.骨折创伤手术后及血栓形成患者血细胞EPCR-mRNA表达水平较正常对照组均明显升高,差异有统计学意义(P0.05)。 2.骨折创伤后组与深静脉血栓形成组EPCR-mRNA表达水平无明显变化,两组间比较P0.05,无统计学意义。 结论： 深静脉血栓形成病人及创伤后患者人血细胞的EPCR基因表达水平较正常对照组明显升高。EPCR基因表达水平升高,提示可能与人深静脉血栓形成有关。
[Abstract]:[Objective]
According to reports, PubMand and Gene Card database query classification of deep vein thrombosis is of significantly differentially expressed gene formation period showed that soluble EPCR in animal experiments and in vitro cell experiment (sEPCR) showed inhibitory effect on PC/APC activity, it inhibited PC activation, which increased thrombin generation and increase the risk of thrombosis. Based on the previous statistical research on the formation of DVT Affymetrix Rat230AcDNA of rat femoral vein in animal models of gene chip screening, locked deep vein thrombosis by endothelial cell protein C receptor (EPCR) gene; the experiment for deep venous thrombosis patients and patients with white cell trauma after surgery, the expression change of EPCR gene in white blood cells was detected by RT-PCR technology the analysis of differences in EPCR endothelial cell injury during gene expression, and its expression product by thrombin - regulating hormone stem thrombosis The activation process of PC was disturbed and the blood sample laboratory coagulation function test was used to further analyze its role in the formation of DVT.
[materials and methods]
The human gene sequences in Ncbi and GeneCard database (2012) were querying, and the endothelial cell protein C receptor gene with significantly up-regulated thrombosis was detected. RT-PCR was used to detect the expression level of EPCR gene in human leukocytes.
Statistical study of DVT rat femoral vein of cDNA Rat230A Affymetrix animal model of gene chip on the formation of early gene sequence of human Ncbi and Gene query in Card database, locked before and after trauma, thrombosis and endothelial cell protein C expression have significant differences of receptor gene. On the basis of "venous thromboembolism prevention guide > (NICE 2012) the experimental study on clinical observation and study of volunteers, the diagnosis and the inclusion criteria (exclude the risk factors such as smoking, family history and the recent acceptance of various types of surgery, trauma or long-term hospitalization history. Identify high-risk groups: critically ill patients in ICU ward, acute myocardial infarction, heart failure, and stroke the severity of lung disease in patients with malignant tumors. In addition, oral contraceptives, hormone replacement therapy, pregnancy and postpartum 6 weeks, cancer chemotherapy, diabetes, acute rheumatic diseases, inflammation Inflammatory bowel disease, nephrotic syndrome, metabolic syndrome and other diseases), the control group data collection experiment, patients admitted to medical history, clinical symptoms and laboratory examination data. According to the research object into the selection criteria of patients with deep vein thrombosis in 18 cases of DVT group, selected 20 cases of postoperative Department of orthopedics trauma patients as the non thrombosis group health examination results, selected 20 volunteers as the control group. Each research object in terms of age, weight and gender were no significant difference between the experimental and control group collected the morning fasting peripheral blood 5m1, non thrombosis group were admitted to hospital after the formation of thrombus group and postoperative diagnosis, and 1 days after the median cubital vein from the early morning fasting peripheral blood 5m1. blood specimens of 3.8% sodium citrate, 3000r/min centrifugal 10 minutes after centrifugation, precipitation component device at -20 deg.c freezing test; detected by RT-PCR on human blood leukocytes The expression of.PCR amplification reaction of endothelial cell protein C cell receptor gene (pre denaturation, denaturation, annealing, extension, final, Mo Yanshen) RT-PCR analysis of PCR products, identification of specific amplification products by 1.5% agarose gel electrophoresis was measured total RNA purity and quality. The operation steps are in strict accordance with the instructions to perform statistics; using SPSS13.0 for statistical analysis, single factor analysis of variance. The inspection level is P0.05.
Result锛，

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