人血细胞中KLF-4表达下调影响DVT形的临床研究

发布时间：2018-02-21 15:27

本文关键词： 深静脉血栓 Kr(u|¨)ppel样因子4 分子标记物　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：在前期对形成DVT大鼠股静脉动物模型Affymetrix Rat230A cDNA基因芯片的统计研究,筛查归纳深静脉血栓形成时期具有明显差异性表达的基因的基础上,通过查阅PubMand和Gene Card数据库,锁定深静脉血栓形成下调基因KLF-4。检测KLF-4在正常人血液样本、血栓未形成患者血液样本及深静脉血栓形成患者血液样本中表达变化,探讨KLF-4的表达情况与深静脉血栓形成的关系,确定人血细胞转录层面KLF-4作为早期诊断人深静脉血栓的候选分子标志物。方法： 1、参考《静脉血栓栓塞症预防的NICE指南》(2012年)和《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》(2012年第9版)制定本研究的DVT诊断标准：DVT的综合诊断=危险因素评估(基础危险因素+专科危险因素)+主要临床表现+下肢深静脉彩色多普勒超声检查(附录2)。于2010年3月-2012年3月共纳入样本数58例,正常对照组(20例,为健康志愿者)、血栓未形成组(20例,为下肢长骨骨折或脊柱骨折7d内行手术后无形成血栓的患者)、血栓形成组(18例,为DVT形成患者),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。收集血样本,应用primer5.0引物设计软件对人KLF-4设计反转录引物,将各组人血细胞总RNA反转录为cDNA,以GAPDH为内参照运用半定量PCR检测KLF-4在临床血液样本白细胞中的表达情况。 2、对实验数据进行处理,分析KLF-4在深静脉血栓形成中的表达情况和作用,确定KLF-4在人血细胞转录层面能够作为早期诊断DVT及预后转归的候选分子标志物。结果从半定量PCR胶图及目的基因与内参基因灰度值比值可以看出：内参对照GAPDH大小均一,亮度一致,C组条带明显较A组及B组条带亮度降低,灰度值：A组(0.9803±0.0740),B组(0.9764±0.0839)C组(0.3751±0.0359),C组与A组相比P0.05,差距有统计学意义,C组与B组比较P0.05,差距有统计学意义,B组与A组相比P0.05,差距无统计学意义。结论 1.KLF-4与深静脉血栓之间存在着非常密切的相关性,很有可能为影响深静脉血栓形成与否的一个主要影响因素。 2.KLF-4在人深静脉血栓患者血液中的表达下调,可以作为分子标记物在血液中进行检测来诊断人深静脉血栓。
[Abstract]:Objective:. On the basis of the statistical study of Affymetrix Rat230A cDNA gene chip in the animal model of femoral vein formation in DVT rats, and the screening of genes with obvious difference expression during deep venous thrombosis, the databases of PubMand and Gene Card were consulted. To investigate the relationship between the expression of KLF-4 and deep venous thrombosis (DVT), we detected the expression of KLF-4 in the blood samples of normal subjects, non-thrombosis patients and deep venous thrombosis patients, and investigated the relationship between the expression of KLF-4 and deep venous thrombosis (DVT). To identify human blood transcriptional KLF-4 as a candidate molecular marker for early diagnosis of human deep venous thrombosis. Methods:. 1. Refer to the NICE guidelines for the Prevention of Venous Thromboembolism (2012) and the American Association of chest Physicians' guidelines for Clinical practice on Antithrombotic and Thrombosis Prevention-diagnosis of Deep Venous Thrombosis (2012 9th Edition) to develop the DVT diagnostic criteria for this study. The main clinical manifestations of quasi: DVT: comprehensive diagnosis = risk factor assessment (basic risk factors specialist risk factors) (appendix 2). From March 2010 to March 2012, 58 samples were included. The normal control group consisted of 20 healthy volunteers, 20 patients without thrombosis, 18 patients with thrombus after 7 days of operation and 18 patients with long bone fracture or spinal fracture of lower extremity. For the patients with DVT, fasting venous blood samples were collected in all three groups. Blood samples were collected on the first day after operation in the thrombus group, and on the first day after the diagnosis in the thrombosis group. Primer5.0 primer design software was used to design reverse transcription primers for human KLF-4. The total RNA of human blood cells in each group was reversed to cDNA. using GAPDH as internal reference, semi-quantitative PCR was used to detect the expression of KLF-4 in the leukocytes of clinical blood samples. 2. The experimental data were processed, and the expression and role of KLF-4 in deep venous thrombosis were analyzed. It was confirmed that KLF-4 could be used as a candidate molecular marker for early diagnosis and prognosis of DVT at the transcriptional level of human blood cells. Results. From the semi-quantitative PCR gel map and the ratio of gray value of target gene to internal reference gene, it can be seen that the size of GAPDH in the control group is uniform, and the brightness of the band in group C is obviously lower than that in group A and group B. The grayscale value of group A was 0.9803 卤0.0740, group B was 0.9764 卤0.0839C, the difference between group C and group A was 0.3751 卤0.0359, the difference between group C and group A was significant (P 0.05), the difference between group B and group A was significant (P 0.05), the difference between group B and group A was not statistically significant. Conclusion. 1. KLF-4 is closely related to deep venous thrombosis, which may be one of the main factors influencing the formation of deep venous thrombosis. 2. The expression of KLF-4 was down-regulated in the blood of patients with deep venous thrombosis (DVT). KLF-4 could be used as a molecular marker to detect human deep venous thrombosis (DVT).
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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