本文選題：深靜脈血栓　切入點(diǎn)：內(nèi)皮細(xì)胞　出處：《昆明醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的] 依據(jù)PubMand和Gene Card數(shù)據(jù)庫分類查詢深靜脈血栓形成時(shí)期具有明顯差異性表達(dá)基因的相關(guān)文獻(xiàn)報(bào)道,表明：在動物實(shí)驗(yàn)及體外細(xì)胞實(shí)驗(yàn)中發(fā)現(xiàn)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風(fēng)險(xiǎn)。結(jié)合前期對形成DVT大鼠股靜脈動物模型Affymetrix Rat230AcDNA基因芯片的統(tǒng)計(jì)研究,篩查鎖定深靜脈血栓形成上調(diào)內(nèi)皮細(xì)胞蛋白C受體(EPCR)基因；本實(shí)驗(yàn)針對深靜脈血栓形成患者及創(chuàng)傷手術(shù)后患者的白細(xì)胞,采用RT-PCR技術(shù)檢測白細(xì)胞中EPCR基因的表達(dá)變化,分析在內(nèi)皮細(xì)胞損傷時(shí)期EPCR基因的差異表達(dá)水平,及其表達(dá)產(chǎn)物通過凝血酶-血栓調(diào)節(jié)素干擾PC活化過程,結(jié)合血液樣本實(shí)驗(yàn)室凝血功能檢查進(jìn)一步分析其在參與DVT形成中所發(fā)揮的作用關(guān)系。 [材料和方法] 查詢Ncbi和GeneCard數(shù)據(jù)庫(2012)中人類的基因序列,鎖定血栓形成期具有顯著上調(diào)的內(nèi)皮細(xì)胞蛋白C受體基因,采用RT-PCR檢測人血白細(xì)胞中EPCR基因的表達(dá)變化水平 對前期形成DVT大鼠股靜脈動物模型Affymetrix Rat230A cDNA基因芯片統(tǒng)計(jì)研究,查詢Ncbi和Gene Card數(shù)據(jù)庫中人類的基因序列,鎖定在創(chuàng)傷前后、血栓形成期具有明顯差異表達(dá)變化的內(nèi)皮細(xì)胞蛋白C受體基因。依據(jù)《靜脈血栓栓塞癥預(yù)防的NICE指南》(2012)制定本實(shí)驗(yàn)研究臨床志愿者及研究對象的觀察、診斷和納入標(biāo)準(zhǔn)(排除危險(xiǎn)因素如：吸煙、家族史及近期接受各類外科手術(shù)、創(chuàng)傷或長期住院治療史。判斷高危人群：ICU病房的危重患者,急性心肌梗死、心肺衰竭、缺血性腦卒中以及嚴(yán)重的肺部疾病患者,惡性腫瘤等。此外,口服避孕藥,激素替代治療,妊娠及產(chǎn)后6周,腫瘤化療,糖尿病、急性風(fēng)濕性疾病、炎癥性腸病、腎病綜合征、代謝綜合征等疾病),收集實(shí)驗(yàn)對照組資料、患者入院病史、臨床癥狀體征及實(shí)驗(yàn)室檢查資料。依據(jù)研究對象納入標(biāo)準(zhǔn)選取深靜脈血栓形成的患者18例為血栓組,選取骨科創(chuàng)傷術(shù)后20例病人為血栓未形成組、選取體檢結(jié)果提示健康的志愿者20人作為對照組。各組研究對象在年齡、體重及性別方面無明顯統(tǒng)計(jì)學(xué)差異,實(shí)驗(yàn)對照組采集晨起空腹外周血5m1,血栓未形成組和血栓形成組均于入院后、術(shù)后和確診后1天經(jīng)肘正中靜脈抽取晨起空腹外周血5m1。血液標(biāo)本經(jīng)3.8%枸櫞酸鈉抗凝,3000r/min離心10分鐘,取離心后沉淀成分分裝置于-20℃凍存待測；采用RT-PCR檢測各組人血白細(xì)胞中內(nèi)皮細(xì)胞蛋白C受體基因的表達(dá)。PCR反應(yīng)擴(kuò)增(預(yù)變性、變性、退火、延伸、終末延伸),對PCR產(chǎn)物進(jìn)行RT-PCR分析,識別特異性擴(kuò)增產(chǎn)物,經(jīng)1.5%瓊脂糖凝膠電泳檢測評估各組總RNA純度及質(zhì)量。操作步驟均嚴(yán)格按照說明書執(zhí)行；采用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析,單因素方差分析。以P0.05為顯著性檢驗(yàn)水準(zhǔn)。 結(jié)果： 1.骨折創(chuàng)傷手術(shù)后及血栓形成患者血細(xì)胞EPCR-mRNA表達(dá)水平較正常對照組均明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 2.骨折創(chuàng)傷后組與深靜脈血栓形成組EPCR-mRNA表達(dá)水平無明顯變化,兩組間比較P0.05,無統(tǒng)計(jì)學(xué)意義。 結(jié)論： 深靜脈血栓形成病人及創(chuàng)傷后患者人血細(xì)胞的EPCR基因表達(dá)水平較正常對照組明顯升高。EPCR基因表達(dá)水平升高,提示可能與人深靜脈血栓形成有關(guān)。
[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錛，

本文編號：1600792