本文選題：P-選擇素　切入點：深靜脈血栓　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] 在課題組前期利用Affymetrix Rat230A cDNA基因芯片技術(shù)篩選TDVT(Traumatic Deep Venous Thrombosis,TDVT)大鼠靜脈內(nèi)皮組織中上調(diào)差異性表達(dá)基因基礎(chǔ)上,查閱Ncibi Gene和Gene Card數(shù)據(jù)庫(2012)確定與血小板活化及內(nèi)皮細(xì)胞粘附密切相關(guān)的選擇素家族成員CD62P (P-selectin,p-選擇素)作為進(jìn)一步研究對象。結(jié)合CD62P主要定位于血小板的a-顆粒和內(nèi)皮細(xì)胞的Weibel-Palade小體內(nèi)的機(jī)制,利用RT-PCR技術(shù)檢測各實驗組血樣白細(xì)胞、血小板中CD62P的轉(zhuǎn)錄表達(dá),初步探討CD62P基因在深靜脈血栓形成過程中轉(zhuǎn)錄表達(dá)情況。為進(jìn)一步研究CD62P參與血小板活化及內(nèi)皮細(xì)胞粘附的分子機(jī)制,以及CD62P是否有望成為臨床中預(yù)測和早期診斷DVT的特異性分子標(biāo)志物提供理論參考。 [材料和方法] 一、確定實驗研究對象,收集各實驗組血液樣本,通過RT—PCR技術(shù)檢測CD62P基因在血液白細(xì)胞、血小板轉(zhuǎn)錄表達(dá)情況 1、參考2012年第9版《美國胸科醫(yī)師協(xié)會抗栓與血栓預(yù)防臨床實踐指南—深靜脈血栓形成的診斷》制定本研究的DVT診斷標(biāo)準(zhǔn),按下列危險因素評估高危人群：血栓病史或家族史、長期臥床或制動、高齡、肥胖；內(nèi)科急性因素：呼吸衰竭、急性腦卒中、急性心力衰竭、急性心肌梗死、急性感染性疾病、急性風(fēng)濕性疾病等；內(nèi)科基礎(chǔ)及慢性因素：慢性心力衰竭、惡性腫瘤、偏癱、慢性肺部疾病、糖尿病、肥胖、膠原血管病及易栓癥、炎癥性腸病、腎病綜合征、代謝綜合征等疾病。此外,中心靜脈置管、永久性起搏器置入、口服避孕藥,激素替代治療,妊娠及產(chǎn)后6周等；外科危險因素包括主要為普通外科手術(shù)(結(jié)、直腸腫瘤)、骨科大手術(shù)、血管外科手術(shù)(涉及下肢靜脈手術(shù))、婦科手術(shù)(分娩、盆腔腫瘤)、神經(jīng)外科手術(shù)(肢體運動障礙及顱高壓脫水治療)；其中骨科大手術(shù)包括人工全髖、膝關(guān)節(jié)關(guān)節(jié)置換術(shù),股骨頸骨折和股骨轉(zhuǎn)子間骨折,股骨干骨折,骨盆骨折,脊柱骨折合并脊髓損傷,手術(shù)時間45min,術(shù)中應(yīng)用止血帶。 評估危險因素后,結(jié)合臨床表現(xiàn)(下肢疼痛、腫脹等、Homans征、Neuhof征)和彩色多普勒超聲檢查綜合診斷。納入研究對象排除其它高危因素(肥胖、懷孕、糖尿病、腫瘤、高血壓、高脂血癥、免疫系統(tǒng)疾病、全身炎性反應(yīng)綜合征等因素)(詳見附錄2),于2010年3月-2012年3月共納入58例病例,正常對照組20例(健康人群)、血栓未形成組20例(創(chuàng)傷行手術(shù)后及骨科大手術(shù)后1天無形成血栓的患者)、血栓形成組18例(為DVT形成患者),三組間的年齡、性別構(gòu)成、體重指數(shù)等差異無統(tǒng)計意義。三組均采集清晨空腹靜脈血液樣本,血栓未形成組于手術(shù)后第1d采集,血栓形成組于確診后第1d采集。提取各組樣本總RNA并進(jìn)行質(zhì)量檢測； 2、應(yīng)用Oligo6.69引物設(shè)計軟件,設(shè)計CD62P基因反轉(zhuǎn)錄引物,將各組人血液總RNA反轉(zhuǎn)錄為cDNA; 3、采用PCR檢測CD62P基因在各個實驗組中的轉(zhuǎn)錄表達(dá)情況,以GAPDH為內(nèi)參,利用光密度進(jìn)行半定量分析； 二、結(jié)果數(shù)據(jù)處理和分析 將所有試驗記錄數(shù)據(jù)分類并統(tǒng)計,統(tǒng)計數(shù)據(jù)采用x±s表示,組間比較采用單因素方差分析,由SPSS13.0統(tǒng)計軟件進(jìn)行數(shù)據(jù)處理,p0.05表示差異有顯著性意義。 [結(jié)果] 與正常對照組(A組)相比,血栓未形成組(B組)有表達(dá),血栓形成組(C組)呈現(xiàn)高表達(dá)。A組與B組相比,P0.05；A組與C組相比,P0.05；C組與B組相比,P0.05。 [結(jié)論] 通過各實驗組人血白細(xì)胞、血小板中CD62P基因轉(zhuǎn)錄表達(dá)量的RT-PCR檢測結(jié)果表明：血栓形成組及創(chuàng)傷后血栓未形成組與健康人群相比CD62P在基因轉(zhuǎn)錄層面呈現(xiàn)明顯上調(diào)表達(dá)趨勢,深靜脈血栓形成組表達(dá)量大于創(chuàng)傷術(shù)后及骨科大手術(shù)后血栓未形成組。據(jù)此,初步推測該基因可能在DVT形成和機(jī)體應(yīng)激反應(yīng)中發(fā)揮著重要作用,與血栓形成密切相關(guān)。
[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錛，

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