急性創(chuàng)傷性深靜脈血栓消退與不消退差異表達(dá)基因研究

1、. 急性創(chuàng)傷性深靜脈血栓消退與不消退差異表達(dá)基因研究中文摘要目的：在建立大鼠急性創(chuàng)傷性肢體深靜脈血栓動(dòng)物模型根底上，應(yīng)用Affymetri*基因芯片技術(shù)，從基因水平研究血栓形成后，消退與不消退兩種不同病理過程間股靜脈中差異表達(dá)基因，探索影響創(chuàng)傷性肢體深靜脈血栓消退的局部關(guān)鍵因素。方法：1. 分組：250只SD大鼠雌雄不限隨機(jī)取20只作為對(duì)照組，余230只據(jù)創(chuàng)傷造模后血栓形成病理變化過程分為：創(chuàng)傷即刻組B組，造模后0.5小時(shí)、血栓形成前期組C組，造模后2.5小時(shí)、血栓形成頂峰期組D組，造模后25小時(shí)、血栓消退期組E組，造模后72小時(shí)、血栓不消退組F組，造模后72小時(shí)和血栓不形成組G組，造模后72

2、小時(shí)。2. 造模：對(duì)照組大鼠不造模，創(chuàng)傷組大鼠造模時(shí)不行麻醉，造模方法為：雙側(cè)腹股溝區(qū)碘伏液消毒后行側(cè)切口，長(zhǎng)約1cm，暴露出股動(dòng)、靜脈及股神經(jīng)，稍作鈍性別離顯露長(zhǎng)約1.5cm的股靜脈，用12.5mm全齒蚊式血管鉗分三段各鉗夾血管1次力量為血管鉗緊1扣，每次持續(xù)3秒后，用1號(hào)絲線全層連續(xù)縫合皮膚切口，不放置引流，除對(duì)照組、創(chuàng)傷即刻組外大鼠均行雙后肢髖人字石膏固定；造模后觀察大鼠雙足顏色和腫脹情況。3. 取材方法：對(duì)照組大鼠不造模即取材，創(chuàng)傷即刻組于造模后0.5小時(shí)，其余各組大鼠在相應(yīng)時(shí)相點(diǎn)沿原切口暴露股靜脈，觀察血栓形成狀態(tài)，符合分組標(biāo)準(zhǔn)后，用3的戊巴比妥鈉溶液按1ml/kg體重，腹腔注射麻醉

3、，仰臥位固定，碘伏液消毒雙后肢前側(cè)皮膚區(qū)域后，沿雙側(cè)股靜脈走行切開皮膚約4.5cm，觀察局部組織反響、股靜脈血栓是否形成、嚴(yán)重程度及消退、不消退情況，分別記錄；顯微鏡下別離并切取雙側(cè)各長(zhǎng)約4.5cm的股靜脈及主要屬支，留長(zhǎng)約0.5cm的血管用甲醛固定，送HE染色組織切片鏡檢；0.9%生理鹽水沖洗干凈血管中的血液或血栓，離體30秒放入凍存管，置入液氮罐中保存，用于總RNA提取。4. 總RNA提?。篢RIzol法分別提取上述7組股靜脈標(biāo)本總RNA；各組總RNA樣品經(jīng)瓊脂糖凝膠電泳檢測(cè)合格后28SRNA和18SRNA條帶整齊，兩者帶寬比值超過2倍分為兩份，一份送以進(jìn)展基因芯片檢測(cè)，另一份備用以行RT

4、-PCR檢測(cè)。5.芯片及RT-PCR檢測(cè)：按Affymetri* RAT 230A表達(dá)譜芯片操作流程，經(jīng)cDNA探針制備、雜交、洗脫、染色、掃描，完成芯片檢測(cè)；應(yīng)用RT-PCR技術(shù)檢測(cè)保存的RNA樣品中IL-1、Cinc2表達(dá)情況，并與芯片數(shù)據(jù)作比擬。6. 數(shù)據(jù)篩選及分析：通過倍數(shù)變化分析方法兩組間同一基因的Signal log2 ratio比擬，差值必須1或-1，常規(guī)認(rèn)為具有差異性，選取血栓消退與不消退組差異表達(dá)基因，應(yīng)用Gene Cluster 3.0分析軟件聚類能更容易地選取共表達(dá)的基因組，提示可能具有相似功能，繪制直觀曲線圖，并行GO功能分類，查詢“NCBI、“KI及期刊文獻(xiàn)，搜尋相應(yīng)

5、背景知識(shí)資料，結(jié)合表達(dá)變化規(guī)律綜合分析。結(jié)果:1. 250只SD大鼠共死亡5只，其中3只因造模過程中股動(dòng)脈破裂，出血死亡，造模后25小時(shí)2只死于肺栓塞，其余大鼠均存活；D、E、F、G四組190只大鼠死亡5只， 25小時(shí)有126只血栓形成，D組取材用去25只，余101只觀察至72小時(shí)，64只血栓消退，37只血栓不消退；至72小時(shí)又有23只發(fā)生血栓，36只一直無血栓形成150；動(dòng)物死亡率2，血栓發(fā)生率80.54，血栓消退率63.37，血栓不形成率19.46；HE染色股靜脈組織切片鏡檢證實(shí)：大體觀察血栓形成狀態(tài)進(jìn)展的分組準(zhǔn)確可靠。2. 各時(shí)相點(diǎn)7份標(biāo)本總RNA經(jīng)瓊脂糖凝膠電泳檢測(cè)，28SRNA和18

6、SRNA條帶整齊，兩者量的比值超過2倍，樣品質(zhì)量好、無降解。3. Affymetri* RAT 230A芯片所能檢測(cè)的15866個(gè)基因中，7389個(gè)基因有差異表達(dá)，占總數(shù)的46.57%，全部時(shí)相點(diǎn)均無變化的有8477個(gè)，占總數(shù)的53.43%；主要涉及促分裂素原活化蛋白激酶、Ca+、黏附斑、刺激神經(jīng)的配體-受體相互作用、細(xì)胞因子-受體相互作用、核糖體、肌動(dòng)蛋白細(xì)胞骨架、Wnt、嘌呤代、白細(xì)胞經(jīng)皮遷移、胰島素、糖酵解/糖異生等信號(hào)通路。4. 消退組與對(duì)照組比擬，Log2 Ratio4.0的上調(diào)表達(dá)基因24個(gè)，無功能描述基因14個(gè)，有局部GO功能描述基因10個(gè)Top2a、Stk6、Slpi、Olr1

7、、Kdap、Itgam、Il6、Cd8a、Brca1、A2m，主要涉及DNA、蛋白質(zhì)代和炎癥反響等生物學(xué)過程；Log2 Ratio-4.0的下調(diào)表達(dá)基因9個(gè)，無功能描述基因4個(gè)，有局部GO功能描述基因5個(gè)Lepr、Gpd3、Atp1b4、Af6、Adh7，主要涉及細(xì)胞間信號(hào)傳遞及能量代等生物學(xué)過程。5不消退組與對(duì)照組比擬，Log2 Ratio4.0的上調(diào)表達(dá)基因26個(gè)，無功能描述基因16個(gè)，有局部GO功能描述基因10個(gè)Slpi、Olr1、Nos2、Mmp9、Kdap、Itgam、Il6、C*cl2、Cinc2、Cd8a，主要涉及炎癥反響與細(xì)胞間作用等生物學(xué)過程；Log2 Ratio-4.0的下

8、調(diào)表達(dá)基因8個(gè)，無功能描述基因6個(gè)，有局部GO功能描述基因2個(gè)Gpd3、Cyp1a1。6. 消退組與不消退組比擬，差異表達(dá)2倍以上Signal log2 ratio1或-1基因共118個(gè)，無功能描述基因70個(gè)，有功能描述的基因48個(gè)，其中凋亡/腫瘤相關(guān)16個(gè)，結(jié)合相關(guān)29個(gè)，代相關(guān)20個(gè)，細(xì)胞周期相關(guān)3個(gè)，信號(hào)傳導(dǎo)相關(guān)11個(gè)，構(gòu)造分子活性相關(guān)3個(gè)，轉(zhuǎn)錄調(diào)控活性相關(guān)3個(gè)，運(yùn)載體活性相關(guān)4個(gè)。7.股靜脈中Mmp-9、Mmp-12、Mmp-13、IL-1、C*cl2、Cinc2、Ccl3、Ptges、Arg1等基因在血栓消退組與不消退組中表達(dá)變化規(guī)律較為突出，Mmp-9差異表達(dá)甚至達(dá)21倍，血栓頂峰

9、期組及不消退組中均呈現(xiàn)較高表達(dá)，而血栓消退組與血栓不形成組中表達(dá)相對(duì)較低；與它們共表達(dá)的三個(gè)未知功能基因，Affymetri*基因芯片探針號(hào)為：1391505_*_at、1379497_at、1377365_at。8. 芯片雜交信號(hào)強(qiáng)度滿意，各時(shí)相點(diǎn)IL-1、Cinc2的RT-PCR檢測(cè)結(jié)果與芯片結(jié)果變化趨勢(shì)根本一致。結(jié)論：1. 股靜脈可通過表達(dá)Mmp-9、Mmp-12、Mmp-13組織修復(fù)、血管形成相關(guān)基因，IL-1、C*cl2、Cinc2、Ccl3等炎癥相關(guān)基因，促進(jìn)損傷血管修復(fù)，發(fā)揮抗凝活性，表達(dá)Arginase、Ptges等血管擴(kuò)、抑制血小板集聚相關(guān)基因，共同改變局部血管狀態(tài)影響血栓消

10、退，其作用在急性創(chuàng)傷性肢體深靜脈血栓形成后，是促進(jìn)還是阻礙血栓消退，還有待于在基因和蛋白水平進(jìn)展功能性研究進(jìn)一步證實(shí)。2. 1391505_*_at、1379497_at、1377365_at三個(gè)未知功能基因與上述基因呈明顯的共表達(dá)趨勢(shì)，推測(cè)其功能與組織修復(fù)、血管形成、炎癥等相關(guān)，在血栓消退中發(fā)揮重要作用，可進(jìn)展深入研究。3.鉗夾股靜脈、髖人字石膏固定雙后肢的方法，能成功建立大鼠急性創(chuàng)傷性肢體深靜脈血栓模型。4大鼠急性創(chuàng)傷性肢體深靜脈血栓形成，是分裂素原活化蛋白激酶、Ca+、細(xì)胞因子-受體相互作用等多種信號(hào)傳導(dǎo)通路參與的過程。5. 大鼠急性創(chuàng)傷性肢體深靜脈血栓模型中，血栓消退組與不消退組差異表

11、達(dá)基因主要與凋亡或腫瘤Itga6, Robo1, Alo*12, Il1b, Ccl3, C*cl2, Ptges, Il1a, Mmp-9、結(jié)合Alo*12, Igfbp3, Oprl, M*2, Mmp12,Il1b, Ccl3, Cinc2, C*cl2, Il1a, Arg1, St*bp5,Il13ra2, Mmp-9、代Alo*12, M*2, Mmp12, Ptges, Arg1, Mmp-9、細(xì)胞循環(huán)Il1b,Il1a、信號(hào)傳導(dǎo)Robo1, Oprl,Il1b, Ccl3, Cinc2, C*cl2, Il1a, Il13ra2、構(gòu)造分子活性Col11a1、運(yùn)載體活性Kj5等功

12、能相關(guān)。6.RT-PCR技術(shù)檢測(cè)IL1、Cinc2表達(dá)，結(jié)果與芯片結(jié)果有較好的一致性，一定程度上驗(yàn)證了Affymetri* 基因芯片數(shù)據(jù)的可靠性。關(guān)鍵詞：急性 創(chuàng)傷性 深靜脈血栓形成 消退 基因To study the differential e*pression genes between thrombi resolution and insolution in the process of acute traumatic deep vein thrombosis by genechipAbstractObjectives:Based on establishing a rat model

13、 of acute traumatic limb deep vein thrombosis. Through the Affymetri* 230A genechip, to study the differential e*pressed genes between the thrombi resolutiongroup and thrombi insolution group after thrombosis. Toe*plorethe partial influence factors ofresolution in traumatic deep vein thrombosis on t

14、he level of gene.Methods: 1.Grouping. 20 SD rats from the total 250 (non-restriction female and male) were divided randomly into the control group; the remained230 were divided into 6 groups: trauma instant group (B, at 0.5h after modeling), thrombosis prophase group (C, at 2.5h after modeling)，thro

15、mbosis crest-time group (D, at 25h after modeling), thrombi resolution group (E, at 72h after modeling), thrombi insolution group (F, at 72h after modeling), non-thrombosis group (G, at 72h after modeling) according to different phases after model being produced and the results of preliminary e*peri

16、ment.2.Modeling. Rats were not anesthetized in model producing process. After inguinal regions sterilized by Iodophors, 1cm long medial inguinal groove incision was adopted to e*pose pro*imal femoral vein, artery and nerve. After blunt dissection, 1.5cm e*posed femoral vein was clamped at three poin

17、ts separately with 12.5mm mosquito-hemostatic forceps, once each point; the clamping strength was fastening one barb of hemostatic forceps, lasting 3 seconds each time, then the incision was sutured, no drainage was set. Rat hibateral posterior limbs were fi*ed with hip spica casts e*cept for group

18、A and B. At different phases after model being produced, color and swelling e*tent of both feet were observed by gross observation.3.Methods for obtaining femoral veins. Rats were anesthetized with 3% pentobarbital sodium (1ml/kg, intraperitoneal injection), supine position fi*ation, sterilizing ant

19、eromedial skin of hibateral posterior limbs through iodophors, e*posing hibateral femoral veins. In group A, 4.5cm femoral vein and related main tributaries were resected. In group B, at 0.5h; group C, at 2.5h; group D, at 25h; group E, F, G, at 72h after model being produced, the same region vascul

20、ar tissue was also resected separately. Part of the 0.5cm vessel separated for HE staining pathological histological analysis, the rest were rinsed by 0.9% physiological saline to clean up blood and thrombi. The vessel specimens were put into nitrogen canister in less than 30 seconds after e* vivo,

21、which would be used for total RNA e*traction.4. E*traction of total RNA. After affirming the status of thrombosis by histological analysis, total mRNAs of femoral vein specimens from the 7 phases were e*tracted separately through TRIzol method. All total RNAsamples were checkedby agarose gel electro

22、phoresis and devided into two portions for being detected through genechip and RT-PCR. 5. RNA detection through genechip and RT-PCR. Through cRNA probes preparation, hybridization, washing, staining and scanning were performed orderly to finish array detecting according to the operation flowsheet.To

23、 validate the accuratissime of genechip through RT-PCR.6. Data screening and analysis. After performing the restrictive conditions: = 1 * GB3 the data of e*perimental group inEvsA and FvsA should not be marked “absent(that is to say, the signal intensity is weak); = 2 * GB3 Signal log2 ratios of the

24、 same gene pared between the resolution group and insolution group must be 1 or -1(representing variability routinely), to search out the differential e*pression genes between the resolution group and insolution group. The screened genes were clustered through software of Gene Cluster 3.0 and drawn

25、visual picture. These genes functions were inquested from web “NCBI, “KI, etc. and aggregate analysis was done. Results: 1. 3 and 2 rats died because of femoral artery rupture and pulmonary embolism respectively. In group A, B, C, no rat presented thrombosis. In the 190 rats of group D,E, F and G, t

26、otal 5 rats died. From 2.5h to 72h after model being built, 149 rats presented thrombosis, 36 were no thrombosis, 64 presented thrombi resolution and 37 presented thrombi insolution.The mortality of rats is 2, incidence rates of thrombosis, non-thrombosis and resolution are 80.54, 19.46% and 63.37 r

27、espectively. 2. The total RNA samples of 7 groups were proved to be high qualities without degradation.occurrence 3. The hybridization signal intensity of arrays was satisfactory. The change tendency of IL-1 and Cinc2 detected by genechip and RT-PCR were in good coincidence.4. In the 15866 rat genes

28、 which can be detected by Affymetri* RAT 230A microarray, 7389 presented differential e*pression, were involved in MAPK, Calcium, Focal adhesion , etc. signaling pathways; 8477 did not presentdifferential e*pression in all phases. 5. After parison between group resolution and group control, 24 genes

29、 upregulated(Log2 Ratio4.0). Among them, 14 genes have no functional description, 10 genes(Top2a、Stk6、Slpi、Olr1、Kdap、Itgam、Il6、Cd8a、Brca1、A2m) with partial GO functional description refer to DNA metabolism, protein metabolism, inflammatory reaction,etc. 9 genes downregulated(Log2 Ratio-4.0). Among t

30、hem, 4 genes have no functional description, 5 genes(Lepr、Gpd3、Atp1b4、Af6、Adh7) with partial GO functional description refer to cell-cell signaling, energy metabolism, etc.6. After parison between group insolution and group control, 26 genes upregulated(Log2 Ratio4.0). Among them, 16 genes have no f

31、unctional description, 10 genes(Slpi、Olr1、Nos2、Mmp9、Kdap、Itgam、Il6、C*cl2、Cinc2、Cd8a) with partial GO functional description refer to DNAinflammatory reaction, cell-cell action etc.; 8 genes downregulated(Log2 Ratio-4.0). Among them, 6 genes have no functional description, 2 genes(Gpd3, Cyp1a1) have

32、partial GO functional description.7. The 118 differential e*pression genes between the resolution and insolution had been searched out. Among them, 48 genes, which with symbols were assigned GO functions by Genebank and were related to the functions of apoptosis, tumor, binding, metabolism, cell cyc

33、ling, signaling, construction molecular activity and transporter. 8. pared the resolution to insolution, the e*pressions of Mmp-9, Mmp-12, Mmp-13, IL-1, C*cl2, Cinc2, Ccl3 , Ptges and Arginase were conspicuous which e*pressed higher in groups of D and F and lower in groups E and G inversely. Conclus

34、ions: 1. The femoral vein e*press Mmp-9, Mmp-12, Mmp-13 related to tissue repair, angiopoiesis, and IL-1, C*cl2, Cinc2, Ccl3 related to inflammation to promote vascular repair and enhance anticoagulation. It e*press Ptges , Arginase related to vasodilatation and inhibition of platelet aggregation to

35、 regulate in mon the thrombi resolution through changing the status of local vessel. Whether the process of thrombi resolution is promoted or prevented by them should be proved through more functional e*periments at gene or/and protein level.2. The three unknown function genes(1391505_*_at、1379497_at、1377365_at) coe*press conspicuously with the above genes. It is presumed that the function of them are related to tissue repair, angiopoiesis, inflammation, vasodilatation and inhibition of platelet aggregation.3. The rat model of acute traumatic deep vein thrombosis can be estab