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 Affymetrix 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部 分關(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

2、，造模后72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)碘伏液消毒后行內(nèi)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)約的股靜脈， 用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)絲線全層間斷縫合皮膚切口，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

3、巴比妥鈉溶液按 1ml/kg 體重，腹腔內(nèi)注射麻醉，仰臥位固 定，碘伏液消毒雙后肢前內(nèi)側(cè)皮膚區(qū)域后， 沿雙側(cè)股靜脈走行切開皮膚約， 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)約的股靜脈及主要屬支，留長(zhǎng)約的血管用甲醛固定，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秒內(nèi)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

4、，一份送以進(jìn)行基因芯片檢測(cè)，另一份備用以行RT-PCR僉測(cè)。5. 芯片及RT-PCF檢測(cè)：按Affymetrix RAT230A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-PCF技術(shù)檢測(cè)保存的RNA羊品中IL-1 B、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Signallog 2 ratio 比擬，差值必須?1或w -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

5、線圖，并行GO功能 分類，查詢“ NCB、“CNK網(wǎng)站及期刊文獻(xiàn)，搜尋相應(yīng)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)內(nèi)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,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)行的分組準(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

6、泳檢測(cè)，28SRNA和18SRNA條 帶整齊，兩者量的比值超過2倍，樣品質(zhì)量好、無降解。3. Affymetrix 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、嘌呤代謝、 白細(xì)胞經(jīng)內(nèi)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 Ratio?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 GC功能描述基因1

7、0個(gè)Top2a Stk6、Slpi、Olr1、Kdap Itgam、116、Cd8a Brcal、A2n，主要涉及DNA蛋白質(zhì)代謝和炎癥反響等生 物學(xué)過程；Log? Ratio w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) 傳遞及能量代謝等生物學(xué)過程。5. 不消退組與對(duì)照組比擬，Log2 Ratio?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GC功能描述基因10個(gè)Slpi、0lr1、Nos2 Mmp、Kdap Itgam、Il6、Cxcl2、Cinc2、Cd8a，主要涉及炎癥反響與細(xì)胞間作

8、用等生物學(xué)過程；Log2 Ratio w的下調(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 log 2 ratio > 1或 W-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)，代謝相關(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)，結(jié)構(gòu)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、Cxcl2、Cinc2、Ccl3、Ptges、Arg1等基因在血栓消

9、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，Affymetrix 基因芯片探針號(hào)為：1391505_x_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 B、Cinc2的RT-PCF檢僉測(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、Cxcl2、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)

10、Arginase、Ptges等血管擴(kuò)張、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)行功能性研究 進(jìn)一步證實(shí)。2. 1391505_x_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)行深入研究。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

11、-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 達(dá)基因主要與凋亡或腫瘤 Itga6, Robol, Alox12, 111b, Ccl3, Cxcl2, Ptges, Illa, Mmp-9、結(jié)合Alox12, Igfbp3, Oprl, Mx2, Mmp12,ll1b, Ccl3, Cinc2, Cxcl2, Il1a, Arg1, Stxbp5,ll13ra2, Mmp-9、代謝Alox12, Mx2, Mmp12,Ptges, Arg1, Mmp-9、細(xì)胞循環(huán)Il1b,Il1a 、信號(hào)傳導(dǎo)Robo1, Oprl,Il1b,

12、 Ccl3, Cinc2, Cxcl2, Il1a, Il13ra2、結(jié)構(gòu)分子活性Col11a1、運(yùn)載體活性Kcnj5 等功能相關(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 Affymetrix 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 expression genes between thrombi resolution and insolution in the process of acute traumatic deep vein thrombosis b

13、y genechipAbstractObjectives: Based on establishing a rat model of acute traumatic limb deep vein thrombosis. Through the Affymetrix 230A gen echip, to study the differe ntial expressed genes betwee n the thrombi resoluti on group and thrombi in soluti on group after thrombosis. To explore the parti

14、al in flue nee factors of resoluti on in traumatic deep vein thrombosis on the 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 remained 230 were divided into 6 groups: trauma in sta nt group (B, at

15、0.5h after modeli ng), thrombosis prophase group (C, at 2.5h after modeling), thrombosis crest-time group (D, at 25h after modeling), thrombi resolution group (E, at 72h after modeling), thrombi insolution group (F, at 72h after modeli ng), non-thrombosis group (G, at 72h after modeli ng) according

16、to different phases after model being produced and the results of prelimi nary experime nt.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 expose proximal femoral vein, artery

17、and n erve. After blu nt dissect ion, exposed femoral vein was clamped at three points separately with mosquito-hemostatic forceps, once each point; the clamp ing stre ngth was faste ning one barb of hemostatic forceps, lasting 3 seconds each time, then the incision was sutured, no drain age was set

18、. Rat hibateral posterior limbs were fixed with hip spica casts except for group A and B. At differe nt phases after model being produced, color and swelli ng exte nt of both feet were observed by gross observati on.3. Methods for obtainingfemoral veins. Rats were anesthetized with 3%pentobarbital s

19、odium (1ml/kg, intraperitoneal injection), supine position fixation, sterilizing anteromedial skin of hibateral posterior limbs through iodophors, exposing hibateral femoral veins. In group A, femoral vein and related main tributaries were resected. In group B, at 0.5h; group C, at 2.5h; group D, at

20、 25h; group E, F, G at 72h after model being produced, the same regi on vascular tissue was also resected separately. Part of the vessel separated for HE stai ning histological an alysis, the rest were rin sed by 0.9% physiological sali ne to clea n up blood and thrombi. The vessel specimens were pu

21、t into nitrogen canister in less than 30 seconds after ex vivo, which would be used for total RNA extract ion.4. Extractio n of total RNA.After affirmi ng the status of thrombosis by histologicalan alysis, total mRNAs of femoral vei n specime ns from the 7 phases were extracted separately through TR

22、Izol method. All total RNA samples were checked by agarose gel electrophoresis and devided into two porti ons for being detected through gen echip and RT-PCR.5. RNA detectio n through gen echip and RT-PCR. Through cRNA probes preparati on, hybridizati on, washi ng, sta ining and sca nning were perfo

23、rmed orderly to finish array detecting according to the operation flowsheet. To validate the accuratissime of gen echip through RT-PCR.(that is6. Data scree ning and an alysis. After perform ing the restrictive con diti ons: thedata of experimental group in EvsA and FvsA should not be marked“ absent

24、 to say, the sig nal inten sity is weak); Sign al log2 ratios of the same gene compared between the resolution group and insolution group must be > 1or <-1(representing variability routinely), to search out the differential expression genes between the resolutio n group and in soluti on group.

25、 The scree ned genes were clustered through software of Gene Cluster 3.0 and draw n visual picture. These genes functions were inquested from web “ NCB ,“ CNKI , etc. and aggregate analysis was done.Results: 1. 3 and 2 rats died because of femoral artery rupture and pulmonary embolism respectively.

26、In group A, B, C, no rat presented thrombosis. In the 190 rats of group D, E, F and G, total 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%,

27、incidenee rates of thrombosis, non-thrombosis and resolution are 80.5%, 19.46% and 63.37% respectively.2. The total RNA samples of 7 groups were proved to be high qualities without degradatio n.3. The hybridizati on sig nal in ten sity of arrays was satisfactory. The eha nge tendency of IL-1 B and C

28、in c0etected by gen echip and RT-PCR were in good coin cide nee.4. In the 15866 rat genes which can be detected by Affymetrix RAT 230A microarray, 7389 presented differential expression, were invoIved in MAPK, Calcium, Focal adhesi on , etc. sig nali ng pathways; 8477 did not prese nt differe ntial

29、expressi on in all phases.5. After comparison between group resolution and group control, 24 genes upregulated(Log2 ). 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, pro

30、tein metabolism, in flammatory reacti on, etc. 9 genes dow nregulated(Log ). Among them, 4 genes have no functional description, 5 genes(Lep、Gpd3、Atp1b4、Af6、Adh7) with partial GO fun cti onal descripti on refer to cell-cell sig nali ng, en ergy metabolism, etc.6. After comparison between group insol

31、ution and group control, 26 genes upregulated(Log2 ). Among them, 16 genes have no functional description, 10 genes(Slp、Olr1、Nos2、Mmp9、Kdap、Itgam、Il6、Cxcl2、Cinc2、Cd8a) with partial GO functional descripti on refer to DNA in flammatory reacti on, cell-cell actio n etc.; 8 genes downregulated(Log2 ).

32、Among them, 6 genes have no functional description, 2 genes(Gpd3, Cyp1a1) have partial GO functional description.7. The 118 differential expression genes between the resolution and insolution had bee n searchedout. Among them, 48 genes, which with symbols were assig nedGO fun cti ons by Gen eba nk a

33、nd were related to the fun cti ons of apoptosis, tumor, binding, metabolism, cell cycli ng, sig nali ng, con struct ion molecular activity and tran sporter.8. Compared the resolution to insolution, the expressions of Mmp-9, Mmp-12, Mmp-13, IL-1, Cxcl2, Cinc2, Ccl3 , Ptges and Arginase were conspicuo

34、us which expressed higher in groups of D and F and lower in groups E and G inv ersely.Conclusions: 1. The femoral vein express Mmp-9, Mmp-12, Mmp-13 related to tissue repair, an giopoiesis, and IL-1, Cxcl2, Cin c2, Ccl3 related to in flammatio n to promote vascular repair and enhance anticoagulation

35、. It express Ptges , Arginase related to vasodilatatio n and in hibiti on of platelet aggregati on to regulate in com mon the thrombi resolution through changing the status of local vessel. Whether the process of thrombi resoluti on is promoted or preve nted by them should be proved through more fun

36、 cti onal experime nts at gene or/and prote in level.2. The three unknown function genes(1391505_x_at 1379497_at、1377365_at) coexpress conspicuously with the above genes. It is presumed that the function of them are related to tissue repair, an giopoiesis, in flammati on, vasodilatati on and in hibiti on of platelet aggregati on.3. The rat model of acute traumatic deep vei n thrombosis can be