急性创伤性深静脉血栓消退与不消退差异表达基因研究

1、急性创伤性深静脉血栓消退与不消退差异表达基因研究中文摘要目的： 在建立大鼠急性创伤性肢体深静脉血栓动物模型根底上，应用 Affymetrix 基因芯片技术，从基因水平研究血栓形成后，消退与不消退两种不 同病理过程间股静脉中差异表达基因， 探索影响创伤性肢体深静脉血栓消退的部 分关键因素。方法：1.分组：250只SD大鼠雌雄不限随机取20只作为对照组，余 230只据创伤造模后血栓形成病理变化过程分为：创伤即刻组B组，造模后0.5小时、血栓形成前期组C组，造模后2.5小时、血栓形成顶峰期组D组， 造模后25小时、血栓消退期组E组，造模后72小时、血栓不消退组F组, 造模后72小时和血栓不形成组G组

2、，造模后72小时。2. 造模： 对照组大鼠不造模，创伤组大鼠造模时不行麻醉，造模方法为： 双侧腹股沟区碘伏液消毒后行内侧切口， 长约1cm暴露出股动、静脉及股神经， 稍作钝性别离显露长约的股静脉， 用全齿蚊式血管钳分三段各钳夹血管 1 次力 量为血管钳紧 1 扣，每次持续 3 秒后，用 1 号丝线全层间断缝合皮肤切口，不 放置引流， 除对照组、 创伤即刻组外大鼠均行双后肢髋人字石膏固定； 造模后观 察大鼠双足颜色和肿胀情况。3. 取材方法： 对照组大鼠不造模即取材，创伤即刻组于造模后 0.5 小时， 其余各组大鼠在相应时相点沿原切口暴露股静脉， 观察血栓形成状态， 符合分组 标准后，用 3的戊

3、巴比妥钠溶液按 1ml/kg 体重，腹腔内注射麻醉，仰卧位固 定，碘伏液消毒双后肢前内侧皮肤区域后， 沿双侧股静脉走行切开皮肤约， 观察 局部组织反响、 股静脉血栓是否形成、 严重程度及消退、 不消退情况， 分别记录； 显微镜下别离并切取双侧各长约的股静脉及主要属支，留长约的血管用甲醛固定，送HE染色组织切片镜检；0.9%生理盐水冲洗干净血管中的血液或血栓，离 体30秒内放入冻存管，置入液氮罐中保存，用于总 RNA提取。4. 总RNA提取：TRIzol法分别提取上述7组股静脉标本总RNA各组总RNA样品经琼脂糖凝胶电泳检测合格后28SRNA和18SRNA条带整齐，两者带宽比值 超过2倍分为两份

4、，一份送以进行基因芯片检测，另一份备用以行RT-PCR佥测。5. 芯片及RT-PCF检测：按Affymetrix RAT230A表达谱芯片操作流程，经cDNA探针制备、杂交、洗脱、染色、扫描，完成芯片检测；应用RT-PCF技术检测保存的RNA羊品中IL-1 B、Cinc2表达情况，并与芯片数据作比拟。6. 数据筛选及分析：通过倍数变化分析方法两组间同一基因的Signallog 2 ratio 比拟，差值必须?1或w -1，常规认为具有差异性，选取血栓消退 与不消退组差异表达基因，应用 Gene Cluster 3.0 分析软件聚类能更容易地 选取共表达的基因组，提示可能具有相似功能，绘制直观曲

5、线图，并行GO功能 分类，查询“ NCB、“CNK网站及期刊文献，搜寻相应背景知识资料，结合表 达变化规律综合分析。结果：1. 250只SD大鼠共死亡5只，其中3只因造模过程中股动脉破裂， 出血死亡，造模后25小时内2只死于肺栓塞，其余大鼠均存活；D E、F、G四组190只大鼠死亡5只，25小时有126只血栓形成，D组取材用去25只，余 101只观察至72小时，64只血栓消退，37只血栓不消退；至72小时又有23只 发生血栓，36只一直无血栓形成,血栓不形成率19.46 %; HE染色股静脉组织 切片镜检证实：大体观察血栓形成状态进行的分组准确可靠。2. 各时相点7份标本总RNA经琼脂糖凝胶电

6、泳检测，28SRNA和18SRNA条 带整齐，两者量的比值超过2倍，样品质量好、无降解。3. Affymetrix RAT 230A 芯片所能检测的15866个基因中，7389个基因有 差异表达，占总数的46.57%,全部时相点均无变化的有8477个,占总数的53.43%; 主要涉及促分裂素原活化蛋白激酶、 Ca+黏附斑、刺激神经的配体-受体相互 作用、细胞因子-受体相互作用、核糖体、肌动蛋白细胞骨架、 Wnt、嘌呤代谢、 白细胞经内皮迁移、胰岛素、糖酵解/糖异生等信号通路。4. 消退组与对照组比拟，Log2 Ratio?的上调表达基因24个，无功能描述 基因14个，有局部 GC功能描述基因1

7、0个Top2a Stk6、Slpi、Olr1、Kdap Itgam、116、Cd8a Brcal、A2n，主要涉及DNA蛋白质代谢和炎症反响等生 物学过程；Log? Ratio w的下调表达基因9个，无功能描述基因4个，有局部GO 功能描述基因5个Lepr、Gpd3 Atp1b4、Af6、Adh7，主要涉及细胞间信号 传递及能量代谢等生物学过程。5. 不消退组与对照组比拟，Log2 Ratio?的上调表达基因26个，无功能描 述基因16个，有局部GC功能描述基因10个Slpi、0lr1、Nos2 Mmp、Kdap Itgam、Il6、Cxcl2、Cinc2、Cd8a，主要涉及炎症反响与细胞间作

8、用等生物学过程；Log2 Ratio w的下调表达基因8个，无功能描述基因6个，有局部GO 功能描述基因2个Gpd3 Cyp1a1。6. 消退组与不消退组比拟，差异表达 2倍以上Signal log 2 ratio > 1或 W-1基因共118个，无功能描述基因70个，有功能描述的基因48个，其中凋 亡/肿瘤相关16个，结合相关29个，代谢相关20个，细胞周期相关3个，信号 传导相关11个，结构分子活性相关3个，转录调控活性相关3个，运载体活性 相关4个。7. 股静脉中 Mmp-9 Mmp-12 Mmp-13 IL-1、Cxcl2、Cinc2、Ccl3、Ptges、Arg1等基因在血栓消

9、退组与不消退组中表达变化规律较为突出，Mmp-9差异表达甚至达21倍，血栓顶峰期组及不消退组中均呈现较高表达，而血栓消退组与血栓不形成组中表达相对较低;与它们共表达的三个未知功能基因，Affymetrix 基因芯片探针号为：1391505_x_at、1379497_at、1377365_at。8. 芯片杂交信号强度满意，各时相点IL-1 B、Cinc2的RT-PCF检佥测结果与 芯片结果变化趋势根本一致。结论：1.股静脉可通过表达 Mmp-9 Mmp-12 Mmp-13组织修复、血管形成 相关基因，IL-1、Cxcl2、Cinc2、Ccl3等炎症相关基因，促进损伤血管修复， 发挥抗凝活性，表达

10、Arginase、Ptges等血管扩张、抑制血小板集聚相关基因， 共同改变局部血管状态影响血栓消退，其作用在急性创伤性肢体深静脉血栓形 成后，是促进还是阻碍血栓消退，还有待于在基因和蛋白水平进行功能性研究 进一步证实。2. 1391505_x_at、1379497_at、1377365_at三个未知功能基因与上述基因 呈明显的共表达趋势，推测其功能与组织修复、血管形成、炎症等相关，在血 栓消退中发挥重要作用，可进行深入研究。3. 钳夹股静脉、髋人字石膏固定双后肢的方法，能成功建立大鼠急性创 伤性肢体深静脉血栓模型。4. 大鼠急性创伤性肢体深静脉血栓形成，是分裂素原活化蛋白激酶、Ca+ 细胞因子

11、-受体相互作用等多种信号传导通路参与的过程。5. 大鼠急性创伤性肢体深静脉血栓模型中，血栓消退组与不消退组差异表 达基因主要与凋亡或肿瘤 Itga6, Robol, Alox12, 111b, Ccl3, Cxcl2, Ptges, Illa, Mmp-9、结合Alox12, Igfbp3, Oprl, Mx2, Mmp12,ll1b, Ccl3, Cinc2, Cxcl2, Il1a, Arg1, Stxbp5,ll13ra2, Mmp-9、代谢Alox12, Mx2, Mmp12,Ptges, Arg1, Mmp-9、细胞循环Il1b,Il1a 、信号传导Robo1, Oprl,Il1b,

12、 Ccl3, Cinc2, Cxcl2, Il1a, Il13ra2、结构分子活性Col11a1、运载体活性Kcnj5 等功能相关。6. RT-PCR技术检测IL1、Cinc2表达，结果与芯片结果有较好的一致性， 一定程度上验证了 Affymetrix 基因芯片数据的可靠性。关键词：急性创伤性深静脉血栓形成 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