急性创伤性深静脉血栓消退与不消退差异表达基因研究.doc

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基础上，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）、血栓形成高峰期组（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，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，稍作钝性分离显露长约1.5cm的股静脉，用12.5mm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4.5cm，观察局部组织反应、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；显微镜下分离并切取双侧各长约4.5cm的股静脉及主要属支，留长约0.5cm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，一份送以进行基因芯片检测，另一份备用以行RT-PCR检测。5. 芯片及RT-PCR检测：按Affymetrix RAT 230A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-PCR技术检测保留的RNA样品中IL-1、Cinc2表达情况，并与芯片数据作比较。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Signal log2 ratio比较，差值必须1或-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），绘制直观曲线图，并行GO功能分类，查询“NCBI”、“CNKI”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150；动物死亡率2，血栓发生率80.54，血栓消退率63.37，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. 消退组与对照组比较，Log2 Ratio4.0的上调表达基因24个，无功能描述基因14个，有部分GO功能描述基因10个（Top2a、Stk6、Slpi、Olr1、Kdap、Itgam、Il6、Cd8a、Brca1、A2m），主要涉及DNA、蛋白质代谢和炎症反应等生物学过程；Log2 Ratio-4.0的下调表达基因9个，无功能描述基因4个，有部分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较，Log2 Ratio4.0的上调表达基因26个，无功能描述基因16个，有部分GO功能描述基因10个（Slpi、Olr1、Nos2、Mmp9、Kdap、Itgam、Il6、Cxcl2、Cinc2、Cd8a），主要涉及炎症反应与细胞间作用等生物学过程；Log2 Ratio-4.0的下调表达基因8个，无功能描述基因6个，有部分GO功能描述基因2个（Gpd3、Cyp1a1）。6. 消退组与不消退组比较，差异表达2倍以上（Signal log2 ratio1或-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，血栓高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、Cinc2的RT-PCR检测结果与芯片结果变化趋势基本一致。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-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, Robo1, Alox12, Il1b, Ccl3, Cxcl2, Ptges, Il1a, Mmp-9）、结合（Alox12, Igfbp3, Oprl, Mx2, Mmp12,Il1b, Ccl3, Cinc2, Cxcl2, Il1a, Arg1, Stxbp5,Il13ra2, Mmp-9）、代谢（Alox12, Mx2, Mmp12, Ptges, Arg1, Mmp-9）、细胞循环（Il1b,Il1a）、信号传导（Robo1, Oprl,Il1b, 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 by genechip AbstractObjectives: Based on establishing a rat model of acute traumatic limb deep vein thrombosis. Through the Affymetrix 230A genechip, to study the differential expressed genes between the thrombi resolution group and thrombi insolution group after thrombosis. To explore the partial influence factors of resolution 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 instant group (B, at 0.5h after modeling), 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 modeling), non-thrombosis group (G, at 72h after modeling) according to different phases after model being produced and the results of preliminary experiment. 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 and nerve. After blunt dissection, 1.5cm exposed femoral vein was clamped at three points 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 fixed with hip spica casts except for group A and B. At different phases after model being produced, color and swelling extent 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 fixation, sterilizing anteromedial skin of hibateral posterior limbs through iodophors, exposing 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 vascular 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 ex vivo, which would be used for total RNA extraction. 4. Extraction of total RNA. After affirming the status of thrombosis by histological analysis, total mRNAs of femoral vein specimens from the 7 phases were extracted separately through TRIzol method. All total RNA samples were checked by agarose gel electrophoresis 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 validate the accuratissime of genechip through RT-PCR. 6. Data screening and analysis. After performing the restrictive conditions: the data of experimental group in EvsA and FvsA should not be marked “absent”(that is to say, the signal intensity is weak); Signal log2 ratios of the same gene compared between the resolution group and insolution group must be 1 or -1(representing variability routinely), to search out the differential expression genes between the resolution group and insolution group. The screened genes were clustered through software of Gene Cluster 3.0 and drawn visual picture. These genes functions were inquested from web “NCBI”, “CNKI”, 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, 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, incidence rates of thrombosis, non-thrombosis and resolution are 80.54, 19.46% and 63.37 respectively. 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 which can be detected by Affymetrix RAT 230A microarray, 7389 presented differential expression, were involved in MAPK, Calcium, Focal adhesion , etc. signaling pathways; 8477 did not present differential expression in all phases. 5. After comparison between group resolution and group control, 24 genes 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 them, 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 comparison between group insolution and group control, 26 genes upregulated(Log2 Ratio4.0). Among them, 16 genes have no functional description, 10 genes(Slpi、Olr1、Nos2、Mmp9、Kdap、Itgam、Il6、Cxcl2、Cinc2、Cd8a) with partial GO functional description refer to DNA inflammatory 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 partial GO functional description.7. The 118 differential expression 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 cycling, signaling, construction molecular activity and transporter. 8. Compared the resolution to insolution, the expressions of Mmp-9, Mmp-12, Mmp-13, IL-1, Cxcl2, Cinc2, Ccl3 , Ptges and Arginase were conspicuous which expressed higher in groups of D and F and lower in groups E and G inversely. Conclusions: 1. The femoral vein express Mmp-9, Mmp-12, Mmp-13 related to tissue repair, angiopoiesis, and IL-1, Cxcl2, Cinc2, Ccl3 related to inflammation to promote vascular repair and enhance anticoagulation. It express Ptges , Arginase related to vasodilatation and inhibition of platelet aggregation to regulate in common 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 experiments at gene or/and protein 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, angiopoiesis, inflammation, vasodilatation and inhibition of platelet aggregation.3. The rat model of acute traumatic deep vein thrombosis can be esta