创伤性股静脉血栓基因表达变化及低分子肝素干预研究
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摘要
目的
通过建立更接近骨科临床实际的创伤性肢体深静脉血栓形成的大鼠模型，动态检测血栓形成各时相点中股静脉血管的基因表达变化，重点关注血栓自然消退与不消退状态的差异表达基因。同时，再采用低分子肝素对该模型进行防治研究，以期找出与血栓预后密切相关的基因和药物作用靶点，为进一步研究深静脉血栓形成的基因网络变化及药物开发利用奠定基础。
材料和方法
(一)创伤性肢体深静脉血栓形成模型的建立及股静脉血管基因表达研究
1、将150只SD大鼠随机分为正常对照组(A组，n=10)和模型组(n=140)。模型组采用大鼠双侧后肢定量击打+髋人字石膏外固定的方法建立创伤性深静脉血栓形成模型，再根据造模后的观察时相点和／或血栓形成状态再分为7组：创伤即刻(B组，造模后0.5h)、血栓形成初始期(C组，造模后72h)、高峰期血栓形成(D组，造模后120h)、高峰期血栓不形成(H组，造模后120h)、血栓消退(E组，造模后168h)、血栓不消退(F组，造模后168h)、血栓不形成(G组，造模后168h)。2、在相应的时相点，先通过肉眼观察初步确定血栓的状态，每组纳入10只大鼠，分别切取双侧长约4～5cm的股静脉，每条股静脉再切取长约0.5cm的组织以进行HE染色、光镜观察确定血栓形成的状态；剩余的血管组织置于液氮罐备用。3、选择符合分组条件的各组股静脉血管同组混合，采用Trizol一步法提取总RNA，获取的8组RNA样品各取1μl用分光光度仪测定其含量，再各取1μl用2％琼脂糖凝胶电泳检测其质量。4、质检合格的8组样品送往上海国家生物工程中心，采用Lab-on质检系统再次确认送检各组RNA质量，质检合格的各组RNA样品进行生物素标记，采用Genechip Rat Genome 230 2.0芯片进行杂交、扫描和芯片数据处理。5、采用倍数变化分析筛选出各时相点与A组比较的差异表达基因(上调基因：Log_2Ratio≥1，且Change标注为Ⅰ；下调基因：Log_2 Ratio≤-1，且Change标注为D)并进行GO分类。
(二)血栓消退组与血栓不消退组差异表达基因分析
以(一)部分中血栓消退组(E组)和血栓不消退(F组)的数据为基础，进行两组间比较，结果以Log_2 Ratio≥1或≤-1为标准，筛查出在两组中表达相悖的差异基因，先将这些基因作GO分类，再采用Gene Cluster 3.0聚类分析软件进行进一步的分析。
(三)低分子肝素防治创伤性深静脉血栓形成药物作用靶点的实验研究
1、150只SD大鼠同法造模，其中50只大鼠用于创伤性深静脉血栓形成的预防；剩余的100只大鼠用于创伤性深静脉血栓形成的治疗。2、用于预防的50只大鼠造模后随机分为药物预防组(n=40)和对照组(Y_0组；n=10)。预防组造模后6h首次给药，药物采用低分子肝素，按500IU／kg体重、腹腔内注射给药，一日一次，再根据取材时间随机分为Y_1组(n=10；首次给药后3h取材)和Y_2组(最后一次给药后3h取材，观察血栓形成情况并与D组血栓发生率相比较)；对照组(Y_0组)在造模后6h首次腹腔内注射相同体积的生理盐水，3h后取材。3、用于治疗的100只SD大鼠同法造模并观察至造模后120h，将有血栓形成的大鼠(n≌50)作为低分子肝素作用机制研究的实验对象，并随机分为治疗组(n=40)和对照组(Z_0组；n=10)。治疗组采用低分子肝素，按600IU／kg体重、腹腔内注射给药，一日一次。再根据取材时间将大鼠分为：Z_1组(首次给药后3h取材)和Z_2组(最后一次给药后3h取材，观察血栓形成情况并与E组血栓消退率相比较)；Z_0组在首次注射生理盐水后3h取材。4、Y_0组、Y_1组、Z_0组和Z_1组每组纳入相应的大鼠10只，分别切取双侧股静脉后进行RNA抽提和芯片检测，方法同(一)相应部分。5、在倍数变化分析的基础上，通过pathway数据库信息对Y_1／Y_0组、Z_1／Z_0组的差异表达基因进行分析。6、将E／F组差异表达基因与Y_1／Y_0组、Z_1／Z_0组的差异表达基因进行交集找出共表达基因。
结果
(一)创伤性肢体深静脉血栓形成模型的建立及股静脉血管基因表达研究结果
1、该模型中，股静脉血栓发生于造模后72h，血栓形成高峰期(D组)在造模后120h，其血栓发生率为50.5％，之后血栓开始自发消退，至造模后168h，血栓消退率(E组)为56.7％，相应血栓不消退率(F组)为43.3％，不消退血栓持续至造模后240h以上。2、各组所获取的股静脉组织，肉眼、光镜观察结果与相应血栓状态基本一致。3、提取的8组股静脉组织总RNA样品无污染、无降解、28s：18s约为2：1，质量均达到芯片检测的要求。4、Genechip Rat Genome 230 2.0表达谱芯片所检测的大鼠31042个基因中，B组与A组比较，349个基因出现差异表达，其中214个上调，135个下调；C组与A组比较，2393个基因出现差异表达，其中1386个上调，1007个下调；D组与A组比较，1743个基因出现差异表达，其中945个上调，798个下调；H组与A组比较，2790个基因出现差异表达，其中1685个上调，1105个下调；E组与A组比较，1913个基因出现差异表达，其中1222个上调，691个下调；F组与A组比较，2564个基因出现差异表达，其中1535个上调，1029个下调；G组与A组比较，1849个基因出现差异表达，其中1235个上调，614个下调；D组与H组比较，805个基因出现差异表达，其中51个上调，755个下调。这些差异表达基因的功能主要涉及细胞凋亡、分子黏附、代谢、细胞周期、信号转导等。
(二)血栓消退组与血栓不消退组差异表达基因分析结果
1、E／F组比较共有229个基因呈现差异性表达，其中上调基因111个，下调基因118个。在已知功能的差异表达基因中主要涉及细胞凋亡、蛋白结合、物质代谢(转运)方面的基因。2、Cluster聚类分析将之分为三簇：第一簇基因包括Mybph、Myf6、Sln、Cox6a2、Alox15等45个基因，它们的变化在B组、C组、D组和F组表达水平较低，而在H组、E组和G组表达水平较高；第二簇基因包括pcyt1b、tfdb2、bpgm、Ca2等76个基因，它们的变化主要在E组表达水平较高，而大部分基因在其余各个时相点均呈现低水平表达；第三簇基因包括mmp12、tnfaip6、lampl、pfkl等108个基因，这簇基因变化的显著特征为在F组呈现显著的差异性表达，小部分基因在B组和H组也出现高表达。
(三)低分子肝素防治创伤性深静脉血栓形成药物作用靶点的实验研究结果
1、造模后第120h，D组血栓形成率为50.5％；运用低分子肝素钠进行预防后Y_2的血栓发生率为16.7％，两者比较差别具有显著的统计学意义(p=0.008＜0.01)。2、造模后第168h，E组血栓消退率为56.7％，运用低分子肝素钠治疗后Z_2组的血栓消退率为91.7％，两者比较差别具有显著的统计学意义(p=0.004＜0.01)。3、Y_1／Y_0组比较共有1193个基因呈现差异性表达，其中上调基因471个，下调基因722个。4、Z_1／Z_0组比较共有1229个基因呈现差异性表达，其中上调基因907个，下调基因322个。5、低分子肝素干预后的差异表达基因涉及多个pathway通路，主要包括MAPK信号通路、粘附斑通路、Ca~(2+)通路、细胞因子—细胞因子受体通路和凋亡通路。5、E／F组差异表达基因与Y_1／Y_0组、Z_1／Z_0组的差异表达基因进行交集后得到15个共表达基因。
结论
1、采用创伤+固定制动的方式可建立更符合临床实际的创伤性深静脉血栓形成动物模型，该模型为TDVT发病机制和防治方面的研究提供了一种较为可靠的模型。2、TDVT是涉及多基因变化的一种疾病，并以上调表达的差异基因发挥主要作用，功能涉及细胞凋亡、分子黏附、代谢、细胞周期、信号转导等方面。3、包括Mybph、Myf6、Sln、Cox6a2、Alox15在内的45个基因差异性表达与创伤后血栓的不形成、血栓的自发消退密切相关；包括pcyt1b、tfdb2、bpgm、Ca2在内的76个基因差异性表达与血栓形成的预后相关，其高表达水平可促进血栓形成后自发消退的过程。包括mmp12、mfaip6、lamp1、pfk1在内的108个基因差异性表达在维持血栓的持续不消退方面起重要作用，为相关领域的进一步研究提供了方向。4、在LWMH防治TDVT的机制中，除凝血酶和因子Fxa途径外，LWMH还通过调节血管内皮细胞的凝血、抗凝及纤溶相关基因的表达发挥其药物作用，其中如Thbd、Plaur等相关基因可能是其作用靶点。5、低分子肝素干预TDVT的分子机制主要涉及MAPK通路、粘附斑信号通路、细胞因子—细胞因子受体通路、钙离子通道信号通路和凋亡信号通路，主要通过调节细胞增殖、分化、炎症反应等影响血栓的形成和预后。6、在自然消退与否以及药物干预差异表达基因交集后所得到的Actn3、RGD：620059及Mbp等15个基因，在血栓消退中发挥重要作用，可作为LWMH防治TDVT的靶点基因进一步研究。
Objective Based on establishing a traumatic limb deep vein thrombosis (DVT) rat model, to dynamically detect femoral vein gene expression changes and screen differential expression genes at different phases in this pathological process. To focus on the differential expression genes between thrombus resolution and insolution groups. Meanwhile, to perform Low molecular weight heparin (LMWH) intervention in this model in order to select genes which could have a close relationship with DVT prognosis and could serve as drug action targets. This will be a basis of further studies about gene network in this pathological process and novel drug development for DVT.
Methods:
1. Establishing a traumatic limb DVT rat model and studying femoral vein gene expression.
(1) 150 SD rats were randomly divided into control (group A, 10 rats) and model rats (140 rats). In the model rats, beating on bilateral posterior limbs without fixation was performed only in post-traumatic group (group B, 10 rats); beating on bilateral posterior limbs combined with hip spica cast fixation was performed in the others. According to different observation phases and/or pathological states in the process of TDVT, the model rats was subdivided into 7 groups: the control, post-traumatic instant (group B, post-traumatic 0.5h), initial period of thrombosis (group C, post-traumatic 72h), thrombogenesis at thrombotic crest-time (group D, post-traumatic 120h), nonthrombogenesis at the thrombotic crest-time (group H, post-traumatic 120h), thrombus resolution (group E, post-traumatic 168h), thrombus insolution (group F, post-traumatic 168h) and nonthrombosis at post-traumatic 168h (group G). (2) At the corresponding phases, according to gross observation, each 10 rats meeting to different pathological features were seleted into corresponding groups. Bilateral femoral veins (around 4～5cm long ) were cut respectively, in whch, 0.5 cm was cut for HE staining and histological observation in order to ensure the reliability of grouping and the rest was stored in nitrogen canister. (3) Femoral veins were mixed in the same groups. And Trizol one-step method was applied for total RNA extraction. And 1μl RNA sample of each group was taken for RNA content determination by spectrophotometer; other 1μ1 was for RNA quality determination by 2% agarose gel electrophoresis (AGE). (4) Valid RNA samples were sent to Shanghai Biochip CO. Ltd and the quality of RNA samples were detected again by Lab-on quality determination system. Then RNA samples were labeled with biotin and for hybridization. Then according to the manipulation process of Genechip Rat Genome 230 2.0, genechips were detected through cDNA probe preparation, hybridization, staining, scanning in order. (5)Combined with Fold Change (FC) analysis, to screen differental expression genes of each group compared with the control (up-regulation: Log_2 Ratio≥1 and Change was marked as I; down-regulation: Log_2 Ratio≤-1 and Change was marked as D). And the differential expression genes were classified according to GO classification.
2. Differential expression genes analysis between thrombus solution and insolution groups.
Based on the data from E and F groups gained from Part 1, to perform group comparison between them, and genes with an opposite expression trend in group E compared with group F (screening criterion: Log2 Ratio≥1 or≤-1) were seleted. Then Gene Cluster 3.0 software was applied in these genes for further analysis.
3. An experimetal study of gene targets in LMWH preventing and curing TDVT.
(1) Another 150 SD rats were modeled through the modeling method mentioned above. And in which, 50 rats were used for the study of TDVT prophylaxis and the rest 100 were for the drug therapy of TDVT. (2) The 50 rats for TDVT prophylaxis studying were modeled and divided into drug prophylaxis group (n=40) and control group (Y_0 group, n=10). In the drug prophylaxis group, at post-traumatic 6h, LMWH intraperitoneal injection was performed initially (500IU/kg, once a day). According to different time of sampling, drug prophylaxis group was subdivided into group Y_1 (n=10, sampling at 3h after initial LMWH injection) and Y_2 group (sampling at 3h after the last LMWH injection, to observe the states of thrombogenesis and to compare the thrombotic rates between Y_2 and D groups). In the control (group Y_0) rats, the same volume physiological saline was injected, and bilateral femoral veins were cut at post-injection 3h. (3) 100 SD rats used for TDVT treatment were modeled as well as observed until post-traumatic 120h. In which, the rats with thrombogenesis (n≌50) was selected for drug medication studing and subdivided into drug therapy group (n=40) and control group (group Z_0, n= 10). In drug therapy group, LMWH intraperitoneal injection was performed (600IU/kg, once a day). According to different time of sampling, drug prophylaxis group rats were subdivided into group Z_1 (sampling at 3h after initial LMWH injection) and Z_2 group (sampling at 171h after successive medication, to observe the states of thrombogenesis and to compare the thrombogenesis rates between Y_2 and E groups). Group Z_0 rats were injected the same volume physiological saline and the bileteral femoral veins were cut at post-injection 3h. (4) Each 10 rats were selected for Y_0, Y_1, Z_0 and Z_1 groups. Their bilateral femoral veins were cut for RNA extraction and genechip detection. (5) Based on FC analysis results, KEGG Pathway Database was applied to analyze the differential expression genes in Y_1/ Y_0 and Z_1/Z_0 conparisons. (6) Through performing the intersection set among the differential expression genes in E/F, Y_1/Y_0, Z_1/Z_0 conparisons, to further select coexpression genes in them.
Results
1. Results of TDVT rat model establishing and femoral vein gene expression analysis.
(1) In this model, femoral vein thrombogenesis started at post-traumatic 72h; the thrombogenesis crest-time was at the post-traumatic 120h and the rate of thrombogenesis was 50.5%. After that, thrombi began to resolve, and to the post-traumatic 168h, the rate of thrombi solution was 56.7%; the rate of thrombusi insolution was 43.3%. And the state of thrombus insolution would be persistence until more than post-traumatic 240h. (2) The macroscopic and histological observations results of femoral vein thrombosis were conincidence with the corresponding thrombi states. (3)The femoral vein total RNA samples from the 8 groups were no pollution or degradation and their qualities met to the conditions of genechip detection. (4) In the 31042 genes which can be detected by Genechip Rat Genome 230 2.0, group B compared with the control (B/A), 349 genes presented differential expression, in which, 214 were up-regulated and 135 was down-regulated; group C compared with the control (C/A), 2393 genes presented differential expression, in which, 1386 genes were up-regulated and 1007 was down-regulated; group D compared with the control (D/A), 1743 genes presented differential expression, in which, 945 genes were up-regulated and 798 were down-regulated; group H compared with the control (H/A), 2790 genes presented differential expression, in which, 1685 genes were up-regulated and 1105 were down-regulated; group E compared with the control (E/A), 1913 genes presented differeential expression, in which, 1222 genes were up-regulated and 691 were down-regulated; group F compared with the control (F/A), 2564 genes presented differential expression, in which, 1535 genes were up-regulated and 1029 were down-regulated; group G compared with the control (G/A), 1849 genes presented differential expression, in which, 1235 genes were up-regulated and 614 were down-regulated; group D compared with group H (D/A), 805 genes presented differential expression, in which, 51 genes were up-regulated and 755 were down-regulated. The function of these genes involved cell apoptosis, binding, metablism, cell cycle, transcription regulator activity, etc..
2. Results of differential expression gene analysis between thrombus solution and insolution groups.
(1) In E/F comparison, 229 genes presented differential expression, in which, 111 were up-regulated and 118 were down-regulated. And the differential expression genes with known functions mainly involved cell apoptosis, binding, metablism, etc.. (2) Through Cluster analysis, the genes were divided into 3 clusters: the first cluster included 45 genes such as Mybph, Myf6, Sln, Cox6a2, Alox15, etc., and their expression levels were lower in B, C, D, F groups, but being higher in H, E, G groups; the second cluster included 76 genes such as pcytlb, tfdb2, bpgm, Ca2, etc., and their expression levels were higher only in group E, but being lower in the other phases; the third cluster genes included 108 genes such as mmpl2, tnfaip6, lamp1, pfk1, etc., and the significant expression feature of them was that they presented significantly differential expression in group F and a part of them also presnted up-regualtion in B, H groups.
3. Results of the experimetal study of target genes in LMWH preventing and curing TDVT.
(1) Post-traumatic 120h, the rate of thrombogenesis was 50.5%; after LMWH prophylaxis, the rate decreased to 16.7%, and there was a significantly statistical difference between them (p=0.008<0.01). (2) At post-traumatic 168h, the rate of thrombi solution was 56.7%; after LMWH therapy, the rate increased to 91.7%, and there was a significantly statistical difference between them (p=0.004<0.01). (3) In Y_1 /Y_0 comparison, 1193 genes presented differential expression, in which, 471 were up-regulated and 722 were down-regulated. (4) In Z_1/D comparison, 1229 genes presented differential expression, in which, 907 were up-regulation and 322 were down-regulation. (5) After LMWH intervention, the diferential expression genes involved several pathways including MAPK, focal adhesion, calcium, cytokine-cytokine receptor interaction signaling pathway, apoptosis, etc.. (6) The intersection set among the differential expression genes in E/F, Y_1/Y_0, Z_1/Z_0 conparisons included 15 genes.
Conclusion:
1. Applying trauma combined with immobilization can establish a TDVT animal model, which is more close to clinical pathological features of TDVT and can serve as a reliable model for studies of the patho genesis, prophylaxis and cure of TDVT.
2. TDVT is a disease related to multiple genes which are mainly involved in cell apoptosis, binding, metablism, cell cycle, signaling transduction, etc...
3. The 45 genes including Mybph, Myf6, Sln, Cox6a2, Alox15, etc are related to nonthrombogenesis and thrombus resolution; the 76 genes including pcyt1b, tfdb2, bpgm, Ca2, etc. are related to thrombogenic prognosis and higher expression levels of them can improve the progress of thrombus resolution; the 108 genes including mmp12, tnfaip6, lamp1, pfk1, etc. play a role in thrombotic persistent insolution states.
4. In the mechanism of LWMH preventing and curing TDVT, besides thrombin and Fxa, LWMH also regulates genes related to blood coagulation, anticoagulation and fibrolysis in vascular endothelial cell so as to perforn drug action. In which, Thbd, Plaur, etc. could be the drug action targets.
5. The molecular mechanism of LMWH intervention in TDVT mainly involves MAPK, focal adhesion, cytokine-cytokine, calcium receptor interaction, apoptosis, etc. signaling pathways. And thrombosis as well as its prognosis is affected by cell proliferation, differentiation, inflammatory reaction, etc..
6. The 15 genes including Actn3, RGD:620059, Mbp, etc., which gained from the intersection set among the differential expression genes in thrombus resolution, thrombus insolution and drug interveion groups, play important roles in the process of thrombus resolution. They will be the drug action targets in LMWH preventing and curing TDVT.
通过建立更接近骨科临床实际的创伤性肢体深静脉血栓形成的大鼠模型，动态检测血栓形成各时相点中股静脉血管的基因表达变化，重点关注血栓自然消退与不消退状态的差异表达基因。同时，再采用低分子肝素对该模型进行防治研究，以期找出与血栓预后密切相关的基因和药物作用靶点，为进一步研究深静脉血栓形成的基因网络变化及药物开发利用奠定基础。
材料和方法
(一)创伤性肢体深静脉血栓形成模型的建立及股静脉血管基因表达研究
1、将150只SD大鼠随机分为正常对照组(A组，n=10)和模型组(n=140)。模型组采用大鼠双侧后肢定量击打+髋人字石膏外固定的方法建立创伤性深静脉血栓形成模型，再根据造模后的观察时相点和／或血栓形成状态再分为7组：创伤即刻(B组，造模后0.5h)、血栓形成初始期(C组，造模后72h)、高峰期血栓形成(D组，造模后120h)、高峰期血栓不形成(H组，造模后120h)、血栓消退(E组，造模后168h)、血栓不消退(F组，造模后168h)、血栓不形成(G组，造模后168h)。2、在相应的时相点，先通过肉眼观察初步确定血栓的状态，每组纳入10只大鼠，分别切取双侧长约4～5cm的股静脉，每条股静脉再切取长约0.5cm的组织以进行HE染色、光镜观察确定血栓形成的状态；剩余的血管组织置于液氮罐备用。3、选择符合分组条件的各组股静脉血管同组混合，采用Trizol一步法提取总RNA，获取的8组RNA样品各取1μl用分光光度仪测定其含量，再各取1μl用2％琼脂糖凝胶电泳检测其质量。4、质检合格的8组样品送往上海国家生物工程中心，采用Lab-on质检系统再次确认送检各组RNA质量，质检合格的各组RNA样品进行生物素标记，采用Genechip Rat Genome 230 2.0芯片进行杂交、扫描和芯片数据处理。5、采用倍数变化分析筛选出各时相点与A组比较的差异表达基因(上调基因：Log_2Ratio≥1，且Change标注为Ⅰ；下调基因：Log_2 Ratio≤-1，且Change标注为D)并进行GO分类。
(二)血栓消退组与血栓不消退组差异表达基因分析
以(一)部分中血栓消退组(E组)和血栓不消退(F组)的数据为基础，进行两组间比较，结果以Log_2 Ratio≥1或≤-1为标准，筛查出在两组中表达相悖的差异基因，先将这些基因作GO分类，再采用Gene Cluster 3.0聚类分析软件进行进一步的分析。
(三)低分子肝素防治创伤性深静脉血栓形成药物作用靶点的实验研究
1、150只SD大鼠同法造模，其中50只大鼠用于创伤性深静脉血栓形成的预防；剩余的100只大鼠用于创伤性深静脉血栓形成的治疗。2、用于预防的50只大鼠造模后随机分为药物预防组(n=40)和对照组(Y_0组；n=10)。预防组造模后6h首次给药，药物采用低分子肝素，按500IU／kg体重、腹腔内注射给药，一日一次，再根据取材时间随机分为Y_1组(n=10；首次给药后3h取材)和Y_2组(最后一次给药后3h取材，观察血栓形成情况并与D组血栓发生率相比较)；对照组(Y_0组)在造模后6h首次腹腔内注射相同体积的生理盐水，3h后取材。3、用于治疗的100只SD大鼠同法造模并观察至造模后120h，将有血栓形成的大鼠(n≌50)作为低分子肝素作用机制研究的实验对象，并随机分为治疗组(n=40)和对照组(Z_0组；n=10)。治疗组采用低分子肝素，按600IU／kg体重、腹腔内注射给药，一日一次。再根据取材时间将大鼠分为：Z_1组(首次给药后3h取材)和Z_2组(最后一次给药后3h取材，观察血栓形成情况并与E组血栓消退率相比较)；Z_0组在首次注射生理盐水后3h取材。4、Y_0组、Y_1组、Z_0组和Z_1组每组纳入相应的大鼠10只，分别切取双侧股静脉后进行RNA抽提和芯片检测，方法同(一)相应部分。5、在倍数变化分析的基础上，通过pathway数据库信息对Y_1／Y_0组、Z_1／Z_0组的差异表达基因进行分析。6、将E／F组差异表达基因与Y_1／Y_0组、Z_1／Z_0组的差异表达基因进行交集找出共表达基因。
结果
(一)创伤性肢体深静脉血栓形成模型的建立及股静脉血管基因表达研究结果
1、该模型中，股静脉血栓发生于造模后72h，血栓形成高峰期(D组)在造模后120h，其血栓发生率为50.5％，之后血栓开始自发消退，至造模后168h，血栓消退率(E组)为56.7％，相应血栓不消退率(F组)为43.3％，不消退血栓持续至造模后240h以上。2、各组所获取的股静脉组织，肉眼、光镜观察结果与相应血栓状态基本一致。3、提取的8组股静脉组织总RNA样品无污染、无降解、28s：18s约为2：1，质量均达到芯片检测的要求。4、Genechip Rat Genome 230 2.0表达谱芯片所检测的大鼠31042个基因中，B组与A组比较，349个基因出现差异表达，其中214个上调，135个下调；C组与A组比较，2393个基因出现差异表达，其中1386个上调，1007个下调；D组与A组比较，1743个基因出现差异表达，其中945个上调，798个下调；H组与A组比较，2790个基因出现差异表达，其中1685个上调，1105个下调；E组与A组比较，1913个基因出现差异表达，其中1222个上调，691个下调；F组与A组比较，2564个基因出现差异表达，其中1535个上调，1029个下调；G组与A组比较，1849个基因出现差异表达，其中1235个上调，614个下调；D组与H组比较，805个基因出现差异表达，其中51个上调，755个下调。这些差异表达基因的功能主要涉及细胞凋亡、分子黏附、代谢、细胞周期、信号转导等。
(二)血栓消退组与血栓不消退组差异表达基因分析结果
1、E／F组比较共有229个基因呈现差异性表达，其中上调基因111个，下调基因118个。在已知功能的差异表达基因中主要涉及细胞凋亡、蛋白结合、物质代谢(转运)方面的基因。2、Cluster聚类分析将之分为三簇：第一簇基因包括Mybph、Myf6、Sln、Cox6a2、Alox15等45个基因，它们的变化在B组、C组、D组和F组表达水平较低，而在H组、E组和G组表达水平较高；第二簇基因包括pcyt1b、tfdb2、bpgm、Ca2等76个基因，它们的变化主要在E组表达水平较高，而大部分基因在其余各个时相点均呈现低水平表达；第三簇基因包括mmp12、tnfaip6、lampl、pfkl等108个基因，这簇基因变化的显著特征为在F组呈现显著的差异性表达，小部分基因在B组和H组也出现高表达。
(三)低分子肝素防治创伤性深静脉血栓形成药物作用靶点的实验研究结果
1、造模后第120h，D组血栓形成率为50.5％；运用低分子肝素钠进行预防后Y_2的血栓发生率为16.7％，两者比较差别具有显著的统计学意义(p=0.008＜0.01)。2、造模后第168h，E组血栓消退率为56.7％，运用低分子肝素钠治疗后Z_2组的血栓消退率为91.7％，两者比较差别具有显著的统计学意义(p=0.004＜0.01)。3、Y_1／Y_0组比较共有1193个基因呈现差异性表达，其中上调基因471个，下调基因722个。4、Z_1／Z_0组比较共有1229个基因呈现差异性表达，其中上调基因907个，下调基因322个。5、低分子肝素干预后的差异表达基因涉及多个pathway通路，主要包括MAPK信号通路、粘附斑通路、Ca~(2+)通路、细胞因子—细胞因子受体通路和凋亡通路。5、E／F组差异表达基因与Y_1／Y_0组、Z_1／Z_0组的差异表达基因进行交集后得到15个共表达基因。
结论
1、采用创伤+固定制动的方式可建立更符合临床实际的创伤性深静脉血栓形成动物模型，该模型为TDVT发病机制和防治方面的研究提供了一种较为可靠的模型。2、TDVT是涉及多基因变化的一种疾病，并以上调表达的差异基因发挥主要作用，功能涉及细胞凋亡、分子黏附、代谢、细胞周期、信号转导等方面。3、包括Mybph、Myf6、Sln、Cox6a2、Alox15在内的45个基因差异性表达与创伤后血栓的不形成、血栓的自发消退密切相关；包括pcyt1b、tfdb2、bpgm、Ca2在内的76个基因差异性表达与血栓形成的预后相关，其高表达水平可促进血栓形成后自发消退的过程。包括mmp12、mfaip6、lamp1、pfk1在内的108个基因差异性表达在维持血栓的持续不消退方面起重要作用，为相关领域的进一步研究提供了方向。4、在LWMH防治TDVT的机制中，除凝血酶和因子Fxa途径外，LWMH还通过调节血管内皮细胞的凝血、抗凝及纤溶相关基因的表达发挥其药物作用，其中如Thbd、Plaur等相关基因可能是其作用靶点。5、低分子肝素干预TDVT的分子机制主要涉及MAPK通路、粘附斑信号通路、细胞因子—细胞因子受体通路、钙离子通道信号通路和凋亡信号通路，主要通过调节细胞增殖、分化、炎症反应等影响血栓的形成和预后。6、在自然消退与否以及药物干预差异表达基因交集后所得到的Actn3、RGD：620059及Mbp等15个基因，在血栓消退中发挥重要作用，可作为LWMH防治TDVT的靶点基因进一步研究。
Objective Based on establishing a traumatic limb deep vein thrombosis (DVT) rat model, to dynamically detect femoral vein gene expression changes and screen differential expression genes at different phases in this pathological process. To focus on the differential expression genes between thrombus resolution and insolution groups. Meanwhile, to perform Low molecular weight heparin (LMWH) intervention in this model in order to select genes which could have a close relationship with DVT prognosis and could serve as drug action targets. This will be a basis of further studies about gene network in this pathological process and novel drug development for DVT.
Methods:
1. Establishing a traumatic limb DVT rat model and studying femoral vein gene expression.
(1) 150 SD rats were randomly divided into control (group A, 10 rats) and model rats (140 rats). In the model rats, beating on bilateral posterior limbs without fixation was performed only in post-traumatic group (group B, 10 rats); beating on bilateral posterior limbs combined with hip spica cast fixation was performed in the others. According to different observation phases and/or pathological states in the process of TDVT, the model rats was subdivided into 7 groups: the control, post-traumatic instant (group B, post-traumatic 0.5h), initial period of thrombosis (group C, post-traumatic 72h), thrombogenesis at thrombotic crest-time (group D, post-traumatic 120h), nonthrombogenesis at the thrombotic crest-time (group H, post-traumatic 120h), thrombus resolution (group E, post-traumatic 168h), thrombus insolution (group F, post-traumatic 168h) and nonthrombosis at post-traumatic 168h (group G). (2) At the corresponding phases, according to gross observation, each 10 rats meeting to different pathological features were seleted into corresponding groups. Bilateral femoral veins (around 4～5cm long ) were cut respectively, in whch, 0.5 cm was cut for HE staining and histological observation in order to ensure the reliability of grouping and the rest was stored in nitrogen canister. (3) Femoral veins were mixed in the same groups. And Trizol one-step method was applied for total RNA extraction. And 1μl RNA sample of each group was taken for RNA content determination by spectrophotometer; other 1μ1 was for RNA quality determination by 2% agarose gel electrophoresis (AGE). (4) Valid RNA samples were sent to Shanghai Biochip CO. Ltd and the quality of RNA samples were detected again by Lab-on quality determination system. Then RNA samples were labeled with biotin and for hybridization. Then according to the manipulation process of Genechip Rat Genome 230 2.0, genechips were detected through cDNA probe preparation, hybridization, staining, scanning in order. (5)Combined with Fold Change (FC) analysis, to screen differental expression genes of each group compared with the control (up-regulation: Log_2 Ratio≥1 and Change was marked as I; down-regulation: Log_2 Ratio≤-1 and Change was marked as D). And the differential expression genes were classified according to GO classification.
2. Differential expression genes analysis between thrombus solution and insolution groups.
Based on the data from E and F groups gained from Part 1, to perform group comparison between them, and genes with an opposite expression trend in group E compared with group F (screening criterion: Log2 Ratio≥1 or≤-1) were seleted. Then Gene Cluster 3.0 software was applied in these genes for further analysis.
3. An experimetal study of gene targets in LMWH preventing and curing TDVT.
(1) Another 150 SD rats were modeled through the modeling method mentioned above. And in which, 50 rats were used for the study of TDVT prophylaxis and the rest 100 were for the drug therapy of TDVT. (2) The 50 rats for TDVT prophylaxis studying were modeled and divided into drug prophylaxis group (n=40) and control group (Y_0 group, n=10). In the drug prophylaxis group, at post-traumatic 6h, LMWH intraperitoneal injection was performed initially (500IU/kg, once a day). According to different time of sampling, drug prophylaxis group was subdivided into group Y_1 (n=10, sampling at 3h after initial LMWH injection) and Y_2 group (sampling at 3h after the last LMWH injection, to observe the states of thrombogenesis and to compare the thrombotic rates between Y_2 and D groups). In the control (group Y_0) rats, the same volume physiological saline was injected, and bilateral femoral veins were cut at post-injection 3h. (3) 100 SD rats used for TDVT treatment were modeled as well as observed until post-traumatic 120h. In which, the rats with thrombogenesis (n≌50) was selected for drug medication studing and subdivided into drug therapy group (n=40) and control group (group Z_0, n= 10). In drug therapy group, LMWH intraperitoneal injection was performed (600IU/kg, once a day). According to different time of sampling, drug prophylaxis group rats were subdivided into group Z_1 (sampling at 3h after initial LMWH injection) and Z_2 group (sampling at 171h after successive medication, to observe the states of thrombogenesis and to compare the thrombogenesis rates between Y_2 and E groups). Group Z_0 rats were injected the same volume physiological saline and the bileteral femoral veins were cut at post-injection 3h. (4) Each 10 rats were selected for Y_0, Y_1, Z_0 and Z_1 groups. Their bilateral femoral veins were cut for RNA extraction and genechip detection. (5) Based on FC analysis results, KEGG Pathway Database was applied to analyze the differential expression genes in Y_1/ Y_0 and Z_1/Z_0 conparisons. (6) Through performing the intersection set among the differential expression genes in E/F, Y_1/Y_0, Z_1/Z_0 conparisons, to further select coexpression genes in them.
Results
1. Results of TDVT rat model establishing and femoral vein gene expression analysis.
(1) In this model, femoral vein thrombogenesis started at post-traumatic 72h; the thrombogenesis crest-time was at the post-traumatic 120h and the rate of thrombogenesis was 50.5%. After that, thrombi began to resolve, and to the post-traumatic 168h, the rate of thrombi solution was 56.7%; the rate of thrombusi insolution was 43.3%. And the state of thrombus insolution would be persistence until more than post-traumatic 240h. (2) The macroscopic and histological observations results of femoral vein thrombosis were conincidence with the corresponding thrombi states. (3)The femoral vein total RNA samples from the 8 groups were no pollution or degradation and their qualities met to the conditions of genechip detection. (4) In the 31042 genes which can be detected by Genechip Rat Genome 230 2.0, group B compared with the control (B/A), 349 genes presented differential expression, in which, 214 were up-regulated and 135 was down-regulated; group C compared with the control (C/A), 2393 genes presented differential expression, in which, 1386 genes were up-regulated and 1007 was down-regulated; group D compared with the control (D/A), 1743 genes presented differential expression, in which, 945 genes were up-regulated and 798 were down-regulated; group H compared with the control (H/A), 2790 genes presented differential expression, in which, 1685 genes were up-regulated and 1105 were down-regulated; group E compared with the control (E/A), 1913 genes presented differeential expression, in which, 1222 genes were up-regulated and 691 were down-regulated; group F compared with the control (F/A), 2564 genes presented differential expression, in which, 1535 genes were up-regulated and 1029 were down-regulated; group G compared with the control (G/A), 1849 genes presented differential expression, in which, 1235 genes were up-regulated and 614 were down-regulated; group D compared with group H (D/A), 805 genes presented differential expression, in which, 51 genes were up-regulated and 755 were down-regulated. The function of these genes involved cell apoptosis, binding, metablism, cell cycle, transcription regulator activity, etc..
2. Results of differential expression gene analysis between thrombus solution and insolution groups.
(1) In E/F comparison, 229 genes presented differential expression, in which, 111 were up-regulated and 118 were down-regulated. And the differential expression genes with known functions mainly involved cell apoptosis, binding, metablism, etc.. (2) Through Cluster analysis, the genes were divided into 3 clusters: the first cluster included 45 genes such as Mybph, Myf6, Sln, Cox6a2, Alox15, etc., and their expression levels were lower in B, C, D, F groups, but being higher in H, E, G groups; the second cluster included 76 genes such as pcytlb, tfdb2, bpgm, Ca2, etc., and their expression levels were higher only in group E, but being lower in the other phases; the third cluster genes included 108 genes such as mmpl2, tnfaip6, lamp1, pfk1, etc., and the significant expression feature of them was that they presented significantly differential expression in group F and a part of them also presnted up-regualtion in B, H groups.
3. Results of the experimetal study of target genes in LMWH preventing and curing TDVT.
(1) Post-traumatic 120h, the rate of thrombogenesis was 50.5%; after LMWH prophylaxis, the rate decreased to 16.7%, and there was a significantly statistical difference between them (p=0.008<0.01). (2) At post-traumatic 168h, the rate of thrombi solution was 56.7%; after LMWH therapy, the rate increased to 91.7%, and there was a significantly statistical difference between them (p=0.004<0.01). (3) In Y_1 /Y_0 comparison, 1193 genes presented differential expression, in which, 471 were up-regulated and 722 were down-regulated. (4) In Z_1/D comparison, 1229 genes presented differential expression, in which, 907 were up-regulation and 322 were down-regulation. (5) After LMWH intervention, the diferential expression genes involved several pathways including MAPK, focal adhesion, calcium, cytokine-cytokine receptor interaction signaling pathway, apoptosis, etc.. (6) The intersection set among the differential expression genes in E/F, Y_1/Y_0, Z_1/Z_0 conparisons included 15 genes.
Conclusion:
1. Applying trauma combined with immobilization can establish a TDVT animal model, which is more close to clinical pathological features of TDVT and can serve as a reliable model for studies of the patho genesis, prophylaxis and cure of TDVT.
2. TDVT is a disease related to multiple genes which are mainly involved in cell apoptosis, binding, metablism, cell cycle, signaling transduction, etc...
3. The 45 genes including Mybph, Myf6, Sln, Cox6a2, Alox15, etc are related to nonthrombogenesis and thrombus resolution; the 76 genes including pcyt1b, tfdb2, bpgm, Ca2, etc. are related to thrombogenic prognosis and higher expression levels of them can improve the progress of thrombus resolution; the 108 genes including mmp12, tnfaip6, lamp1, pfk1, etc. play a role in thrombotic persistent insolution states.
4. In the mechanism of LWMH preventing and curing TDVT, besides thrombin and Fxa, LWMH also regulates genes related to blood coagulation, anticoagulation and fibrolysis in vascular endothelial cell so as to perforn drug action. In which, Thbd, Plaur, etc. could be the drug action targets.
5. The molecular mechanism of LMWH intervention in TDVT mainly involves MAPK, focal adhesion, cytokine-cytokine, calcium receptor interaction, apoptosis, etc. signaling pathways. And thrombosis as well as its prognosis is affected by cell proliferation, differentiation, inflammatory reaction, etc..
6. The 15 genes including Actn3, RGD:620059, Mbp, etc., which gained from the intersection set among the differential expression genes in thrombus resolution, thrombus insolution and drug interveion groups, play important roles in the process of thrombus resolution. They will be the drug action targets in LMWH preventing and curing TDVT.
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5、Wang X, Li X, Xu L, et al. gulation of secretory leukocyte protease inhibitor (SLPI) in the brain after ischemic stroke: adenoviral expression of SLPI protects brain from ischemic injury. Mol Pharmacol 2003; 64(4):833-40.
6、Ota Y, Shim0ya K, Zhang Q, et al. The expression of secretory leukocyte protease inhibitor (SLPI) in the fallopian tube: SLPI protects the acrosome reaction of sperm from inhibitory effects of elastase. Hum Reprod 2002; 17(10):2517-22.
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9 Hollander C, Nystrom M, Janciauskiene S, et al. Human mast cells decrease SLPI levels in type II - like alveolar cell model, in vitro.Cancer Cell Int 2003; 3(1): 14.
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11 Jana NK, Gray LR, Shugars DC. Human immunodeficiency virus type 1 stimulates the expression and production of secretory leukocyte protease inhibitor (SLPI) in oral epithelial cells: a role for SLPI in innate mucosal immunity. J Virol 2005; 79(10):6432-40.
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14 Wada H, Kagoshima M, Ito K, et al. 5-Azacytidine suppresses RNA polymerase II recruitment to the SLPI gene. Biochem Biophys Res Commun 2005; 331(1):93-9.
15 van Wetering S, van der Linden AC, van Sterkenburg MA, et al. Regulation of secretory leukocyte proteinase inhibitor (SLPI) production by human bronchial epithelial cells: increase of cell-associated SLPI by neutrophil elastase. J Investig Med 2000; 48(5):359-66.
16 van Wetering S, van der Linden AC, van Sterkenburg MA, et al. Regulation of SLPI and elafin release from bronchial epithelial cells by neutrophil defensins . Am J Physiol Lung Cell Mol Physiol 2000; 278(1):L51-8.
17 Tian X, Shigemasa K, Hirata E, et al. Expression of human kallikrein 7 (hK7/SCCE) and its inhibitor antileukoprotease (ALP/SLPI) in uterine endocervical glands and in cervical adenocarcinomas . Oncol Rep 2004v; 12(5):1001-6.
18 Westin U, Nystrom M, Ljungcrantz I, et al. The presence of elafin, SLPI, IL1-RA and STNF alpha RI in head and neck squamous cell carcinomas and their relation to the degree of tumour differentiation. Mediators Inflamm 2002; 11(1):7-12.