创伤性深静脉血栓形成及低分子肝素

1、创伤性深静脉血栓形成及低分子肝素预防的基因表达变化研究中文摘要目的： 检测大鼠肢体创伤性深静脉血栓形成过程中股静脉血管的基因表达变化情况，筛选与血栓形成和不形成两种状态密切相关的差异表达基因，寻找预防血栓形成的药物作用靶点。材料和方法： 第一部分：创伤性深静脉血栓形成中基因表达变化的研究1、150只SD大鼠随机分为正常对照组（A组，0h）和模型组。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进行造模，根据造模后的不同生物学状态分为七组：创伤即刻组（B组，0.5h）、血栓形成初始期组（C组，72h）、120h血栓形成高峰组（D组，120h）、120h血栓不形成组（H组，120h）、血栓消

2、退组（E组，168h）、血栓不消退组（F组，168h）和创伤后持续无血栓组（G组，168h），上述各分组均纳入10只大鼠。其中，创伤即刻组（B组）由于造模后即取材而不行石膏固定。2、在相应时相点切取4-5cm股静脉血管组织，每条血管取近端0.5 cm用于HE染色光镜观察以确认分组的准确性，剩余血管组织同组混合后抽取总RNA，检测合格后用GeneChip® Rat Genome 230 2.0芯片进行检测。3、在倍数变化分析基础上，对差异表达基因进行GO分类。并对D vs H组差异表达基因进行pathway分析。第二部分：创伤性深静脉血栓形成与不形成差异表达基因的功能集群以D vs H

3、组差异表达基因为基础，采用基因功能集群分析方法对芯片数据作进一步分析，判断导致血栓形成或不形成状态的差异表达基因中变化最显著、最集中的基因功能。第三部分：低分子肝素预防创伤性深静脉血栓形成的基因表达时间序列研究1、另取50只SD大鼠同法造模，造模后随机分为药物预防组（n40）和对照组（Y0组；n10）。预防组造模后6h首次给药，按500 IU/ kg腹腔内注射低分子肝素,一日一次。根据不同取材时间将大鼠分为两组：Y1组（9h药物预防组，9h）：从药物预防的40只大鼠中随机取10只纳入本组，于造模后6h给予药物预防剂量注射，3h后取材；Y2组（120h药物预防组，120h）：剩余的30只SD大鼠

4、每天按时给药一次，持续至造模后第120h观察血栓发生率并取材。进行HE染色光镜观察确认分组准确性、RNA抽提和芯片检测。2、应用基因表达时间序列分析影响实验性状的主流基因群，进行基于聚类分析结果的基因功能显著性分析，分析有相似表达规律的基因主要体现的功能。结果： 第一部分：创伤性深静脉血栓形成中基因表达变化的研究结果1、造模后第120h血栓形成率约为50.5，血栓不形成率约为49.5；第168h，有血栓的大鼠中大概有56.7发生消退，43.3的血栓持续存在不消退。2、各组血管组织肉眼和光镜观察结果对血栓状态的判断基本一致。3、创伤性深静脉血栓形成过程中众多基因差异表达，涉及的GO分类有凋亡、分

5、子粘附、代谢、细胞周期、酶调节、信号转导、转录调节等。4、涉及的通路主要有MAPK、Wnt、JAK-STAT、黏附斑和凋亡信号通路等。第二部分：创伤性深静脉血栓形成与不形成差异表达基因的功能集群结果1、D vs H组共有806个基因呈现差异性表达，其中上调基因51个，下调基因755个。在已知功能的基因中涉及了分子粘附、催化活性、信号通路激活、酶调节活性和物质转运等方面的功能。2、在D vs H组比较的基因功能集群分析提示补体系统激活、细胞的发生、生长、形态发生、定位、运动、蛋白代谢等功能，以及所对应的C4bp、Bf、Serpine1和Plaur等基因与创伤性深静脉血栓的形成与不形成状态相关。第

6、三部分：低分子肝素预防创伤性深静脉血栓形成的基因表达时间序列研究结果1、造模后第120h，D组血栓形成率为50.5；应用低分子肝素进行预防后Y2组血栓形成率为16.7，两者比较差别具有显著统计学意义(P<0.01)。2、药物预防组基因表达时间序列分析，具有显著意义的主流基因群主要涉及的GO功能有：离子通道；酰基转移酶活性；酶结合；除氨酰(基)以外的因子转移酶活性；生血素/人白细胞干扰素分级, 细胞因子受体活性；糖结合；跨膜受体活性等。所对应基因有：Kcna5、Clcn3、Kcnk3、Acat1、Rgd1305719-predicted、Fez1、Rab3ip、Agpat2-predict

7、ed、Tg、Axin2、Mgl1、Ifngr2-predicted、Loc304091、Il6r、Sell、Cspg2、Loc498276、Fcgr2b等。3、在应用低分子肝素进行预防后，Sell、Loc498276、Mgl1、Cspg2、Serpinb2、Ddr1、Aebp1-predicted、Pabpc4-predicted、Plaur和Nrp1等基因表达丰度改变。结论： 1、TDVT演变过程中，差异表达的基因主要涉及了纤溶系统、凝血系统、炎症因子、补体系统等多个系统中。2、凋亡、黏附斑、MAPK、JAK-STAT、Wnt等信号通路通过影响细胞增殖、分化、细胞周期等而参与深静脉血栓状态的

8、调控。3、众多细胞周期、结合、代谢、凋亡和信号转导等相关基因参与了TDVT演变过程。4、Kcna5、Clcn3、Kcnk3、Acat1、RGD1305719_predicted、Agpat_predicted、 Fez1、Rab3ip、Axin2等基因表达下调及Bf、C4bp等基因表达相对上调可致创伤后机体抗血栓能力下降促进血栓形成。5、经在NCBI(美国国立生物技术信息中心)的PubMed数据库中对离子通道相关基因Kcna5、Kcnk3、Clcn3进行逐个检索，发现其在以前的由于创伤/手术因素引起深静脉血栓的文献中未有提及。6、Plaur、Serpine1、Il6r、Tg、Loc304091

9、、Fcgr2b、Ifngr2-predicted等基因表达上调为创伤性深静脉血栓不形成创造了条件。7、除了传统的凝血酶、FXa以外，Sell、Loc498276、Mgl1、Cspg2、Serpinb2、Aebp1_predicted、Ddr1、Pabpc4_predicted、Plaur和Nrp1等在LMWH预防创伤性深静脉血栓形成的分子机制中发挥作用，可作为预防创伤性深静脉血栓形成的药物靶点加以进一步研究。8、本实验首次对低分子肝素预防TDVT形成的基因表达变化这一动态过程进行时间序列分析，通过数学计算模式，能从庞大的数据中筛选与血栓形成与不形成两种状态密切相关的基因，这对于研究TDVT的分

10、子机制是一个新的思路。关键词：创伤和损伤/并发症 静脉血栓形成/预防与控制 肝素，低分子量 基因表达Study on the Gene Expression Changes of Traumatic Deep Vein Thrombosis and Prevention with Low Molecular Weight HeparinAbstractObjective：Using rats as the model, detect the expression changes of genes during the development of traumatic deep vein thr

11、ombosis (TDVT), and identify the differentially expressed genes between thrombosis and non-thrombosis veins. Especially, screen the therapeutic drug target genes which could prevent TDVT. Materials and Methods：Part：Study on the Gene Expression Changes of Traumatic Deep Vein Thrombosis 1. 150 SD rats

12、 were divided into control (Group A, 0h) and experiment groups randomly. In model rats, beating on bilateral posterior limbs combined with hip spica cast fixation were performed.The experiment group was divided into 7 subgroups according to the different biological phases, i.e. the post-traumatic in

13、stant (Group B, 0.5h), the initial period of thrombosis (Group C, 72h), the crest-time of thrombosis (Group D, 120h), non-thrombosis in post-traumatic 120h (Group H, 120h), thrombi solution (Group E, 168h), thrombi insolution (Group F, 168h) and post-traumatic non-thrombosis sequentially (Group G, 1

14、68h). Each 10 individuals were selected into corresponding group randomly. In which, fixation was not performed in post-traumatic instant group (Group B) because of their femoral veins would be cut as soon as modeling.2. Incise 4 to 5 cm femoral vein of the rats in those different biological phases.

15、 About 0.5 cm of each proximate femoral vein was cut for HE staining to confirm the reliability of the previous grouping. The rest of each femoral vein was used to extract total RNA respectively. After RNA quality was assessed, each sample was hybridized to GeneChip® Rat Genome 230 2.0 array (A

16、ffymetrix) to detect the mRNA expression profiles. 3. Based on the Fold Change analysis, the differential expression genes were classified according to GO classification. And the differentially-expressed genes of D vs H group were identified through pathway analysis.Part ：The differential expression

17、 genes function assembly analysis between thrombosis and non-thrombosis in traumatic deep vein thrombosis GO enrichment test was further analysed using these differentially-expressed genes of D vs H group, and functional overrepresentation of these genes was identified. Part ：Study on the gene expre

18、ssion of low molecular weight heparin preventing traumatic deep vein thrombosis by time series1. Besides the 150 rats, another 50 rats were selected to modeling by the same method. Then they were divided into drug prophylaxis group (n=40) and control group（Group Y0, n=10). The drug prophylaxis group

19、 was injected into abdominal cavity with corresponding dose of LMWH (500 IU/kg) in the sixth hour after modeling, then they were injected with same medicine and by the same method one time a day. The drug prophylaxis group rats were divided into two groups according to the time of sampling. 3 hours

20、after the first LMWH injection, the group Y1 (random choose 10 individuals in drug prophylaxis group) were sampled，and the groups Y2 (n=30) were sampled in the 120th hour. HE staining was done in these samples to confirm the reliability, then the RNA was extracted for array preparation. 2. Gene expr

21、ession tendency analytical method was used to detect the gene set which could affect the phenotype significantly. Then the functional significance of these genes was investigated through cluster analysis, finally GO annotation was used to identify the function catalog of the genes with same expressi

22、on pattern.Results：Part：Results of the Gene Expression Changes of Traumatic Deep Vein Thrombosis1. In this model, the rate of thrombogenesis was 50.5% at 120 hours after trauma.The rate of non-thrombogensis was 49.5% at 120 hours after trauma. The rate of thrombi solution was 56.7% at 168 hours afte

23、r trauma, and the other 43.3% remained insoluted. 2. The results of the observations of the femoral vein thrombosis through naked eyes and microscope were consistent. 3. During TDVT, many genes were differentially-expressed, which were related to apoptosis, binding, metabolism, cell cycle, enzyme re

24、gulation, signal transduction and transcription regulation, etc. 4. Besides these, the differentially-expressed genes were mainly involved in MAPK, Wnt, JAK-STAT, focal adhension, apoptosis signal pathways etc. Part ：Results of the differential expression genes function assembly analysis between thr

25、ombosis and non-thrombosis in traumatic deep vein thrombosis 1. In D vs H group, 806 of them were differentially expressed genes, in which, 51 up-regulated and 755 down-regulated. And the differential expression genes with known functions mainly related to binding, catalytic activity, signal transdu

26、cer activity, enzyme regulator activity, transporter activity, etc.2. The gene function assembly analysis of group D vs H indicated that complement activation, development, growth, morphogenesis, cell motility, localization，protein metabolism etc. and the genes of C4bp, Bf, Serpine1 and Plaur etc. w

27、ere related to the state of thrombosis and non-thrombosis.Part ：Results of the gene expression of low molecular weight heparin preventing traumatic deep vein thrombosis analysis by time series1. 120 hours after the modeling, the rate of thrombogenesis of group D was 50.5%, while the rate of group Y2

28、 treated with LMWH was 16.7%. The comparison of these rate had statistical significance (P<0.01). 2. GO annotation of the genes, which were analyzed by time series in drug prophylaxis groups, identified several GO classification which had significant effect. They were related to voltage-gated ion

29、 channel activity, acyltransferase activity, enzyme binding, transferring groups other than amino-acyl groups, hematopoietin/interferon-class (D200-domain) cytokine receptor activity, sugar binding, transmembrane receptor activity etc. And the corresponding genes were Kcna5, Clcn3, Kcnk3, Acat1, Rgd

30、1305719-predicted, Agpat2-predicted, Fez1, Rab3ip, Axin2, Ifngr2-predicted, Loc304091, Il6r, Mgl1, Sell, Cspg2, Loc498276 and Fcgr2b. 3. The expression of Sell, Loc498276, Mgl1, Cspg2, Serpinb2, Aebp1_predicted, Ddr1, Pabpc4_predicted, Plaur, Nrp1 etc. were changed after prevention with low molecula

31、r weight heparin.Conclusion：1. The differentially-expressed genes during TDVT were related to inflammation, fibrolysis/anti-fibrolysis, blood coagulation/anticoagulation, complement etc. 2. The signaling pathways of apoptosis, focal adhension, MAPK, Wnt, JAK-STAT etc. might influence the biological

32、states of TDVT through regulated cell cycle, proliferation, differentiation etc. 3. Many genes which were related to cell cycle, binding, metabolism, apoptosis, signal transduction etc. were involved in the process of TDVT. 4. The up regulation of Bf, C4bp and down regulation of Kcna5, Clcn3, Kcnk3, Acat1, RGD1305719_predicted, Agpat_predicted, Fez1, Rab3ip and Axin2 might decrease the ability of anti-thrombosis, an