創(chuàng)傷性深靜脈血栓形成及低分子肝素

1、創(chuàng)傷性深靜脈血栓形成及低分子肝素預(yù)防的基因表達(dá)變化研究中文摘要目的： 檢測(cè)大鼠肢體創(chuàng)傷性深靜脈血栓形成過(guò)程中股靜脈血管的基因表達(dá)變化情況，篩選與血栓形成和不形成兩種狀態(tài)密切相關(guān)的差異表達(dá)基因，尋找預(yù)防血栓形成的藥物作用靶點(diǎn)。材料和方法： 第一部分：創(chuàng)傷性深靜脈血栓形成中基因表達(dá)變化的研究1、150只SD大鼠隨機(jī)分為正常對(duì)照組（A組，0h）和模型組。模型組采用大鼠雙側(cè)后肢定量擊打髖人字石膏外固定的方法進(jìn)行造模，根據(jù)造模后的不同生物學(xué)狀態(tài)分為七組：創(chuàng)傷即刻組（B組，0.5h）、血栓形成初始期組（C組，72h）、120h血栓形成高峰組（D組，120h）、120h血栓不形成組（H組，120h）、血栓消

2、退組（E組，168h）、血栓不消退組（F組，168h）和創(chuàng)傷后持續(xù)無(wú)血栓組（G組，168h），上述各分組均納入10只大鼠。其中，創(chuàng)傷即刻組（B組）由于造模后即取材而不行石膏固定。2、在相應(yīng)時(shí)相點(diǎn)切取4-5cm股靜脈血管組織，每條血管取近端0.5 cm用于HE染色光鏡觀察以確認(rèn)分組的準(zhǔn)確性，剩余血管組織同組混合后抽取總RNA，檢測(cè)合格后用GeneChip® Rat Genome 230 2.0芯片進(jìn)行檢測(cè)。3、在倍數(shù)變化分析基礎(chǔ)上，對(duì)差異表達(dá)基因進(jìn)行GO分類。并對(duì)D vs H組差異表達(dá)基因進(jìn)行pathway分析。第二部分：創(chuàng)傷性深靜脈血栓形成與不形成差異表達(dá)基因的功能集群以D vs H

3、組差異表達(dá)基因?yàn)榛A(chǔ)，采用基因功能集群分析方法對(duì)芯片數(shù)據(jù)作進(jìn)一步分析，判斷導(dǎo)致血栓形成或不形成狀態(tài)的差異表達(dá)基因中變化最顯著、最集中的基因功能。第三部分：低分子肝素預(yù)防創(chuàng)傷性深靜脈血栓形成的基因表達(dá)時(shí)間序列研究1、另取50只SD大鼠同法造模，造模后隨機(jī)分為藥物預(yù)防組（n40）和對(duì)照組（Y0組；n10）。預(yù)防組造模后6h首次給藥，按500 IU/ kg腹腔內(nèi)注射低分子肝素,一日一次。根據(jù)不同取材時(shí)間將大鼠分為兩組：Y1組（9h藥物預(yù)防組，9h）：從藥物預(yù)防的40只大鼠中隨機(jī)取10只納入本組，于造模后6h給予藥物預(yù)防劑量注射，3h后取材；Y2組（120h藥物預(yù)防組，120h）：剩余的30只SD大鼠

4、每天按時(shí)給藥一次，持續(xù)至造模后第120h觀察血栓發(fā)生率并取材。進(jìn)行HE染色光鏡觀察確認(rèn)分組準(zhǔn)確性、RNA抽提和芯片檢測(cè)。2、應(yīng)用基因表達(dá)時(shí)間序列分析影響實(shí)驗(yàn)性狀的主流基因群，進(jìn)行基于聚類分析結(jié)果的基因功能顯著性分析，分析有相似表達(dá)規(guī)律的基因主要體現(xiàn)的功能。結(jié)果： 第一部分：創(chuàng)傷性深靜脈血栓形成中基因表達(dá)變化的研究結(jié)果1、造模后第120h血栓形成率約為50.5，血栓不形成率約為49.5；第168h，有血栓的大鼠中大概有56.7發(fā)生消退，43.3的血栓持續(xù)存在不消退。2、各組血管組織肉眼和光鏡觀察結(jié)果對(duì)血栓狀態(tài)的判斷基本一致。3、創(chuàng)傷性深靜脈血栓形成過(guò)程中眾多基因差異表達(dá)，涉及的GO分類有凋亡、分

5、子粘附、代謝、細(xì)胞周期、酶調(diào)節(jié)、信號(hào)轉(zhuǎn)導(dǎo)、轉(zhuǎn)錄調(diào)節(jié)等。4、涉及的通路主要有MAPK、Wnt、JAK-STAT、黏附斑和凋亡信號(hào)通路等。第二部分：創(chuàng)傷性深靜脈血栓形成與不形成差異表達(dá)基因的功能集群結(jié)果1、D vs H組共有806個(gè)基因呈現(xiàn)差異性表達(dá)，其中上調(diào)基因51個(gè)，下調(diào)基因755個(gè)。在已知功能的基因中涉及了分子粘附、催化活性、信號(hào)通路激活、酶調(diào)節(jié)活性和物質(zhì)轉(zhuǎn)運(yùn)等方面的功能。2、在D vs H組比較的基因功能集群分析提示補(bǔ)體系統(tǒng)激活、細(xì)胞的發(fā)生、生長(zhǎng)、形態(tài)發(fā)生、定位、運(yùn)動(dòng)、蛋白代謝等功能，以及所對(duì)應(yīng)的C4bp、Bf、Serpine1和Plaur等基因與創(chuàng)傷性深靜脈血栓的形成與不形成狀態(tài)相關(guān)。第

6、三部分：低分子肝素預(yù)防創(chuàng)傷性深靜脈血栓形成的基因表達(dá)時(shí)間序列研究結(jié)果1、造模后第120h，D組血栓形成率為50.5；應(yīng)用低分子肝素進(jìn)行預(yù)防后Y2組血栓形成率為16.7，兩者比較差別具有顯著統(tǒng)計(jì)學(xué)意義(P<0.01)。2、藥物預(yù)防組基因表達(dá)時(shí)間序列分析，具有顯著意義的主流基因群主要涉及的GO功能有：離子通道；?；D(zhuǎn)移酶活性；酶結(jié)合；除氨酰(基)以外的因子轉(zhuǎn)移酶活性；生血素/人白細(xì)胞干擾素分級(jí), 細(xì)胞因子受體活性；糖結(jié)合；跨膜受體活性等。所對(duì)應(yīng)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、在應(yīng)用低分子肝素進(jìn)行預(yù)防后，Sell、Loc498276、Mgl1、Cspg2、Serpinb2、Ddr1、Aebp1-predicted、Pabpc4-predicted、Plaur和Nrp1等基因表達(dá)豐度改變。結(jié)論： 1、TDVT演變過(guò)程中，差異表達(dá)的基因主要涉及了纖溶系統(tǒng)、凝血系統(tǒng)、炎癥因子、補(bǔ)體系統(tǒng)等多個(gè)系統(tǒng)中。2、凋亡、黏附斑、MAPK、JAK-STAT、Wnt等信號(hào)通路通過(guò)影響細(xì)胞增殖、分化、細(xì)胞周期等而參與深靜脈血栓狀態(tài)的

8、調(diào)控。3、眾多細(xì)胞周期、結(jié)合、代謝、凋亡和信號(hào)轉(zhuǎn)導(dǎo)等相關(guān)基因參與了TDVT演變過(guò)程。4、Kcna5、Clcn3、Kcnk3、Acat1、RGD1305719_predicted、Agpat_predicted、 Fez1、Rab3ip、Axin2等基因表達(dá)下調(diào)及Bf、C4bp等基因表達(dá)相對(duì)上調(diào)可致創(chuàng)傷后機(jī)體抗血栓能力下降促進(jìn)血栓形成。5、經(jīng)在NCBI(美國(guó)國(guó)立生物技術(shù)信息中心)的PubMed數(shù)據(jù)庫(kù)中對(duì)離子通道相關(guān)基因Kcna5、Kcnk3、Clcn3進(jìn)行逐個(gè)檢索，發(fā)現(xiàn)其在以前的由于創(chuàng)傷/手術(shù)因素引起深靜脈血栓的文獻(xiàn)中未有提及。6、Plaur、Serpine1、Il6r、Tg、Loc304091

9、、Fcgr2b、Ifngr2-predicted等基因表達(dá)上調(diào)為創(chuàng)傷性深靜脈血栓不形成創(chuàng)造了條件。7、除了傳統(tǒng)的凝血酶、FXa以外，Sell、Loc498276、Mgl1、Cspg2、Serpinb2、Aebp1_predicted、Ddr1、Pabpc4_predicted、Plaur和Nrp1等在LMWH預(yù)防創(chuàng)傷性深靜脈血栓形成的分子機(jī)制中發(fā)揮作用，可作為預(yù)防創(chuàng)傷性深靜脈血栓形成的藥物靶點(diǎn)加以進(jìn)一步研究。8、本實(shí)驗(yàn)首次對(duì)低分子肝素預(yù)防TDVT形成的基因表達(dá)變化這一動(dòng)態(tài)過(guò)程進(jìn)行時(shí)間序列分析，通過(guò)數(shù)學(xué)計(jì)算模式，能從龐大的數(shù)據(jù)中篩選與血栓形成與不形成兩種狀態(tài)密切相關(guān)的基因，這對(duì)于研究TDVT的分

10、子機(jī)制是一個(gè)新的思路。關(guān)鍵詞：創(chuàng)傷和損傷/并發(fā)癥 靜脈血栓形成/預(yù)防與控制 肝素，低分子量 基因表達(dá)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