【作者】 徐军；

【导师】 赵学凌； 王兵； 赵宏斌； 张春强；

【作者基本信息】 昆明医学院 ， 外科学， 2009， 硕士

【摘要】 【目的】1.应用恒古骨伤愈合剂治疗兔创伤性深静脉血栓（TDVT）,通过观察血栓形成情况评估其疗效;2.应用ELISA技术检测D-二聚体（D-Dimer）、抗磷脂抗体（APA）、凝血酶-抗凝血酶复合物（TAT）在恒古骨伤愈合剂治疗兔TDVT三个关键时相点的表达量;3.通过观察上述三个指标在不同组别的表达变化,探究恒古骨伤愈合剂治疗兔TDVT过程中对凝血系统的影响。【方法】一、恒古骨伤愈合剂治疗兔TDVT的疗效观察及血液、血管组织采集1.实验动物及分组:本实验共64只SPF级新西兰大白兔,按给药不同随机分为正常对照组（A组共8只）、恒古骨伤愈合剂组（B组共28只）、生理盐水对照组（C组共28只）;再根据观察时间不同分别将B、C组分为造模后24小时观察组（B₁、C₁组,每组各8只）,造模后72小时观察组（B₂、C₂组,每组各8只）及造模后120小时观察组（B₃、C₃组,每组各8只）;2.造模方法:实验采用左侧股静脉钳夹+髋人字石膏固定方法对B、C组兔进行造模。实验兔麻醉后,取腹股沟内侧沿股动脉走行方向作一长4-5cm纵行切口,暴露约2.5cm的左侧股静脉,用12.5mm全齿蚊式血管钳分五段各钳夹1次,力量为血管钳紧2扣,每次持续3秒;用1号丝线全层间断缝合皮肤切口,不放置引流,均行石膏固定;3.给药时间及剂量:B组兔造模后立刻灌喂恒古骨伤愈合剂,以后每隔24h喂药一次（剂量为1.17ml/kg）;C组兔在对应时间灌喂生理盐水（剂量为1.17ml/kg）;4.观察、取材时间及方法:造模前对A组各兔抽取耳缘静脉血1.5ml,装入标准枸橼酸钠抗凝管,混匀、离心、取血浆-20℃保存;造模后24小时,B组随机取8只作为B₁组,C组随机取8只作为C₁组,剪开切口缝线,肉眼观察并比较两组血栓发生率,并取长约1.0cm的股静脉进行HE染色、光镜观察确定血栓形成状态,同时,由耳缘静脉抽取每只兔各1.5ml静脉血,装入标准枸橼酸钠抗凝管,混匀,3000rpm/min离心20min后取血浆-20℃保存;造模后72小时和120小时分别随机取8只作为B₂、C₂、B₃、C₃组,以相同方式取材、采血;5.血栓发生率统计学处理:对各组资料数据进行x²检验,以P＜0.05为有统计学意义,P＜0.01为有显著的统计学意义。二、ELISA检测D-Dimer、APA、TAT探讨恒古骨伤愈合剂治疗TDVT对凝血系统的影响1.ELISA检测:采用ELISA方法分别检测A、B₁、B₂、B₃、C₁、C₂、C₃各组大白兔血液中D-Dimer、APA、TAT的表达量;2.统计学处理:使用SPSS11.5统计软件包对所得表达量进行统计学处理。各组计量资料数据用均数士标准差（（?）±s）表示,两组数据间比较用单因素方差分析（One-way ANOVA）和S-N-K检验,以P＜0.05为差别有统计学意义,P＜0.01为差别有显著的统计学意义。【结果】一、恒古骨伤愈合剂治疗兔TDVT的疗效观察及血液样本采集1.实验动物一般情况:B、C两组兔共死亡5只,动物死亡率8.93%2.血栓发生情况:该模型中,股静脉血栓形成高峰期在造模后24h,血栓发生率分别为:B组66.7%（16/24）,C组79.2%（22/24）,差别有统计学意义（P＜0.05）;之后血栓开始消退,在恒古骨伤愈合剂治疗72h,血栓可基本消退,此时血栓发生率为:B组25.0%（4/16）,C组75.0%（12/16）,差别有统计学意义（P＜0.05）;造模后120h,B₃组血栓已完全消退,C₃组血栓发生率为50%（4/8）,两者比较差别有显著的统计学意义（p＜0.01）。3.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一致。二、ELISA检测D-Dimer、APA、TAT探讨恒古骨伤愈合剂治疗TDVT对凝血系统的影响1.C组（9.11±1.11ng/ml）与A组（1.70±0.30ng/ml）相比,D-dimer、APA、TAT的表达量存在统计学差异2.C组（9.11±1.11ng/ml）与B组（2.27±0.62 ng/ml）相比,D-dimer、APA、TAT的表达量存在统计学差异【结论】1.恒古骨伤愈合剂能有效降低兔TDVT发生率,促进血栓消退;2.恒古骨伤愈合剂可能通过影响凝血系统,降低血液高凝状态治疗兔TDVT。更多还原

【Abstract】 Objective.1.To explore therapeutic effect of Osteoking in treatment of rabbits TDVT by observe thrombosis situation.2.After treatment with Osteoking in the rabbits TDVT.ELISA applied to detected expression levels of D-Dimmer、APA、TAT at different phases of rabbits TDVT.3.By observing the changes of D-Dimmer,APA,TAT during the different group of traumatic deep vein thrombosis（TDVT）,to explore the role of fibrinolytic and coagulation systems during the treatment with Osteoking.Methods.1.Therapeutic effect observation with Osteoking in rabbits TDVT and collection of blood and vascular tissue samplesAnimal and grouping:According to different drug treated with Osteoking,64 SPF rabbits were divided randomly into control group（group A,total 8）,Osteoking group（group B,total 28）,physiological saline group（group B,total 28）.According to different observation phases B,C groups were divided into other 6 groups: post-modeling 24h group（B₁,C₁ group,respectively 8）,post-modeling 72h group（B₂,C₂ group,respectively 8） and post-modeling 120h group（B₃,C₃ group, respectively 8）.Modeling:Directly clamping left femoral vein combined with hip spica fixation was performed to establish acute TDVT rabbits models with B,C groups.Rabbits were anesthetized,supine position fixation,sterilization,exposing left femoral veins.2.5cm isolated femoral vein was clamped at five points separately with 12.5mm mosquito-hemostatic forceps,once each point;the clamping strength was fastening two barb of hemostatic forceps,lasting 3 seconds each time,then the incision was sutured,no drainage was set,left posterior limbs were fixed with hip spica casts.Administration and dosage:Once every 24 hours,administrate in group B with Osteoking about 1.17ml/kg,while C group with normal saline with the same dosage.Observation,methods and time for obtaining blood and vein tissue:To collect A group rabbits blood about 1.5ml from ear-edge vein at 24 hours after modeling 8 rabbits were selected randomly from group B to establish B₁ group,while the rabbits were selected randomly from group B to establish B₁ group,then resect the suture of the wound gross observe ratio of the left femoral vein thrombosis between the two groups.Resected 1.0cm vascular tissue put into stationary liquid for the further HE staining histological analysis and light microscope.8 rabbits were selected randomly at post-modeling 72h and 120h to establish group B₂,C₂,B₃,C₃ respectively,then obtain blood and vascular specimen for the same method of group B₁.Statistical analysis about TDVT incidence rate:Information on the data in each group using x² test,P＜0.05 for statistically significant,P＜0.01 for significant statistical significance.2.ELISA detection of D-dimer,APA,TAT to explore Osteoking treatmentTDVT impact on the coagulation systemELISA were used to detect the blood D-dimer,APA,TAT expression of rabbits in A、B₁、B₂、B₃、C₁、C₂、C₃ group.Using SPSS 11.5 statistical software package analyze the expression of the statistical volume.Measurement data in each group are demonstrate by the standard deviation（±s）,compare with two sets of data using one-way ANOVA and SNK test, P＜0.05 for the differences were statistically significant,P＜0.01 for the significant difference in statistical significance.Results1.The condition of rabbits:5 rabbits died in group B,C,and mortality is 8.93%.Thrombosis:In this model,post-modeling 24h is the thrombotic crest-time of femoralvein,the average incidence of TDVT between the two groups is 80.8%,then thrombosus begin spontaneous to resolution.After Osteoking treatment thrombosus almost resolution.After modeling 24h left femoral vein DVT incidence rate:Group B are 66.7%,Group C 79.2%,This had statistical significance（p＜0.05）;After modeling 72h Group B are25.0%,Group C are 75.0%also had statistical significance（p＜0.05）. After modeling 72h Group B₃ almost resolution.Group B₃ are 50%the two groups comparison with each other has significant statistical significance.（p＜0.01）Gross observation and light microscope observation of vascular specimens of each group consistent with the corresponding state thrombosis.2.ELISA detection of D-dimer,APA,TAT to explore Osteoking treatmentTDVT impact on the coagulation system.D-dimer,APA,TAT expression of group C（9.11±1.11ng/ml） comparison with group A（1.70±0.30ng/ml） exsist significant statistical different.D-dimer,APA,TAT expression of group C（9.11±1.11ng/ml） comparison with group B（2.27±0.62 ng/ml）exsist significant statistical different.Conlusion.1.Treatment with Osteoking in the rabbits TDVT could decrease the incidence of thrombosis and promote thrombolysis.2.Osteoking may affect the coagulation system,reducing blood hypercoagulation in treated rabbits TDVT.更多还原