【摘要】：[目的]1.利用異氟烷麻醉方法和下腔靜脈狹窄法構建DVT機化再通模型；使用血紅素加氧酶-1的激動劑(CoPPIX)及抑制劑(SnPPIX)干預C57小鼠DVT的溶解再通過程,并觀察各組小鼠DVT模型的死亡率、成栓率,下腔靜脈血栓的長度、濕重和病理切片的變化差異。2.檢測C57小鼠下腔靜脈壁和血栓的HO-1、VEGF和SDF-1的表達情況,探討HO-1、 VEGF和SDF-1在DVT溶解中后期的作用及相互關系。[材料與方法]實驗一：下腔靜脈狹窄法構建C57小鼠DVT模型及觀察HO-1激動劑及抑制劑對DVT機化再通過程的影響1.實驗分組及DVT模型的構建：C57小鼠200只,體重25-30g,雌性。按隨機分組原則分A組(空白對照組,n=20)、B組(正常DVT組,n=60)、C組(CoPPIX干預組,n=60)組和D組(SnPPIX干預組,n=60)。C組和D組在造模前24h分別腹腔注射CoPPIX和SnPPXI,5mg/kg; B組腹腔注射等量的生理鹽水。給藥24小時后B、C、D采用異氟烷麻醉配合下腔靜脈狹窄法構建DVT模型。2.取材：在造模后4、7、10、13天,采取頸椎脫臼法處死小鼠,采用游標卡尺測量血栓長度,取下結扎點至髂總靜脈匯合處之間下腔靜脈及其內容物,每個時間點每組中隨機取3只小鼠DVT標本,使用4%多聚甲醛浸泡固定,保存行病理檢查；其余標本分離血栓與靜脈壁,稱取血管內的血栓濕重,靜脈壁和內容物分裝后-80°保存?zhèn)溆谩?.指標測定和統(tǒng)計學分析：統(tǒng)計每組死亡率、成栓率、血栓長度和濕重變化情況。統(tǒng)計學軟件采用SPSS19.0,計量資料采用均數±標準差(x±s)；同一時間點,組間兩兩比較采用單因素方差分析、用LSD法(最小二乘法),*p0.05有統(tǒng)計學意義。實驗二：不同時間點各組小鼠DVT模型靜脈壁組織中HO-1、VEGF和SDF-1的mRNA達變化1.Trizol法提取每組小鼠不同時間點下腔靜脈中總RNA, RT-PCR將各組小鼠靜脈組織RNA逆轉錄為cDNA;以GAPDH為內參照,用Real-time PCR法檢測不同時間點HO-1、VEGF和SDF-1 mRNA的表達變化；2.統(tǒng)計學分析：統(tǒng)計學軟件采用SPSS19.0o計量資料采用均數±標準差(x±s)；同一時間點,組間兩兩比較采用單因素方差分析,用LSD法(最小二乘法),*p0.05有統(tǒng)計學意義。[結果]實驗一：下腔靜脈狹窄法構建C57小鼠DVT模型及觀察HO-1激動劑及抑制劑對DVT機化再通過程的影響1.麻醉效果：實驗開始使用3%-5%異氟烷對C57小鼠進行快速誘導,術中使用0.5%-1.0%濃度的異氟烷能維持滿意的麻醉效果。誘導達到麻醉滿意時間和術后清醒時間均很快,約1-2min,麻醉過程中小鼠出現的最大不良反應為呼吸抑制,但停止麻醉藥或加大空氣流量后,小鼠呼吸抑制的情況可迅速緩解.2.小鼠存活情況和死亡率：A、D兩組死亡率為0%,B、C兩組總死亡率分別為1.67%3.各組模型的總成栓率：A組小鼠成栓率為0%,B、C、D組總成栓率分別為69.4%、62.7%、83.3%4.不同時間點各組模型中的DVT長度變化情況：各組的DVT長度經單因素方差分析比較后顯示：第4天時,C組、D組的DVT長度和B組相比無明顯統(tǒng)計學差異,但C組與D組之間比較時P=0.018,P0.05,兩組之間存在統(tǒng)計學差異；第7天時,B、C、D各組間兩兩比較無統(tǒng)計學差異；第10天時,C、D組的DVT長度和正常組相比無明顯統(tǒng)計學差異, C組與D組之間比較時P=0.003,P0.05,兩組之間存在統(tǒng)計學差異；第13天時,B、C、D各組間兩兩比較均存存明顯的統(tǒng)計學差異,尤其是C組和D組之間,P0.001。最后,該實驗比較了各組第4天到第13天的DVT長度的平均值減少比例,結果顯示,B、C、D組DVT長度平均減少量分別為2.19mm、2.74mm、2.16mm,各組的減少比例分別為37.6%、49.5%和31.9%。5.不同時間點各組模型中的DVT濕重變化情況：各組的DVT濕重經單因素方差分析比較后顯示：第4天時,B、C組之間無明顯統(tǒng)計學差異,P=0.719,但B、C組與D組之間存在明顯統(tǒng)計學差異,P值分別為0.013和0.01,均小于0.05；第7天時,B組與C、D組之間比較無統(tǒng)計學差異,而C組和D組之間比較,P=0.019,兩組之間DVT濕重存在明顯統(tǒng)計學差異；第10天時,B組和C組之間無明顯統(tǒng)計學差異,P=0.365,B、C組與D組之間存在明顯統(tǒng)計學差異,P值分別為0.008和0.001,第13天時,第13天時B、C、D組之間兩兩比較均存在明顯的統(tǒng)計學差異,P值均0.001。最后,該實驗比較了了各組第4天到第13天的DVT濕重的平均值減少比例,結果顯示,B、C、D組DVT濕重平均減少量分別為7.1mg、8.14mg、6.2mg,各組的減少比例分別為55.5%、72.0%和41.1%。6.各組各時間點的DVT病理學改變：A組中均未見DVT形成。B、C、D分別于術后第7、10、13天取材,有DVT形成小鼠的下腔靜脈內可見紅白相間的固體質塊,即血栓,且隨著術后飼養(yǎng)的時間推移,DVT的長度逐漸變短,直徑也逐漸變細。切片行HE染色后在顯微鏡下可見：隨著時間推移,各組DVT機化面積逐漸曾加,機化的血栓邊緣與血管壁之間存在粘連。第13天時可見,C組DVT已全部機化,并且可見DVT內有血管樣結構形成,且可見機化區(qū)域有大量的裂隙產生；C組與B組相比,DVT內炎癥細胞明顯較少,且B組可見DVT機化面積已經超過橫截面積的一半,而D組其DVT機化面積明顯較小,機化面積僅接近DVT橫截面積的一半。實驗二：不同時間點各組小鼠DVT模型靜脈壁組織中HO-1、VEGF和SDF-1的mRNA表達變化PCR結果以A組為空白對照組,檢測各組HO-1、VEGF和SDF-1的mRNA的相對表達量,結果經統(tǒng)計學分析后得出,在DVT形成后第4、7、10、13天,B、C、D組的HO-1的表達均存在顯著統(tǒng)計學差異,兩兩之間比較,P0.05；相應的,第4、7、10天VEGF的表達也均存在統(tǒng)計學差異,P0.05,第13天時B、C組之間無統(tǒng)計學差異,P0.05,B、C與D組之間比較P0.05,存在統(tǒng)計學差異；第4、7、10、13天,B、C、D組的SDF-1的表達均存在顯著統(tǒng)計學差異,兩兩之間比較,P0.05。[結論]1.異氟烷在小鼠麻醉過程中安全有效,誘導快,復蘇快,不增加動物模型死亡率。2.下腔靜脈狹窄法能夠實現DVT機化再通模型的構建；3.HO-1在血栓形成過程中具有保護作用；在DVT溶解中后期HO-1可能通過誘導VEGF和SDF-1高表達促進DVT再通。
[Abstract]:[Objective]1. to construct the DVT repass model by isoflurane anesthesia and inferior vena cava stenosis method; use the heme oxygenase -1 agonist (CoPPIX) and inhibitor (SnPPIX) to intervene the dissolution and repassage process of DVT in C57 mice, and observe the mortality, thrombus rate, the length of inferior vena cava thrombus, wet weight and pathological section of the mice DVT model in each group. The variation of.2. in the inferior vena cava and thrombus in C57 mice was detected by.2., and the expression of VEGF and SDF-1 were detected. The effect and relationship of HO-1, VEGF and SDF-1 in the middle and late stages of DVT dissolution. [materials and methods] Experiment 1: the inferior vena cava stenosis method was used to construct DVT model of C57 mice and observe HO-1 agonists and inhibitors. 1. experimental group and DVT model were constructed: 200 C57 mice, weight 25-30g, female. According to the principle of random grouping, A group (blank control group, n=20), group B (normal DVT group, n=60), C group (CoPPIX intervention group, n=60) group and D group The same amount of physiological saline. After 24 hours of administration, B, C, D used isoflurane anesthesia combined with the inferior vena cava stenosis method to construct DVT model.2. material: after 4,7,10,13 days after the model, the cervical dislocations were taken to kill the mice, the vernier caliper was used to measure the thrombus length, and the inferior vena cava and its contents between the ligation point and the general iliac vein were taken down, each time was taken. 3 DVT specimens of mice in each group were randomly selected to be soaked with 4% polyformaldehyde and preserved for pathological examination. The remaining specimens were separated from the thrombus and venous wall, and the thrombus was weighed in the blood vessels. The venous wall and the contents of the contents were measured and analyzed by the.3. index of -80 degrees after the separation of the venous walls and contents: the mortality, the thrombus rate, the length of thrombus and the length of the thrombus were statistically analyzed. The change of wet weight. The statistical software used SPSS19.0, the measurement data used mean standard deviation (x + s); the same time point, 22 comparison between the groups using single factor analysis of variance, the LSD method (least square method), *p0.05 has statistical significance. Experiment two: HO-1, VEGF and SDF-1 mRNA in the DVT model of each group of mice at different time points. The 1.Trizol method was used to extract the total RNA in the inferior vena cava of each group of mice at different time points, and RT-PCR was used to reverse the reverse transcription of RNA in each group of mice to cDNA, and GAPDH as the internal reference. The Real-time PCR method was used to detect the changes of HO-1, VEGF and SDF-1 mRNA at different time points. Statistical analysis was used for statistical analysis. Mean standard deviation standard deviation (x + s); the same time point, 22 comparison between groups using single factor analysis of variance, LSD method (least square method), *p0.05 has statistical significance. [results] Experiment 1: inferior vena cava stenosis method to construct DVT model of C57 mice and observe the effect of HO-1 agonist and inhibitor on DVT mechanical repassage process of DVT: 1. anesthesia effect: experimental opening 3%-5% isoflurane was used for rapid induction of C57 mice. The 0.5%-1.0% concentration of isoflurane could maintain a satisfactory anesthetic effect. The induction of anesthesia satisfaction time and postoperative waking time were very fast, about 1-2min. The biggest adverse reaction in the anesthetic process was repression, but stopped anaesthetized or increased air flow. After the respiratory inhibition, the survival and mortality of.2. mice were quickly relieved: the mortality of A, D two groups was 0%, B, and the total mortality of C two groups was the total thrombus rate of each group of 1.67%3. models, respectively: the thrombus rate of the A group was 0%, B, C, and D group was 69.4%, 62.7%, and 83.3%4. in the different time points of each group were changed to the length of DVT length. Condition: the DVT length of each group was compared with the single factor analysis of variance. At fourth days, there was no significant difference in the length of DVT in group C and group D compared with B group, but there was a statistical difference between the C group and the D group in P=0.018, P0.05 and two groups; at seventh days, B, C, and there was no statistical difference between the groups of D, and tenth days. There was no significant difference in length compared with the normal group. There was a statistical difference between group C and group D, P=0.003, P0.05, and two groups. At thirteenth days, B, C, and D were statistically different among 22 groups, especially between the C group and the D group, P0.001. last, and the experiment compared the DVT length of each group from fourth to thirteenth days. The average decrease in the mean value of the B, C and D groups was 2.19mm, 2.74mm, 2.16mm, respectively, and the decrease ratio of each group was 37.6%, 49.5% and 31.9%.5. at different time points, respectively. The wet weight of DVT in each group of different time points of each group showed that the wet weight of DVT in each group was compared with one factor analysis of variance: fourth days, B, no obvious series between C groups. There were significant differences in P=0.719, but there were significant statistical differences between B, C group and D group, P value was 0.013 and 0.01, respectively less than 0.05. At seventh days, there was no statistical difference between group B and C, D group, and C group and D group, P=0.019, there was significant difference between the two groups. Tenth days, there was no significant statistics between the group and the group. There were significant differences between P=0.365, B, C group and D group, P values were 0.008 and 0.001 respectively. At thirteenth days, thirteenth days, B, C, D groups were all statistically significant differences, and P values were all 0.001. last. The experiment compared the average value of DVT wet weight in each group from fourth days to thirteenth days. The average decrease in wet weight of VT was 7.1mg, 8.14mg, 6.2mg. The reduction ratio of each group was 55.5%, 72% and 41.1%.6. in each group. The DVT pathological changes of each time point were found in group A: no DVT formed.B, C, D were taken from the postoperative 7,10,13 days respectively. With the passage of time after operation, the length of DVT gradually became shorter and the diameter was gradually thinning. The slice line HE staining was visible under the microscope: as time went on, the area of DVT in each group gradually increased, and there was a adhesion between the edge of the thrombus and the wall of the blood vessel. The DVT of group C was all computerized in the thirteenth day, and there was a vascular like in DVT. The structure was formed, and there was a large number of cracks in the visible area. Compared with the B group, the C group was obviously less inflammatory cells in DVT, and the area of DVT in the B group was more than half of the cross section area, while the DVT area of the D group was smaller and the area was only half of the DVT cross section. Experiment two: mice DVT at different time points. The mRNA expression changes of HO-1, VEGF and SDF-1 in the venous wall tissue of the model were compared with the A group as the blank control group, and the relative expression of HO-1, VEGF and SDF-1 mRNA in each group was detected. The results were statistically analyzed after statistical analysis, and there were significant differences in the expression of the 4,7,10,13 days after the formation of DVT. 22 Correspondingly, there were statistical differences in the expression of VEGF in day 4,7,10. P0.05, B at thirteenth days, there was no statistical difference between group C and P0.05, B, C and D, and there were significant statistical differences between P0.05, C and D group. In the process of intoxication, it is safe, effective, fast inducement, rapid recovery, no increase of animal model mortality,.2. inferior vena cava stenosis method can realize the construction of DVT repass model, 3.HO-1 has protective effect in the process of thrombosis, and HO-1 may promote DVT repass by inducing the high expression of VEGF and SDF-1 in the middle and late stages of DVT dissolution.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R543.6

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