HO-1诱导静脉内皮细胞VEGF、SDF-1表达促进DVT机化再通的研究
发布时间：2018-07-17 17:48
【摘要】：[目的]1.利用异氟烷麻醉方法和下腔静脉狭窄法构建DVT机化再通模型；使用血红素加氧酶-1的激动剂(CoPPIX)及抑制剂(SnPPIX)干预C57小鼠DVT的溶解再通过程,并观察各组小鼠DVT模型的死亡率、成栓率,下腔静脉血栓的长度、湿重和病理切片的变化差异。2.检测C57小鼠下腔静脉壁和血栓的HO-1、VEGF和SDF-1的表达情况,探讨HO-1、 VEGF和SDF-1在DVT溶解中后期的作用及相互关系。[材料与方法]实验一：下腔静脉狭窄法构建C57小鼠DVT模型及观察HO-1激动剂及抑制剂对DVT机化再通过程的影响1.实验分组及DVT模型的构建：C57小鼠200只,体重25-30g,雌性。按随机分组原则分A组(空白对照组,n=20)、B组(正常DVT组,n=60)、C组(CoPPIX干预组,n=60)组和D组(SnPPIX干预组,n=60)。C组和D组在造模前24h分别腹腔注射CoPPIX和SnPPXI,5mg/kg; B组腹腔注射等量的生理盐水。给药24小时后B、C、D采用异氟烷麻醉配合下腔静脉狭窄法构建DVT模型。2.取材：在造模后4、7、10、13天,采取颈椎脱臼法处死小鼠,采用游标卡尺测量血栓长度,取下结扎点至髂总静脉汇合处之间下腔静脉及其内容物,每个时间点每组中随机取3只小鼠DVT标本,使用4%多聚甲醛浸泡固定,保存行病理检查；其余标本分离血栓与静脉壁,称取血管内的血栓湿重,静脉壁和内容物分装后-80°保存备用。3.指标测定和统计学分析：统计每组死亡率、成栓率、血栓长度和湿重变化情况。统计学软件采用SPSS19.0,计量资料采用均数±标准差(x±s)；同一时间点,组间两两比较采用单因素方差分析、用LSD法(最小二乘法),*p0.05有统计学意义。实验二：不同时间点各组小鼠DVT模型静脉壁组织中HO-1、VEGF和SDF-1的mRNA达变化1.Trizol法提取每组小鼠不同时间点下腔静脉中总RNA, RT-PCR将各组小鼠静脉组织RNA逆转录为cDNA;以GAPDH为内参照,用Real-time PCR法检测不同时间点HO-1、VEGF和SDF-1 mRNA的表达变化；2.统计学分析：统计学软件采用SPSS19.0o计量资料采用均数±标准差(x±s)；同一时间点,组间两两比较采用单因素方差分析,用LSD法(最小二乘法),*p0.05有统计学意义。[结果]实验一：下腔静脉狭窄法构建C57小鼠DVT模型及观察HO-1激动剂及抑制剂对DVT机化再通过程的影响1.麻醉效果：实验开始使用3%-5%异氟烷对C57小鼠进行快速诱导,术中使用0.5%-1.0%浓度的异氟烷能维持满意的麻醉效果。诱导达到麻醉满意时间和术后清醒时间均很快,约1-2min,麻醉过程中小鼠出现的最大不良反应为呼吸抑制,但停止麻醉药或加大空气流量后,小鼠呼吸抑制的情况可迅速缓解.2.小鼠存活情况和死亡率：A、D两组死亡率为0%,B、C两组总死亡率分别为1.67%3.各组模型的总成栓率：A组小鼠成栓率为0%,B、C、D组总成栓率分别为69.4%、62.7%、83.3%4.不同时间点各组模型中的DVT长度变化情况：各组的DVT长度经单因素方差分析比较后显示：第4天时,C组、D组的DVT长度和B组相比无明显统计学差异,但C组与D组之间比较时P=0.018,P0.05,两组之间存在统计学差异；第7天时,B、C、D各组间两两比较无统计学差异；第10天时,C、D组的DVT长度和正常组相比无明显统计学差异, C组与D组之间比较时P=0.003,P0.05,两组之间存在统计学差异；第13天时,B、C、D各组间两两比较均存存明显的统计学差异,尤其是C组和D组之间,P0.001。最后,该实验比较了各组第4天到第13天的DVT长度的平均值减少比例,结果显示,B、C、D组DVT长度平均减少量分别为2.19mm、2.74mm、2.16mm,各组的减少比例分别为37.6%、49.5%和31.9%。5.不同时间点各组模型中的DVT湿重变化情况：各组的DVT湿重经单因素方差分析比较后显示：第4天时,B、C组之间无明显统计学差异,P=0.719,但B、C组与D组之间存在明显统计学差异,P值分别为0.013和0.01,均小于0.05；第7天时,B组与C、D组之间比较无统计学差异,而C组和D组之间比较,P=0.019,两组之间DVT湿重存在明显统计学差异；第10天时,B组和C组之间无明显统计学差异,P=0.365,B、C组与D组之间存在明显统计学差异,P值分别为0.008和0.001,第13天时,第13天时B、C、D组之间两两比较均存在明显的统计学差异,P值均0.001。最后,该实验比较了了各组第4天到第13天的DVT湿重的平均值减少比例,结果显示,B、C、D组DVT湿重平均减少量分别为7.1mg、8.14mg、6.2mg,各组的减少比例分别为55.5%、72.0%和41.1%。6.各组各时间点的DVT病理学改变：A组中均未见DVT形成。B、C、D分别于术后第7、10、13天取材,有DVT形成小鼠的下腔静脉内可见红白相间的固体质块,即血栓,且随着术后饲养的时间推移,DVT的长度逐渐变短,直径也逐渐变细。切片行HE染色后在显微镜下可见：随着时间推移,各组DVT机化面积逐渐曾加,机化的血栓边缘与血管壁之间存在粘连。第13天时可见,C组DVT已全部机化,并且可见DVT内有血管样结构形成,且可见机化区域有大量的裂隙产生；C组与B组相比,DVT内炎症细胞明显较少,且B组可见DVT机化面积已经超过横截面积的一半,而D组其DVT机化面积明显较小,机化面积仅接近DVT横截面积的一半。实验二：不同时间点各组小鼠DVT模型静脉壁组织中HO-1、VEGF和SDF-1的mRNA表达变化PCR结果以A组为空白对照组,检测各组HO-1、VEGF和SDF-1的mRNA的相对表达量,结果经统计学分析后得出,在DVT形成后第4、7、10、13天,B、C、D组的HO-1的表达均存在显著统计学差异,两两之间比较,P0.05；相应的,第4、7、10天VEGF的表达也均存在统计学差异,P0.05,第13天时B、C组之间无统计学差异,P0.05,B、C与D组之间比较P0.05,存在统计学差异；第4、7、10、13天,B、C、D组的SDF-1的表达均存在显著统计学差异,两两之间比较,P0.05。[结论]1.异氟烷在小鼠麻醉过程中安全有效,诱导快,复苏快,不增加动物模型死亡率。2.下腔静脉狭窄法能够实现DVT机化再通模型的构建；3.HO-1在血栓形成过程中具有保护作用；在DVT溶解中后期HO-1可能通过诱导VEGF和SDF-1高表达促进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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R543.6
本文编号：2130457
[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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R543.6
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