不同缺血预处理方案对大鼠肢体缺血再灌注损伤保护效应的实验研究
发布时间：2018-07-20 14:14
【摘要】：目的缺血再灌注损伤在临床较为常见，防治措施众多，但多数效果不确切，本实验旨在探讨缺血预处理不同时间方案对肢体缺血再灌注损伤保护作用的差异性，选择合理的缺血预处理时间，为临床开展缺血预处理技术提供充足的理论依据。 方法取40只健康SPF级SD大鼠，随机分成5组（n=8）。术前禁食12h，禁水2h。腹腔10%水合氯醛麻醉后分离显露腹主动脉下段和下腔静脉，在肠系膜下动脉与双髂动脉分叉间用微血管夹阻断腹主动脉血流2h、复通血流2h为缺血再灌注损伤模型。假手术组（A组，仅行开腹，分离腹主动脉不阻断血流）；缺血再灌注组（B组，，夹闭腹主动脉缺血2h后再灌注2h）；C、D、E组：分别阻断腹主动脉1、5和10min，再灌注1、5和10min，如此重复3个循环后再进行2h缺血2h再灌注。各组均在实验程序结束复通血流后经下腔静脉采血34ml，3000r/min离心15min后-80℃保存。采用硫代巴比妥酸(TBA)法测定MDA浓度，黄嘌呤氧化酶法测定SOD活性，使用双抗体一步夹心酶联免疫吸附试验法（ELISA）测定血清超氧化物歧化酶（SOD）、丙二醛（MDA）、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、白细胞介素-10（IL-10）水平，观察各组氧化/抗氧化指标及炎症因子表达的差异性。 实验结果采用SPSS19.0统计软件进行分析，计量资料以x_±s表示，所有分组均进行K-S检验、方差齐性检验，组间两两比较采用LSD法单因素方差分析。 结果 1.与A组比较，B组的SOD活力下降，MDA含量升高，差异有统计学意义（P＜0.05）；与B组比较，C、D、E组的SOD活力升高，MDA含量下降，其中C组与B组比较的差异无统计学意义（P＞0.05），D、E组与B组比较差异有统计学意义（P＜0.05）；C、D、E进行组间比较，E组的SOD活力最高、MDA含量最低，C组SOD活力最低、MDA含量最高，各组间差异有统计学意义（P＜0.05）。 2.与A组比较，B组的IL-6、TNF-α、IL-10含量升高，差异均有统计学意义（P＜0.05）；与B组比较，C、D、E组的IL-6、TNF-α含量明显升高，差异均有统计学意义（P＜0.05）；C、D、E进行组间比较，各数值均呈递增趋势，IL-6的含量D组和C组比较、E组和D组比较差异均无统计学意义（P＞0.05）。TNF-α含量D组和C组差异无统计学意义（P＞0.05），E组和D组差异有统计学意义（P＜0.05）。IL-10含量各组间进行比较差异均有统计学意义（P＜0.05）。 结论 1.缺血预处理对缺血再灌注诱发的氧化损伤存在保护作用，在IprC1、5、10min/3个循环分组内显示IprC时间与其保护作用呈平行关系。 2.机体在缺血预处理时会发生炎症反应，IprC时间的增加促进了炎症因子的表达和蓄积，显示IprC在肢体缺血所致的炎症反应上无明显保护作用，甚至可增加肢体缺血最终造成全身性炎症反应。 3.本实验显示，1min/3个循环缺血预处理方案发挥的抗氧化损伤作用无统计学意义，诱导的炎症反应轻微。10min/3个循环方案抗氧化损伤作用最强，但是炎症反应最重，不适宜选用。5min/3个循环方案有明显的抗氧化损伤作用，炎症反应适中，综合来看临床选作缺血预处理策略较为适宜。
[Abstract]:Objective ischemia reperfusion injury is more common in clinical practice, and the prevention and treatment measures are numerous, but most of the effects are not accurate. The purpose of this experiment is to explore the difference in the protective effect of ischemic preconditioning on the protection of ischemia reperfusion injury of limb, and choose the reasonable time of ischemic preconditioning to provide sufficient theoretical basis for the development of ischemic preconditioning in bed. According to it.
Methods 40 healthy SPF grade SD rats were randomly divided into 5 groups (n=8). Before operation, 12h was fasted and the lower abdominal aorta and inferior vena cava were exposed after 2h. abdominal 10% hydrated chloral anaesthesia. The blood flow 2H was blocked by microvascular clamp between the inferior mesenteric artery and the double iliac artery, and the revascularised 2H was the model of ischemia reperfusion injury. The sham operation was performed. Group A (group B, isolated abdominal aorta without blocking blood flow), ischemia reperfusion group (group B, occlusion of abdominal aorta ischemia 2H reperfusion 2H); C, D, E group: block the abdominal aorta 1,5 and 10min, reperfusion 1,5 and 10min, then repeat the 3 cycles and repeat the 2H ischemia-reperfusion. The blood sampling of the inferior vena cava was 34ml, 3000r/min was preserved at -80 C after 15min. The concentration of MDA was measured by thiobarbituric acid (TBA), the activity of SOD was measured by xanthine oxidase method, and the serum superoxide dismutase (SOD), malondialdehyde (MDA), -6 (IL-6), and the cause of the tumor's bad death were determined by the double antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sub - alpha (TNF- alpha) and interleukin -10 (IL-10) levels were observed to observe the difference of oxidation / antioxidant index and inflammatory factors expression in each group.
The experimental results were analyzed with SPSS19.0 statistical software, and the measurement data were expressed in x_ + s. All the groups were tested with K-S test, variance homogeneity test, and 22 of groups were compared by LSD single factor analysis of variance.
Result
1. compared with the A group, the activity of SOD in the B group decreased and the MDA content increased, and the difference was statistically significant (P < 0.05). Compared with the B group, the SOD activity of C, D and E decreased, and the difference between the C group and the group was not statistically significant (0.05). D activity was the highest, MDA content was the lowest, C group SOD activity was lowest, MDA content was the highest, the difference was statistically significant among all groups (P < 0.05).
2. compared with the A group, the content of IL-6, TNF- a, IL-10 in group B increased, the difference was statistically significant (P < 0.05). Compared with the B group, C, D, E group were significantly higher, the difference was statistically significant (0.05). There was no statistical significance (P > 0.05).TNF- alpha content in D group and C group (P > 0.05), and there was significant difference between group E and D group (P < 0.05), and there was significant difference between each group (P < 0.05).
conclusion
1. ischemic preconditioning has a protective effect on the oxidative damage induced by ischemia-reperfusion, and the IprC time in the IprC1,5,10min/3 cycle grouping is parallel to its protective effect.
The 2. body will have an inflammatory response during ischemic preconditioning. The increase of IprC time promotes the expression and accumulation of inflammatory factors. It shows that IprC has no obvious protective effect on the inflammatory response to limb ischemia, and may even increase limb ischemia and eventually cause systemic inflammatory response.
3. the experiment showed that the anti oxidative damage effect of 1min/3 cyclic ischemic preconditioning regimen was not statistically significant. The mild.10min/3 cycle regimen induced by the induced inflammatory reaction was the strongest, but the inflammatory reaction was the heaviest. The.5min/3 cycle scheme was not suitable for anti oxidative damage. The inflammatory reaction was moderate, and the inflammatory reaction was moderate. In conclusion, the ischemic preconditioning strategy is more suitable.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R54
本文编号：2133802
[Abstract]:Objective ischemia reperfusion injury is more common in clinical practice, and the prevention and treatment measures are numerous, but most of the effects are not accurate. The purpose of this experiment is to explore the difference in the protective effect of ischemic preconditioning on the protection of ischemia reperfusion injury of limb, and choose the reasonable time of ischemic preconditioning to provide sufficient theoretical basis for the development of ischemic preconditioning in bed. According to it.
Methods 40 healthy SPF grade SD rats were randomly divided into 5 groups (n=8). Before operation, 12h was fasted and the lower abdominal aorta and inferior vena cava were exposed after 2h. abdominal 10% hydrated chloral anaesthesia. The blood flow 2H was blocked by microvascular clamp between the inferior mesenteric artery and the double iliac artery, and the revascularised 2H was the model of ischemia reperfusion injury. The sham operation was performed. Group A (group B, isolated abdominal aorta without blocking blood flow), ischemia reperfusion group (group B, occlusion of abdominal aorta ischemia 2H reperfusion 2H); C, D, E group: block the abdominal aorta 1,5 and 10min, reperfusion 1,5 and 10min, then repeat the 3 cycles and repeat the 2H ischemia-reperfusion. The blood sampling of the inferior vena cava was 34ml, 3000r/min was preserved at -80 C after 15min. The concentration of MDA was measured by thiobarbituric acid (TBA), the activity of SOD was measured by xanthine oxidase method, and the serum superoxide dismutase (SOD), malondialdehyde (MDA), -6 (IL-6), and the cause of the tumor's bad death were determined by the double antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sub - alpha (TNF- alpha) and interleukin -10 (IL-10) levels were observed to observe the difference of oxidation / antioxidant index and inflammatory factors expression in each group.
The experimental results were analyzed with SPSS19.0 statistical software, and the measurement data were expressed in x_ + s. All the groups were tested with K-S test, variance homogeneity test, and 22 of groups were compared by LSD single factor analysis of variance.
Result
1. compared with the A group, the activity of SOD in the B group decreased and the MDA content increased, and the difference was statistically significant (P < 0.05). Compared with the B group, the SOD activity of C, D and E decreased, and the difference between the C group and the group was not statistically significant (0.05). D activity was the highest, MDA content was the lowest, C group SOD activity was lowest, MDA content was the highest, the difference was statistically significant among all groups (P < 0.05).
2. compared with the A group, the content of IL-6, TNF- a, IL-10 in group B increased, the difference was statistically significant (P < 0.05). Compared with the B group, C, D, E group were significantly higher, the difference was statistically significant (0.05). There was no statistical significance (P > 0.05).TNF- alpha content in D group and C group (P > 0.05), and there was significant difference between group E and D group (P < 0.05), and there was significant difference between each group (P < 0.05).
conclusion
1. ischemic preconditioning has a protective effect on the oxidative damage induced by ischemia-reperfusion, and the IprC time in the IprC1,5,10min/3 cycle grouping is parallel to its protective effect.
The 2. body will have an inflammatory response during ischemic preconditioning. The increase of IprC time promotes the expression and accumulation of inflammatory factors. It shows that IprC has no obvious protective effect on the inflammatory response to limb ischemia, and may even increase limb ischemia and eventually cause systemic inflammatory response.
3. the experiment showed that the anti oxidative damage effect of 1min/3 cyclic ischemic preconditioning regimen was not statistically significant. The mild.10min/3 cycle regimen induced by the induced inflammatory reaction was the strongest, but the inflammatory reaction was the heaviest. The.5min/3 cycle scheme was not suitable for anti oxidative damage. The inflammatory reaction was moderate, and the inflammatory reaction was moderate. In conclusion, the ischemic preconditioning strategy is more suitable.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R54
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