缺氧预适应对大鼠肺缺血再灌注损伤保护作用的研究

发布时间：2019-07-09 09:18

【摘要】： 第一部分 缺氧预适应对肺缺血再灌注损伤保护模型的建立 目的建立在体大鼠全身缺氧预适应模型,观察该模型是否对在体大鼠肺缺血再灌注损伤具有保护作用,并初步探讨其作用机制。 方法 24只雄性成年SD大鼠随机分为三组:对照组(Control),肺缺血再灌注组(I/R),缺氧预适应组(WHPC)。每组大鼠8只。I/R组予夹闭左侧肺门60 min后再灌注120 min复制原位肺缺血再灌注损伤模型;WHPC组大鼠置于密闭的常压缺氧箱内2 h建立全身缺氧预适应模型后,再按上述方法复制大鼠原位肺缺血再灌注损伤模型;Control组仅完成除夹闭肺门以外的手术操作。 处理完毕后分别观察各组大鼠肺组织形态学,测定肺组织湿干重比值及髓过氧化物酶(MPO)活性,并分别检测各组大鼠血浆中丙二醛(MDA)和超氧化物歧化酶(SOD)水平。 结果 (1)与Control组相比,I/R组大鼠肺组织湿干重比值、肺泡白细胞计数及肺泡损伤数、肺组织MPO活性均显著升高(P0.01);I/R组大鼠血浆MDA含量随着再灌注时间延长而逐渐升高,各时间点均高于Control组(P0.01);血浆SOD含量则随着再灌注时间延长而逐渐下降,各时间点均低于Control组(P0.01)。(2)与I/R组相比,WHPC组大鼠肺组织湿干重比值、肺泡白细胞计数及肺泡损伤数、肺组织MPO活性均明显改善(P0.01);血浆MDA含量较I/R组明显降低;血浆SOD含量则较I/R组明显升高(P0.01)。 结论 缺氧预适应对大鼠在体肺缺血再灌注损伤具有一定的保护作用,其机制可能与抑制肺缺血再灌后脂质过氧化反应减轻肺水肿、抑制炎症反应和中性粒细胞活化有关。 第二部分 缺氧预适应对肺缺血再灌注细胞凋亡的影响 目的 观察缺氧预适应对在体大鼠肺缺血再灌注细胞凋亡的影响,尤其是肺泡Ⅱ型上皮细胞凋亡的动态变化。 方法 缺氧预适应模型和大鼠原位肺缺血再灌注模型同第一部分。54只成年雄性SD大鼠随机分为三组,即对照组(Control组),肺缺血再灌注组(I/R组),缺氧预适应组(WHPC组),WHPC组和IfR组根据再灌注时间又分别再分为3个亚组,分别再灌注30分钟(P_1,R_1组)、60分钟(P_2,R_2组)和90分钟(P_3,R_3组)。Control组大鼠18只,其余每组大鼠6只。 处理结束后用TdT酶介导的dUTP缺口末端标记法(TUNEL)检测各组大鼠肺组织细胞凋亡情况,并计算出各组肺组织细胞凋亡指数;分离出缺血再灌注后的肺泡Ⅱ型上皮细胞,用流式细胞仪检测各组肺泡Ⅱ型上皮细胞凋亡率。 结果 (1)I/R组较Control组肺组织细胞凋亡指数明显升高(P0.05),并且随着再灌注时间的延长,细胞凋亡的数目逐渐增多;而WHPC组虽亦存在细胞凋亡的现象,但细胞凋亡指数较I/R组明显下降(P0.01)。(2)用流式细胞仪检测各组肺泡Ⅱ型上皮细胞凋亡率,结果显示control组肺泡Ⅱ型上皮细胞凋亡率很低,与之比较相同时间点的I/R组和WHPC组均有不同程度的增高,尤以I/R组升高明显(P0.01)。与I/R组相比较,各个时间点的WHPC组肺泡Ⅱ型上皮细胞凋亡率明显减少(P0.01)。 结论 (1)缺氧预适应能够减轻肺缺血再灌注细胞的凋亡。 (2)缺氧预适应可能通过减少肺泡Ⅱ型上皮细胞的凋亡对肺缺血再灌注损伤起保护作用。 第三部分 HIF-1α在缺氧预适应对肺保护中的作用机制 目的 探讨HIF-1α在缺氧预适应对在体肺保护中的作用及其机制,脯氨酸羟化酶的抑制剂DMOG能否模拟缺氧预适应对在体肺缺血再灌注损伤的延迟性保护作用。 方法 54只成年雄性SD大鼠随机分为五组,即空白对照组(Control组),生理盐水干预组(Saline组),缺氧预适应组(WHPC组),NS-398干预组(WHPC+NS组)和DMOG干预组(DMOG组)。缺氧预适应模型和大鼠原位肺缺血再灌注模型同第一部分。Saline组在缺血再灌注损伤模型建立前24h进行腹腔内注射生理盐水0.5ml;NS-398干预组在缺氧预适应前15分钟进行腹腔内注射NS-398(10mg/kg);DMOG组在缺血再灌注损伤模型建立前24h进行腹腔注射DMOG(20mg/kg)。 处理结束后用逆转录—聚合酶链反应检测各组大鼠肺组织中HIF-1αmRNA、HO-1 mRNA的表达;用蛋白免疫印记法检测各组大鼠肺组织中caspase-3蛋白的表达;用免疫组化法观察各组大鼠肺组织中HIF-1α、HO-1、caspase-3蛋白表达情况及表达部位,用TUNEL法检测肺组织细胞凋亡情况,并测定肺湿干重比值。 结果 (1)RT-PCR结果显示:各组大鼠肺组织内均有HIF-1αmRNA表达,但表达水平间无统计学差异。与Control组比较,其余各组肺组织中的HO-1 mRNA表达均显著升高(P0.01);而WHPC组和DMOG组与Saline组相比表达水平却是升高的;WHPC+NS组则与WHPC组相比表达有所下降(P0.05)。(2)免疫组化结果和caspaSe-3的western-blot结果显示:与Saline组比较,WHPC组和DMOG组肺组织中的HIF-1α和HO-1蛋白表达水平升高,caspase-3蛋白表达水平是下降的;WHPC+NS组则与WHPC组相比肺组织中HIF-1α和HO-1蛋白表达水平下降,caspase-3蛋白表达水平是上调的,其差异均具有统计学意义。(3)TUNEL和肺组织湿干重比值结果显示:与Saline组比较,WHPC组和DMOG组肺组织细胞凋亡指数减少,肺湿干重比值降低;WHPC+NS组与WHPC组相比则肺组织细胞凋亡指数和肺湿干重比值增高,其差异均具有统计学意义。 结论 (1)HIF-1α在缺氧预适应对在体大鼠肺缺血再灌注损伤的保护中起着重要作用。 (2)缺氧预适应通过诱导HIF-1α蛋白的稳定表达,诱导保护性靶基因HO-1的过表达,降低caspase-3的表达水平,从而减轻在体肺缺血再灌注损伤细胞凋亡的程度,起到对肺的保护作用。 (3)脯氨酸羟化酶抑制剂DMOG能够模拟缺氧预适应对在体肺缺血再灌注损伤的延迟性保护作用,NS-398能取消这种保护作用。 创新与意义 (1)本研究在建立大鼠左肺原位缺血再灌注模型的基础上,在国内首次研究了缺氧预适应对缺血再灌注肺损伤的保护作用。 (2)本研究观察了缺氧预适应对肺缺血再灌注后肺组织细胞凋亡的动态变化的影响,并首次在缺血再灌注后分离出肺泡Ⅱ型上皮细胞,运用流式细胞技术观察缺氧预适应对肺泡Ⅱ型上皮细胞凋亡率的影响。 (3)在研究缺氧预适应保护机制中,首次发现HIF-1α在缺氧预适应对在体肺缺血再灌注损伤保护作用中亦起着重要作用;HIF-1α蛋白的稳定表达,诱导保护性靶基因HO-1过表达,来降低caspase-3的蛋白表达水平,从而减轻在体肺缺血再灌注损伤细胞凋亡的程度,起到对肺的保护作用。 (4)本研究首次运用脯氨酸羟化酶抑制剂DMOG在大鼠左肺原位缺血再灌注模型上模拟缺氧预适应延迟性保护作用,可能为防治肺缺血再灌注损伤提供了新的治疗策略。不足 缺氧预适应的内源性保护性机制十分复杂,有些机制仍未阐明,因为经费和时间的限制,本研究只在在体动物模型上进行了其保护作用的观察和机制的研究,并未在体外模型上进行进一步论证。并且本研究只涉及了HIF-1在缺氧预适应保护机制中的作用,只观察了其靶基因HO-1的表达情况,目前研究表明其靶基因EPO,VEGF和NOS等亦在保护机制中亦起到一定作用。我们将在以后的研究中进一步深入和完善。
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图片说明：各组大鼠肺湿/干重比值
[Abstract]:the first part Protective model of hypoxic preconditioning on lung ischemia-reperfusion injury The purpose of this model was to establish an in-vivo hypoxic preconditioning model in rats, and to observe whether the model has a protective effect on the ischemia-reperfusion injury of the lung in the body. probe into Methods 24 male adult SD rats were randomly divided into three groups: control group (control), lung ischemia-reperfusion group (I/ C). R), Presuitable for hypoxia In group (WHPC),8 rats of each group were treated with 1/ R group to close the left lung valve for 60 min, and then the in-situ lung ischemia-reperfusion injury model was reperfused for 120 min; and the WHPC group rats were placed in a closed normal-pressure hypoxia box for 2 hours to establish a whole-body hypoxia pre-adaptive model, and then the rats were re-treated according to the above method. A model of in-situ lung ischemia-reperfusion injury in rats The lung tissue morphology, the dry weight ratio of the lung tissue and the activity of myeloperoxidase (MPO) were measured, and the malondialdehyde (MDA) in the plasma of each group was measured. MD Results (1) Compared with the control group, the ratio of dry weight and dry weight of lung tissue, the number of alveolar white blood cell count and the number of alveolar damage and MPO activity of the lung tissue were significantly increased (P0.01). The plasma MDA content in the I/ R group increased gradually with the time of reperfusion, and the time point was higher than that in the control group (P0.01), and the content of SOD in plasma increased with the time of reinfusion. Compared with the control group (P0.01), the ratio of wet and dry weight of lung tissue, the number of alveolar white cells and the number of alveolar damage and MPO activity in the lung of WHPC group were significantly improved (P0.01), and the content of MDA in plasma was significantly lower than that of group I/ R (P0.01). reduce Conclusion The hypoxic preconditioning has a certain protective effect on the ischemia-reperfusion injury of the rats, and the mechanism may be related to the inhibition. lung ischemia After the filling, the lipid peroxidation reaction is used to relieve the pulmonary edema and inhibit the inflammation. reaction The effect of the second partial hypoxia preconditioning on the apoptosis of the lung ischemia-reperfusion cells order Objective To observe the effects of hypoxic preconditioning on the apoptosis of rat lung ischemia-reperfusion cells, especially the dynamic changes of the apoptosis of the alveolar type II epithelial cells. 4 adult male SD rats were randomly divided into three groups: the control group (control group), the lung ischemia-reperfusion group (I/ R group), the hypoxic preconditioning group (WHPC group), the WHPC group and the IfR group according to the reperfusion time. Three sub-groups were divided into 3 subgroups, respectively, and reperfusion for 30 minutes (P _ 1). (R _ 1),60 minutes (P _ 2, R _ 2) and 90 minutes (P _ 3, R _ 3). Detection of lung tissue in each group by the end-end labeling (TUNEL) cell Results (1) The apoptosis index of lung cells in group I/ R group was significantly higher than that of control group (P0 (.05), and with the extension of reperfusion time, the number of cell apoptosis increased gradually, while the apoptosis index of WHPC was significantly lower than that in the I/ R group (P0.01). (2) The apoptosis rate of the alveolar type II epithelial cells in each group was detected by flow cytometry. The apoptosis rate of the control group alveolar type II epithelial cells is very low, and the I/ R group and WHPC of the same time point are compared with the control group. the group There was a different degree of increase, especially in the I/ R group (P0.05). 1) Compared with the I/ R group, the apoptosis rate of the type 鈪，

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