OSAS模式间歇低氧大鼠PMN凋亡及与血管内皮细胞交互影响机制的研究

发布时间：2018-07-10 12:20

本文选题：阻塞性睡眠呼吸暂停综合征 + 慢性间歇低氧　；参考：《天津医科大学》2014年博士论文

【摘要】：OSAS是一种以慢性间歇低氧(CIH)为核心病理生理学基础伴发心、脑血管等多系统损伤的慢性疾病。CIH可以诱发一系列炎症-氧化应激反应和机体细胞凋亡周期的改变,影响细胞功能及与周边细胞间的相互作用,导致器官损伤及功能异常。血管内皮损伤是OSAS相关血管性疾病如高血压、冠心病、脑卒中等的基本病理损伤基础。外周血细胞是和血管内皮细胞接触及相互作用最主要的体液成分。PMN是重要的炎症细胞,其生存周期、功能状态及与血管内皮细胞的交互作用直接影响血管内皮的功能。本研究从不同间歇低氧暴露时间对PMN凋亡及其以不同方式与血管内皮细胞相互作用对细胞间粘附和内皮凋亡的影响和抗氧化剂Tempol干预的角度进一步探讨OSAS相关血管性并发症的病理生理机制,从另一视角为OSAS的临床诊疗提供新的策略和试验依据。内容1.研究不同间歇低氧暴露时间对大鼠PMN凋亡的影响 2.研究间歇低氧大鼠PMN与血管内皮细胞在直接共培养与transwell间接共培养相互作用下,对细胞间粘附指标ICAM-1、E-选择素浓度及内皮细胞凋亡指标bcl-2/bax、caspase-3的影响及抗氧化剂Tempol的干预效果方法第一部分：48只成年雄性Wistar大鼠随机分为常氧(NIH)和间歇低氧(IH)4周组、6周组、8周组,每组8只；暴露结束,腹主动脉取血,双层Ficoll-Histopaque双密度梯度离心法分离PMN。流式细胞技术Annexin-V和7-AAD双标法检测PMN凋亡率。第二部分：32只大鼠随机分为NIH组、IH组、间歇低氧生理盐水干预组(IHN组)、间歇低氧Tempol干预组(IHT组),暴露6周后腹主动脉取血分离PMN,分别与大鼠主动脉内皮细胞行直接共培养和transwell间接共培养4小时,ELISA法测定上清液ICAM-1和E-选择素浓度；内皮细胞分为：与NIH组PMN直接共培养内皮(NIHE)组和间接共培养内皮(T-NIHE)组,与IH组PMN直接共培养内皮(IHE)组和间接共培养内皮(T-IHE)组,与IHN组PMN直接共培养内皮(IHNE)组和间接共培养内皮(T-IHNE)组和与IHT组PMN直接共培养内皮(NIHE)组和间接共培养内皮(T-NIHE)组,免疫印记(Western blot)法测定内皮细胞bcl-2、bax、caspase-3表达。结果第一部分： 1.IH组大鼠PMN凋亡率低于NIH组：4周NIH组vs IH组(22.35±2.26vs16.23±3.00；P0.05)；6周NIH组vs IH组(23.56±1.26vs16.71±1.97;P0.05)；8周NIH组vs IH (25.48±1.93vs20.16±1.11; P0.05)。 2.随着IH时间的延长,PMN凋亡率有上升趋势：NIH组4周、6周、8周组间存在差异(F=4.297,P=0.033),8周组高于4周组(P=0.011),IH组4周、6周、8周组间存在差异(F=5.845,P=0.013),8周组高于4周组(P=0.007)和6周组(P=0.015)。第二部分： 1.大鼠PMN与大鼠主动脉内皮细胞直接共培养对细胞间粘附和内皮细胞凋亡的影响 1)直接共培养上清液ICAM-1浓度升高,IHE组、IHNE组(P0.001)和IHTE组(P=0.023)高于NIHE组；E-选择素浓度升高,IHE组、IHNE组(P0.001)高于NIHE组,IHTE组低于IHE组(P0.001)和IHNE组,与NIHE组无差别(P=0.557) 2)直接共培养内皮细胞Bcl-2表达增加,IHE组、IHNE组(p0.001)和IHTE组(P=0.029)高于NIHE组；Bax表达增高,IHE组、IHNE组(P0.001)高于NIHE组; Bcl-2/Bax比值降低,IHE组、IHNE组(P0.001)和IHTE组(P=0.014)低于NIHE组；Caspase-3表达升高,IHE组、IHNE组(P0.001)高于NIHE组, 2.大鼠PMN与大鼠主动脉内皮细胞transwell间接共培养对细胞间粘附和内皮细胞凋亡的影响 1) transwell下室上清液ICAM-1、E选择素浓度升高,T-IHE组、T-IHNE组高于T-NIHE组(P0.001), T-IHTE组低于T-IHE组和T-IHNE组(P0.05),与T-NIHE组无差别(P0.05) 2)间接共培养内皮细胞Bcl-2表达增加,T-IHE组(P=0.001)和T-IHNE组(P0.0001)高于T-NIHE组,T-IHTE组与T-NIHE组无差别(P=0.649)；Bax表达增加,T-IHE组、T-IHNE组(P0.0001)和T-IHTE组(P=0.009)高于T-NIHE组；Bcl-2/Bax比值降低,T-IHE组、T-IHNE组(P0.0001)和T-IHTE组(P=0.001)低于T-NIHE组；Caspase-3表达增高,T-IHE组、T-IHNE组(P0.0001)高于T-NIHE组,T-IHTE与T-NIHE组无差别(P=0.602) 3.直接共培养组的ICAM-1、E选择素、Bcl-2、Bax、Caspase-3均高于间接共培养组,P均0.05; Bcl-2/Bax比值低于间接共培养组,P均0.05 结论 1.不同间歇低氧暴露时间均会引起PMN凋亡延迟；随着间歇低氧暴露时间的延长,PMN凋亡有增加趋势。 2.间歇低氧大鼠PMN与内皮细胞直接共培养与间接共培养均可引起粘附作用增强,直接共培养增强程度重于间接共培养。 3.间歇低氧大鼠PMN与内皮细胞直接共培养与间接共培养均促进内皮细胞凋亡,直接共培养重于间接共培养。 4.抗氧化剂Tempol可部分改善PMN对内皮细胞的粘附和致凋亡损伤作用；控制粘附作用后效果更加明显。 5.抗粘附和抗氧化,克服炎症细胞凋亡延迟、保护内皮细胞等措施是OSAS模式间歇低氧血管相关性并发症防治的重要策略。
[Abstract]:OSAS is a chronic disease with chronic intermittent hypoxia (CIH) as the core pathophysiological basis. Chronic diseases such as cerebral vascular injury,.CIH can induce a series of inflammatory oxidative stress reactions and changes in cell apoptosis cycle, affect cell function and interaction with peripheral cells, resulting in organ damage and dysfunction. Vascular endothelial injury is the basic pathological damage basis of OSAS related vascular diseases such as hypertension, coronary heart disease and cerebral pawns. Peripheral blood cells are the most important humoral components of contact and interaction with vascular endothelial cells,.PMN is an important inflammatory cell, and its life cycle, function state and interaction with vascular endothelial cells are directly affected. The function of the vascular endothelial cells. This study further explored the pathophysiological mechanism of PMN related vascular complications from the angle of different intermittent hypoxia exposure and the effect of intercellular interaction on intercellular adhesion and endothelial apoptosis in different ways and vascular endothelial cell interaction and the intervention of antioxidant Tempol, from another perspective of OSAS. The clinical diagnosis and treatment provide a new strategy and experimental basis.
Content 1.. The effects of different intermittent hypoxia exposure time on PMN apoptosis in rats were studied.
2. study the interaction between PMN and vascular endothelial cells in intermittent hypoxic rats and vascular endothelial cells in direct co culture and indirect co culture of Transwell, the effect of ICAM-1, E- selectin concentration and bcl-2/bax, caspase-3 on the apoptosis index of endothelial cells and the effect of antioxidant Tempol on the effect of intercellular adhesion.
Method
The first part: 48 adult male Wistar rats were randomly divided into 4 weeks of normal oxygen (NIH) and intermittent hypoxia (IH), 6 weeks group and 8 week group, 8 in each group. The exposure ended, the abdominal aorta took blood, and the double density gradient centrifugation of double layer Ficoll-Histopaque was used to separate the PMN. flow cytometry, Annexin-V and 7-AAD double standard method to detect the apoptosis rate of PMN.
The second part: 32 rats were randomly divided into group NIH, group IH, intermittent hypoxic saline intervention group (group IHN), intermittent hypoxic Tempol intervention group (IHT group). After 6 weeks of exposure, the abdominal aorta was removed from the aorta, and PMN was extracted from the aorta, and the aorta endothelial cells were co cultured directly with the rat aorta and the indirect co culture of Transwell was 4 hours respectively. ELISA method was used to determine ICAM-1 and E- selection of the supernatant. Endothelial cells were divided into two groups: endothelial (NIHE) group and indirect co culture endothelium (T-NIHE) group with group NIH PMN, and IH group PMN directly co culture endothelial (IHE) group and indirect co culture endothelium (T-IHE) group. The endothelial (IHNE) group and indirect co culture endothelium (T-IHNE) group and indirect co culture endothelium (T-IHNE) group and co culture endothelial cells were co cultured directly with IHN group PMN. (NIHE) group and indirect co cultured endothelial (T-NIHE) group. The expression of Bcl-2, Bax and Caspase-3 in endothelial cells was detected by Western blot.
Result
Part one:
The apoptosis rate of PMN in group 1.IH was lower than that in group NIH: group vs IH (22.35 + 2.26vs16.23 + 3; P0.05) in group NIH for 4 weeks, and vs IH group (23.56 + 1.97, 1.97) in group NIH weeks, and 8 weeks (25.48 + 1.97 + 1.11;).
2. with the prolongation of IH time, the apoptosis rate of PMN had a rising trend: 4 weeks, 6 weeks and 8 weeks in group NIH (F=4.297, P=0.033), 8 weeks group higher than 4 weeks group (P=0.011), 4 weeks, 6 weeks and 8 weeks in group IH (F=5.845, P=0.013), 8 weeks group was higher than 4 week group (P= 0.007) and 6 weeks group (P=0.015).
The second part:
Effects of direct co culture of PMN and rat aortic endothelial cells on intercellular adhesion and endothelial cell apoptosis in 1. rats
1) the concentration of ICAM-1 in direct co culture supernatant was higher than that in group IHE, group IHNE (P0.001) and IHTE group (P=0.023) higher than that of NIHE group; E- selectin concentration increased, IHE group and IHNE group (P0.001) were higher than those of NIHE group.
2) the expression of Bcl-2 in direct co cultured endothelial cells increased, IHE group, IHNE group (p0.001) and IHTE group (P=0.029) were higher than those in NIHE group; Bax expression was higher, IHE group and IHNE group (P0.001) were higher than those of NIHE group;
Effects of indirect co culture of Transwell and PMN on intercellular adhesion and endothelial cell apoptosis in 2. rat aorta endothelial cells
1) Transwell inferior room supernatant ICAM-1, E selectin concentration increased, T-IHE group, T-IHNE group was higher than T-NIHE group (P0.001), T-IHTE group was lower than T-IHE group and T-IHNE group (P0.05), and no difference from T-NIHE group.
2) the expression of Bcl-2 in the indirect co cultured endothelial cells increased, the T-IHE group (P=0.001) and the T-IHNE group (P0.0001) were higher than the T-NIHE group, and there was no difference between the T-IHTE group and the T-NIHE group (P=0.649), the Bax expression increased, the T-IHE group, the T-IHNE group and the T-NIHE group were higher than those in the group; In group T-NIHE, Caspase-3 expression increased in group T-IHE, T-IHNE group (P0.0001) was higher than T-NIHE group, T-IHTE and T-NIHE group had no difference (P=0.602).
3. the ICAM-1, E selectin, Bcl-2, Bax and Caspase-3 in the direct co culture group were higher than those in the indirect co culture group, P 0.05, Bcl-2/Bax ratio lower than that in the indirect co culture group, P 0.05.
conclusion
1. different intermittent hypoxic exposure time will delay the apoptosis of PMN. With the prolongation of intermittent hypoxia exposure time, PMN apoptosis will increase.
2. the direct co culture and indirect co culture of PMN and endothelial cells in intermittent hypoxic rats can cause the enhancement of adhesion, and the degree of direct co culture enhancement is heavier than that of indirect co culture.
3. direct co culture and indirect co culture of PMN and endothelial cells in intermittent hypoxia rats promoted endothelial cell apoptosis, and direct co culture was more important than indirect co culture.
4. the antioxidant Tempol can partly improve the adhesion and apoptosis damage of PMN to endothelial cells, and the effect is more obvious after controlling adhesion.
5. anti adhesion and antioxidation, to overcome the delayed apoptosis of inflammatory cells, and to protect endothelial cells are important strategies for the prevention and treatment of complications associated with OSAS mode intermittent hypoxia.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R766

【参考文献】