本文選題：阻塞性睡眠呼吸暫停綜合征 + 慢性間歇低氧��； 參考：《天津醫(yī)科大學(xué)》2014年博士論文

【摘要】：OSAS是一種以慢性間歇低氧(CIH)為核心病理生理學(xué)基礎(chǔ)伴發(fā)心、腦血管等多系統(tǒng)損傷的慢性疾病。CIH可以誘發(fā)一系列炎癥-氧化應(yīng)激反應(yīng)和機體細胞凋亡周期的改變,影響細胞功能及與周邊細胞間的相互作用,導(dǎo)致器官損傷及功能異常。血管內(nèi)皮損傷是OSAS相關(guān)血管性疾病如高血壓、冠心病、腦卒中等的基本病理損傷基礎(chǔ)。外周血細胞是和血管內(nèi)皮細胞接觸及相互作用最主要的體液成分。PMN是重要的炎癥細胞,其生存周期、功能狀態(tài)及與血管內(nèi)皮細胞的交互作用直接影響血管內(nèi)皮的功能。本研究從不同間歇低氧暴露時間對PMN凋亡及其以不同方式與血管內(nèi)皮細胞相互作用對細胞間粘附和內(nèi)皮凋亡的影響和抗氧化劑Tempol干預(yù)的角度進一步探討OSAS相關(guān)血管性并發(fā)癥的病理生理機制,從另一視角為OSAS的臨床診療提供新的策略和試驗依據(jù)。 內(nèi)容1.研究不同間歇低氧暴露時間對大鼠PMN凋亡的影響 2.研究間歇低氧大鼠PMN與血管內(nèi)皮細胞在直接共培養(yǎng)與transwell間接共培養(yǎng)相互作用下,對細胞間粘附指標ICAM-1、E-選擇素濃度及內(nèi)皮細胞凋亡指標bcl-2/bax、caspase-3的影響及抗氧化劑Tempol的干預(yù)效果 方法 第一部分：48只成年雄性Wistar大鼠隨機分為常氧(NIH)和間歇低氧(IH)4周組、6周組、8周組,每組8只；暴露結(jié)束,腹主動脈取血,雙層Ficoll-Histopaque雙密度梯度離心法分離PMN。流式細胞技術(shù)Annexin-V和7-AAD雙標法檢測PMN凋亡率。 第二部分：32只大鼠隨機分為NIH組、IH組、間歇低氧生理鹽水干預(yù)組(IHN組)、間歇低氧Tempol干預(yù)組(IHT組),暴露6周后腹主動脈取血分離PMN,分別與大鼠主動脈內(nèi)皮細胞行直接共培養(yǎng)和transwell間接共培養(yǎng)4小時,ELISA法測定上清液ICAM-1和E-選擇素濃度；內(nèi)皮細胞分為：與NIH組PMN直接共培養(yǎng)內(nèi)皮(NIHE)組和間接共培養(yǎng)內(nèi)皮(T-NIHE)組,與IH組PMN直接共培養(yǎng)內(nèi)皮(IHE)組和間接共培養(yǎng)內(nèi)皮(T-IHE)組,與IHN組PMN直接共培養(yǎng)內(nèi)皮(IHNE)組和間接共培養(yǎng)內(nèi)皮(T-IHNE)組和與IHT組PMN直接共培養(yǎng)內(nèi)皮(NIHE)組和間接共培養(yǎng)內(nèi)皮(T-NIHE)組,免疫印記(Western blot)法測定內(nèi)皮細胞bcl-2、bax、caspase-3表達。 結(jié)果 第一部分： 1.IH組大鼠PMN凋亡率低于NIH組：4周NIH組vs IH組(22.35±2.26vs16.23±3.00；P0.05)；6周NIH組vs IH組(23.56±1.26vs16.71±1.97;P0.05)；8周NIH組vs IH (25.48±1.93vs20.16±1.11; P0.05)。 2.隨著IH時間的延長,PMN凋亡率有上升趨勢：NIH組4周、6周、8周組間存在差異(F=4.297,P=0.033),8周組高于4周組(P=0.011),IH組4周、6周、8周組間存在差異(F=5.845,P=0.013),8周組高于4周組(P=0.007)和6周組(P=0.015)。 第二部分： 1.大鼠PMN與大鼠主動脈內(nèi)皮細胞直接共培養(yǎng)對細胞間粘附和內(nèi)皮細胞凋亡的影響 1)直接共培養(yǎng)上清液ICAM-1濃度升高,IHE組、IHNE組(P0.001)和IHTE組(P=0.023)高于NIHE組；E-選擇素濃度升高,IHE組、IHNE組(P0.001)高于NIHE組,IHTE組低于IHE組(P0.001)和IHNE組,與NIHE組無差別(P=0.557) 2)直接共培養(yǎng)內(nèi)皮細胞Bcl-2表達增加,IHE組、IHNE組(p0.001)和IHTE組(P=0.029)高于NIHE組；Bax表達增高,IHE組、IHNE組(P0.001)高于NIHE組; Bcl-2/Bax比值降低,IHE組、IHNE組(P0.001)和IHTE組(P=0.014)低于NIHE組；Caspase-3表達升高,IHE組、IHNE組(P0.001)高于NIHE組, 2.大鼠PMN與大鼠主動脈內(nèi)皮細胞transwell間接共培養(yǎng)對細胞間粘附和內(nèi)皮細胞凋亡的影響 1) transwell下室上清液ICAM-1、E選擇素濃度升高,T-IHE組、T-IHNE組高于T-NIHE組(P0.001), T-IHTE組低于T-IHE組和T-IHNE組(P0.05),與T-NIHE組無差別(P0.05) 2)間接共培養(yǎng)內(nèi)皮細胞Bcl-2表達增加,T-IHE組(P=0.001)和T-IHNE組(P0.0001)高于T-NIHE組,T-IHTE組與T-NIHE組無差別(P=0.649)；Bax表達增加,T-IHE組、T-IHNE組(P0.0001)和T-IHTE組(P=0.009)高于T-NIHE組；Bcl-2/Bax比值降低,T-IHE組、T-IHNE組(P0.0001)和T-IHTE組(P=0.001)低于T-NIHE組；Caspase-3表達增高,T-IHE組、T-IHNE組(P0.0001)高于T-NIHE組,T-IHTE與T-NIHE組無差別(P=0.602) 3.直接共培養(yǎng)組的ICAM-1、E選擇素、Bcl-2、Bax、Caspase-3均高于間接共培養(yǎng)組,P均0.05; Bcl-2/Bax比值低于間接共培養(yǎng)組,P均0.05 結(jié)論 1.不同間歇低氧暴露時間均會引起PMN凋亡延遲；隨著間歇低氧暴露時間的延長,PMN凋亡有增加趨勢。 2.間歇低氧大鼠PMN與內(nèi)皮細胞直接共培養(yǎng)與間接共培養(yǎng)均可引起粘附作用增強,直接共培養(yǎng)增強程度重于間接共培養(yǎng)。 3.間歇低氧大鼠PMN與內(nèi)皮細胞直接共培養(yǎng)與間接共培養(yǎng)均促進內(nèi)皮細胞凋亡,直接共培養(yǎng)重于間接共培養(yǎng)。 4.抗氧化劑Tempol可部分改善PMN對內(nèi)皮細胞的粘附和致凋亡損傷作用；控制粘附作用后效果更加明顯。 5.抗粘附和抗氧化,克服炎癥細胞凋亡延遲、保護內(nèi)皮細胞等措施是OSAS模式間歇低氧血管相關(guān)性并發(fā)癥防治的重要策略。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R766

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