【摘要】： 第一部分 缺氧預(yù)適應(yīng)對肺缺血再灌注損傷保護模型的建立 目的建立在體大鼠全身缺氧預(yù)適應(yīng)模型,觀察該模型是否對在體大鼠肺缺血再灌注損傷具有保護作用,并初步探討其作用機制。 方法 24只雄性成年SD大鼠隨機分為三組:對照組(Control),肺缺血再灌注組(I/R),缺氧預(yù)適應(yīng)組(WHPC)。每組大鼠8只。I/R組予夾閉左側(cè)肺門60 min后再灌注120 min復(fù)制原位肺缺血再灌注損傷模型;WHPC組大鼠置于密閉的常壓缺氧箱內(nèi)2 h建立全身缺氧預(yù)適應(yīng)模型后,再按上述方法復(fù)制大鼠原位肺缺血再灌注損傷模型;Control組僅完成除夾閉肺門以外的手術(shù)操作。 處理完畢后分別觀察各組大鼠肺組織形態(tài)學(xué),測定肺組織濕干重比值及髓過氧化物酶(MPO)活性,并分別檢測各組大鼠血漿中丙二醛(MDA)和超氧化物歧化酶(SOD)水平。 結(jié)果 (1)與Control組相比,I/R組大鼠肺組織濕干重比值、肺泡白細胞計數(shù)及肺泡損傷數(shù)、肺組織MPO活性均顯著升高(P0.01);I/R組大鼠血漿MDA含量隨著再灌注時間延長而逐漸升高,各時間點均高于Control組(P0.01);血漿SOD含量則隨著再灌注時間延長而逐漸下降,各時間點均低于Control組(P0.01)。(2)與I/R組相比,WHPC組大鼠肺組織濕干重比值、肺泡白細胞計數(shù)及肺泡損傷數(shù)、肺組織MPO活性均明顯改善(P0.01);血漿MDA含量較I/R組明顯降低;血漿SOD含量則較I/R組明顯升高(P0.01)。 結(jié)論 缺氧預(yù)適應(yīng)對大鼠在體肺缺血再灌注損傷具有一定的保護作用,其機制可能與抑制肺缺血再灌后脂質(zhì)過氧化反應(yīng)減輕肺水腫、抑制炎癥反應(yīng)和中性粒細胞活化有關(guān)。 第二部分 缺氧預(yù)適應(yīng)對肺缺血再灌注細胞凋亡的影響 目的 觀察缺氧預(yù)適應(yīng)對在體大鼠肺缺血再灌注細胞凋亡的影響,尤其是肺泡Ⅱ型上皮細胞凋亡的動態(tài)變化。 方法 缺氧預(yù)適應(yīng)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大鼠隨機分為三組,即對照組(Control組),肺缺血再灌注組(I/R組),缺氧預(yù)適應(yīng)組(WHPC組),WHPC組和IfR組根據(jù)再灌注時間又分別再分為3個亞組,分別再灌注30分鐘(P_1,R_1組)、60分鐘(P_2,R_2組)和90分鐘(P_3,R_3組)。Control組大鼠18只,其余每組大鼠6只。 處理結(jié)束后用TdT酶介導(dǎo)的dUTP缺口末端標(biāo)記法(TUNEL)檢測各組大鼠肺組織細胞凋亡情況,并計算出各組肺組織細胞凋亡指數(shù);分離出缺血再灌注后的肺泡Ⅱ型上皮細胞,用流式細胞儀檢測各組肺泡Ⅱ型上皮細胞凋亡率。 結(jié)果 (1)I/R組較Control組肺組織細胞凋亡指數(shù)明顯升高(P0.05),并且隨著再灌注時間的延長,細胞凋亡的數(shù)目逐漸增多;而WHPC組雖亦存在細胞凋亡的現(xiàn)象,但細胞凋亡指數(shù)較I/R組明顯下降(P0.01)。(2)用流式細胞儀檢測各組肺泡Ⅱ型上皮細胞凋亡率,結(jié)果顯示control組肺泡Ⅱ型上皮細胞凋亡率很低,與之比較相同時間點的I/R組和WHPC組均有不同程度的增高,尤以I/R組升高明顯(P0.01)。與I/R組相比較,各個時間點的WHPC組肺泡Ⅱ型上皮細胞凋亡率明顯減少(P0.01)。 結(jié)論 (1)缺氧預(yù)適應(yīng)能夠減輕肺缺血再灌注細胞的凋亡。 (2)缺氧預(yù)適應(yīng)可能通過減少肺泡Ⅱ型上皮細胞的凋亡對肺缺血再灌注損傷起保護作用。 第三部分 HIF-1α在缺氧預(yù)適應(yīng)對肺保護中的作用機制 目的 探討HIF-1α在缺氧預(yù)適應(yīng)對在體肺保護中的作用及其機制,脯氨酸羥化酶的抑制劑DMOG能否模擬缺氧預(yù)適應(yīng)對在體肺缺血再灌注損傷的延遲性保護作用。 方法 54只成年雄性SD大鼠隨機分為五組,即空白對照組(Control組),生理鹽水干預(yù)組(Saline組),缺氧預(yù)適應(yīng)組(WHPC組),NS-398干預(yù)組(WHPC+NS組)和DMOG干預(yù)組(DMOG組)。缺氧預(yù)適應(yīng)模型和大鼠原位肺缺血再灌注模型同第一部分。Saline組在缺血再灌注損傷模型建立前24h進行腹腔內(nèi)注射生理鹽水0.5ml;NS-398干預(yù)組在缺氧預(yù)適應(yīng)前15分鐘進行腹腔內(nèi)注射NS-398(10mg/kg);DMOG組在缺血再灌注損傷模型建立前24h進行腹腔注射DMOG(20mg/kg)。 處理結(jié)束后用逆轉(zhuǎn)錄—聚合酶鏈反應(yīng)檢測各組大鼠肺組織中HIF-1αmRNA、HO-1 mRNA的表達;用蛋白免疫印記法檢測各組大鼠肺組織中caspase-3蛋白的表達;用免疫組化法觀察各組大鼠肺組織中HIF-1α、HO-1、caspase-3蛋白表達情況及表達部位,用TUNEL法檢測肺組織細胞凋亡情況,并測定肺濕干重比值。 結(jié)果 (1)RT-PCR結(jié)果顯示:各組大鼠肺組織內(nèi)均有HIF-1αmRNA表達,但表達水平間無統(tǒng)計學(xué)差異。與Control組比較,其余各組肺組織中的HO-1 mRNA表達均顯著升高(P0.01);而WHPC組和DMOG組與Saline組相比表達水平卻是升高的;WHPC+NS組則與WHPC組相比表達有所下降(P0.05)。(2)免疫組化結(jié)果和caspaSe-3的western-blot結(jié)果顯示:與Saline組比較,WHPC組和DMOG組肺組織中的HIF-1α和HO-1蛋白表達水平升高,caspase-3蛋白表達水平是下降的;WHPC+NS組則與WHPC組相比肺組織中HIF-1α和HO-1蛋白表達水平下降,caspase-3蛋白表達水平是上調(diào)的,其差異均具有統(tǒng)計學(xué)意義。(3)TUNEL和肺組織濕干重比值結(jié)果顯示:與Saline組比較,WHPC組和DMOG組肺組織細胞凋亡指數(shù)減少,肺濕干重比值降低;WHPC+NS組與WHPC組相比則肺組織細胞凋亡指數(shù)和肺濕干重比值增高,其差異均具有統(tǒng)計學(xué)意義。 結(jié)論 (1)HIF-1α在缺氧預(yù)適應(yīng)對在體大鼠肺缺血再灌注損傷的保護中起著重要作用。 (2)缺氧預(yù)適應(yīng)通過誘導(dǎo)HIF-1α蛋白的穩(wěn)定表達,誘導(dǎo)保護性靶基因HO-1的過表達,降低caspase-3的表達水平,從而減輕在體肺缺血再灌注損傷細胞凋亡的程度,起到對肺的保護作用。 (3)脯氨酸羥化酶抑制劑DMOG能夠模擬缺氧預(yù)適應(yīng)對在體肺缺血再灌注損傷的延遲性保護作用,NS-398能取消這種保護作用。 創(chuàng)新與意義 (1)本研究在建立大鼠左肺原位缺血再灌注模型的基礎(chǔ)上,在國內(nèi)首次研究了缺氧預(yù)適應(yīng)對缺血再灌注肺損傷的保護作用。 (2)本研究觀察了缺氧預(yù)適應(yīng)對肺缺血再灌注后肺組織細胞凋亡的動態(tài)變化的影響,并首次在缺血再灌注后分離出肺泡Ⅱ型上皮細胞,運用流式細胞技術(shù)觀察缺氧預(yù)適應(yīng)對肺泡Ⅱ型上皮細胞凋亡率的影響。 (3)在研究缺氧預(yù)適應(yīng)保護機制中,首次發(fā)現(xiàn)HIF-1α在缺氧預(yù)適應(yīng)對在體肺缺血再灌注損傷保護作用中亦起著重要作用;HIF-1α蛋白的穩(wěn)定表達,誘導(dǎo)保護性靶基因HO-1過表達,來降低caspase-3的蛋白表達水平,從而減輕在體肺缺血再灌注損傷細胞凋亡的程度,起到對肺的保護作用。 (4)本研究首次運用脯氨酸羥化酶抑制劑DMOG在大鼠左肺原位缺血再灌注模型上模擬缺氧預(yù)適應(yīng)延遲性保護作用,可能為防治肺缺血再灌注損傷提供了新的治療策略。不足 缺氧預(yù)適應(yīng)的內(nèi)源性保護性機制十分復(fù)雜,有些機制仍未闡明,因為經(jīng)費和時間的限制,本研究只在在體動物模型上進行了其保護作用的觀察和機制的研究,并未在體外模型上進行進一步論證。并且本研究只涉及了HIF-1在缺氧預(yù)適應(yīng)保護機制中的作用,只觀察了其靶基因HO-1的表達情況,目前研究表明其靶基因EPO,VEGF和NOS等亦在保護機制中亦起到一定作用。我們將在以后的研究中進一步深入和完善。
文內(nèi)圖片：
圖片說明：各組大鼠肺濕/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，

本文編號：2512052