卡立泊來德對離體肺缺血再灌注損傷的初步實驗研究

1、卡立泊來德對離體肺缺血再灌注損傷的初步實驗研究 作者：周榮華，陳果，蒲江濤，陳艷芳，劉馨燭，張艷菊，龍村，劉斌 【摘要】 目的 研究選擇性鈉/氫離子(Na+/H+ )交換阻斷劑卡立泊來德(Cariporide)對離體大鼠肺缺血/再灌注損傷的防治效果。 方法 建立離體大鼠肺缺血再灌注損傷模型。18只SD成年大鼠隨機分為3組，每組6只：對照組:采用含5羥乙基淀粉的KHHB(Krebs - Henseleit hydroxyethylamylopectine buffer)灌注液持續(xù)平衡灌注、通氣120 min；缺血再灌注組:平衡30 min后，缺血60 min、再灌注30 min； Caripor

2、ide組:平衡15 min，給予Cariporide預(yù)處理15 min，缺血60 min、再灌注30 min。持續(xù)監(jiān)測并記錄肺動脈壓(PAP)；留取再灌注末灌注液,測定超氧化物歧化酶(SOD) 活性和丙二醛(MDA) 含量；再灌注結(jié)束，測肺濕/干重比(W/D)；光、電鏡觀察肺病理學(xué)改變。 結(jié)果 缺血再灌注組PAP、MDA含量、SOD活性和W/D 較對照組明顯升高(P0.05)，SOD 活性顯著下降(P0.05)。Cariporide能明顯減少PAP、MDA和W/D的增加，提高SOD活性(P0.05)，顯微、超微結(jié)構(gòu)證實Cariporide組肺血管內(nèi)皮及肺泡上皮的病理變化明顯減輕。結(jié)論 Cari

3、poride 能降低離體大鼠肺缺血再灌注損傷。 【關(guān)鍵詞】 Na+/H+ 交換； 卡立伯來德；肺；缺血再灌注損傷Abstract: OBJECTIVE To investigate the protective effects of selective Na+/H+ exchanger cariporide on isolated rat lung ischemia and reperfusion injury. METHODS The isolated lung ischemia/reperfusion models were established. After equilibration

4、, eighteen adult S.D rats were randomly divided into three groups: the control group(n=6),the ischemia/reperfusion group (n=6) and the cariporide group (n=6). In the control group, the isolated rat lungs were continuously perfused with the Krebs - Henseleit hydroxyethylamylopectine buffer (KHHB) sol

5、ution for 120min. In the ischemia/reperfusion group, the isolated rat lungs were reperfused for 30min after 60min of ischemiaIn the cariporide group, cariporide was administered in the KHHB solution before ischemia. Pulmonary artery pressure (PAP) were continuously monitored. The KHHB solution at 30

6、min of repefusions was collected to measure the malondialdehyde(MDA) content and superoxide dismutase (SOD) activity. After reperfusion, wet to dry lung weight ratio(W/D) was measured. The morphologic changes were also observed under light and electronic microscopes. RESULTS PAP,MDA and W/D in the i

7、schemia/reperfusion group were significantly higher than those in the control group (P0.05), and the SOD activity was lower than that in the control group (P0.05). While, compared with the ischemia/reperfusion group, cariporide significantly reduced the PAP, MDA and W/D(P0.05), and significantly inc

8、reased the SOD activity(P0.05). The morphologic and ultrastructural damages in the cariporide group were milder than those in ischemia/reperfusion group. CONCLUSION Cariporide can significantly protect lung injury induced by ischemia and reperfusion in the isolated rat.Key words： Na+/H+ exchanger; C

9、ariporide; Lung; Ischemia/reperfusion injury肺缺血再灌注損傷廣泛存在于臨床，例如：各種體外循環(huán)手術(shù)、肺移植術(shù)、心肺聯(lián)合移植術(shù)后、肺血栓內(nèi)膜剝除術(shù)及肺栓塞溶栓治療，以肺水腫、肺動脈高壓為主要表現(xiàn)1，直接影響患者預(yù)后，但無特效防治方法。已有研究表明，選擇性鈉氫離子交換（Na+/H+ exchanger isoform-1, NHE1）阻斷劑卡立泊來德（Cariporide）能減輕心肌缺血再灌注損傷2-3。而肺組織中也同樣存在NHE14-5，Cariporide是否對肺缺血再灌注損傷存在作用，尚不清楚。本研究通過建立離體大鼠肺缺血再灌注模型，探討Carip

10、oride對肺缺血再灌注損傷的防治效果。1 材料和方法1.1 離體肺缺血再灌注模型 SD成年大鼠（雌雄不限，體重275345 g），用3%戊巴比妥鈉(40 mg/kg)腹腔注射麻醉，腹腔注入肝素(1000 U/kg)行全身肝素化，待后爪反射消失，氣管切開插管，接微型人工呼吸機（江灣型）行控制呼吸(呼吸頻率60次/min，潮氣量6 ml/min)。正中劈開胸骨開胸，游離肺動脈，套線備用。經(jīng)右心室插管至肺動脈主干（20G Y型套管針），結(jié)扎固定。迅速完整取下整個心肺組織，置于Langendorff灌流裝置（Bridge AD Instrument PTY Ltd, Australia）， 37、9

11、5%O2、5%CO2飽和的含5羥乙基淀粉的改良KHHB(Krebs - Henseleit hydroxyethylamylopectine buffer)灌注液6、以30cm的高度落差經(jīng)肺動脈插管非循環(huán)灌注肺臟，流量10 ml/min，并迅速剪開左心耳，以利于肺灌注液的引流。停止人工灌注和機械通氣定義為：37常溫肺缺血,保濕，缺血期肺內(nèi)注入1.5 ml空氣后夾閉氣管；同時恢復(fù)灌注和機械通氣則為再灌注，建立大鼠離體肺缺血再灌注損傷模型6。1.2 實驗分組 采用完全隨機雙盲的研究方法，成年SD大鼠18只，隨機分為三組，每組6只：對照組，KHHB灌注液持續(xù)灌注離體肺120 min；缺血再灌注組，K

12、HHB灌注液平衡灌注30 min后，常溫37缺血60 min，再灌注30 min；Cariporide組，缺血前用加入Cariporide（德國Aventis Pham公司惠贈）的KHHB灌注液預(yù)處理15 min，然后常溫缺血60 min，再灌注30 min。KHHB灌注液中Cariporide濃度為10mol/L。1.3 檢測指標 在整個灌流過程中，用Powerlab多道生理記錄儀（Bridge AD Instrument PTY Ltd, Australia, Chart v 5.0）持續(xù)監(jiān)測并記錄肺動脈壓(PAP)。取再灌注30 min的KHHB灌注液，以硫代巴比妥酸法測定丙二醛(MDA)含量，黃嘌呤氧化酶法測定超氧化物歧化酶(SOD) 活性，SOD、MDA 試劑盒購于南京建成公司。再灌注結(jié)束，結(jié)扎左肺門，用分析天平稱量左肺濕重，70 24 h 烘干后稱干重，兩者之比為肺組織濕/干重比(W/D)。取右下肺約1cm1cm1cm小塊肺組織，采用4%多聚甲醛固定, 脫水，石蠟包埋，切片后HE染色，光鏡下觀察肺形態(tài)學(xué)變化；同時取1mm1mm1mm 小塊肺組織，3戊二醛固定，常規(guī)脫水、包埋和切片，醋酸鈾-枸櫞酸鉛復(fù)染，透射電鏡下觀察肺組織超微結(jié)構(gòu)變化。1.4 統(tǒng)