白介素8對(duì)兔腹主動(dòng)脈粥樣硬化狹窄的影響

1、白介素8對(duì)兔腹主動(dòng)脈粥樣硬化狹窄的影響         10-09-10 10:04:00     編輯：studa20                         作者：齊曉勇 孟存良 李英肖 黨懿 劉惠良   &

2、#160;                     王天紅 袁華兵 倪艷輝 劉 光 武振朝 郜利會(huì)【摘要】  目的 觀察白介素8（IL8）水平對(duì)兔腹主動(dòng)脈粥樣硬化狹窄的影響。方法 新西蘭大白兔36只隨機(jī)分為實(shí)驗(yàn)組、IL8單克隆抗體治療組、對(duì)照組。實(shí)驗(yàn)組和治療組動(dòng)物通過穿刺股動(dòng)脈送入球囊拉傷腹主動(dòng)脈。治療組動(dòng)物通過耳緣靜脈注射IL8單克隆抗體進(jìn)行干預(yù)。對(duì)照組動(dòng)物只進(jìn)行股動(dòng)脈穿刺和留置鞘管，

3、不進(jìn)行球囊擴(kuò)張拉傷。分別于實(shí)驗(yàn)前,實(shí)驗(yàn)后4 h、1 d、3 d，1 w、2 w、4 w，耳緣靜脈采血4 ml，檢測(cè)三組動(dòng)物血清IL8水平，4 w后處死動(dòng)物進(jìn)行病理組織學(xué)檢查，應(yīng)用光學(xué)顯微鏡和計(jì)算機(jī)病理圖像分析系統(tǒng)分析測(cè)定以下指標(biāo)：內(nèi)膜中膜厚度、面積，計(jì)算血管狹窄程度。結(jié)果 實(shí)驗(yàn)組動(dòng)物球囊損傷4 h后IL8水平即開始升高，術(shù)后1 d達(dá)到峰值，持續(xù)增高4 w，且明顯高于治療組和對(duì)照組。治療組和對(duì)照組動(dòng)物IL8水平無(wú)上述變化規(guī)律。實(shí)驗(yàn)組動(dòng)物腹主動(dòng)脈明顯狹窄，組織學(xué)檢查內(nèi)膜面積和中膜面積、血管狹窄程度等指標(biāo)大于治療組和對(duì)照組（P<0.01），治療組管腔狹窄，對(duì)照組動(dòng)物管腔無(wú)明顯變化，治療組與對(duì)照

4、組比較無(wú)顯著性差異(P>0.05)。相關(guān)分析顯示，IL8水平與內(nèi)膜厚度、內(nèi)膜面積、中膜厚度、中膜面積等呈正相關(guān)(相關(guān)系數(shù)r分別為0.894，0.783，0.801，0.912，均P<0.01)。結(jié)論 IL8水平高表達(dá)引起腹主動(dòng)脈粥樣硬化狹窄的產(chǎn)生，經(jīng)IL8單克隆抗體治療后動(dòng)脈狹窄程度明顯減輕。IL8水平與動(dòng)脈狹窄程度呈正相關(guān)，是發(fā)生腹主動(dòng)脈粥樣硬化狹窄的獨(dú)立危險(xiǎn)因素。 【關(guān)鍵詞】  腹主動(dòng)脈；動(dòng)脈粥樣硬化；炎性反應(yīng)；白介素8；單克隆抗體    【Abstract】  Objective  To observe the ef

5、fects of interleukin8 (IL8) on atherosclerosis(AS) of abdominal aorta in rabbits. Methods  Thirtysix New Zealand white rabbits were randomly divided into experiment, treatment and control groups, each group 12 cases. Rabbits in experiment and treatment groups were injuried in abdominal aorta by

6、 balloon inflation after punctured in femoral artery. Monoclonal antibody of IL8 was injected by venous in rabbits of treatment group. Rabbits in control group were punctured only in femoral artery. Peripheral blood was collected before experiment and 4 h, 1, 3 d, 1, 2, 4 w later. The level of IL8 w

7、as measured by enzyme linked immunosorbent assay (ELISA) respectively. The luminals of abdomial aorta in three groups were observed 4 w later. Histopathologic examination was performed. Such contents as follows were assayed by light microscope and computer image analysis system: luminal area, intima

8、 and tunica media area, angiostenosis of blood vessel. Results  The level of IL8 was raised in 4 h and achieved to peak in 1 d after balloon inflation in rabbits of experiment group. The higher level of this inflammatory factor lasted 4 w. There was no variation above in treatment and control g

9、roups. It was observed that abdominal aorta became stenosis obviously in experiment group. The area of intima and tunica media as well as the extent of stenosis in experiment group were bigger than those in treatment and control groups. There was no statistical difference between treatment and contr

10、ol groups. Correlation analysis indicated that there were positive relations between IL，0.783，0.801，0.912，P<0.01). Multiple regression analysis of Logistic indicated that IL8 was an independent risk for the AS（P<0.01）. The high levels of IL8 leaded to AS in abdominal aorta of rabbits. The seve

11、rity of stenosis became lessen after intervention by monoclonal antibody of IL8. Positive corelation was found between the level of IL8 and severity of arterial stenosis. IL8 was one of independent risks factor leading occurrence and development of AS. Conclusions  The high levels of IL8 leads

12、to AS in abdominal aorta of rabbits. The severity of stenosis becomes lessen after intervention by monoclonal antibody of IL8. Positive corelation is found between the level of IL8 and severity of arterial stenosis. IL8 is one of independent risks factor leading occurrence and development of AS.

13、0;   【Key words】  Abdominal aorta; Atherosclerosis; Inflammatory reaction; Interleukin8; Monoclonal antibody目前研究已經(jīng)證實(shí)，炎性反應(yīng)在動(dòng)脈粥樣硬化（AS）的發(fā)生與發(fā)展中起到重要的作用。參與AS形成的炎性因子眾多，且互相影響，因此觀察炎性因子的變化規(guī)律，了解炎性因子與AS病變的關(guān)系與作用機(jī)制，有助于尋找臨床干預(yù)AS的新方法。白細(xì)胞介素8（Interleukin8，IL8）是體內(nèi)重要的炎性因子，可促使血液中單核細(xì)胞聚集到內(nèi)膜上及向內(nèi)膜下轉(zhuǎn)移。本研究擬采用IL8

14、單克隆抗體進(jìn)行干預(yù)治療，觀察干預(yù)治療后IL8水平變化以及對(duì)腹主動(dòng)脈AS狹窄的影響，為臨床治療AS性疾病提供新思路。1  材料與方法1.1  動(dòng)物與分組選取812月齡新西蘭大白兔36只，雌雄不拘，體質(zhì)量2.503.25 kg，由河北醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供(許可證號(hào)：SCXK(冀)20031003)。以上實(shí)驗(yàn)動(dòng)物隨機(jī)分為球囊拉傷組（實(shí)驗(yàn)組,n=12）和IL8單克隆抗體干預(yù)組（治療組,n=12）以及對(duì)照組（n=12）。1.2  方法1.2.1  兔腹主動(dòng)脈AS模型的建立 實(shí)驗(yàn)動(dòng)物術(shù)前12 h禁食，不禁水，稱體重。常規(guī)建立耳緣靜脈輸液通路，以10 g

15、/L戊巴比妥鈉3.03.5 ml/kg耳緣靜脈注射麻醉實(shí)驗(yàn)動(dòng)物，麻醉后動(dòng)物仰臥固定于手術(shù)臺(tái)，以20%硫化鈉雙側(cè)腹股溝脫毛。常規(guī)消毒鋪巾后，seldinger技術(shù)穿刺股動(dòng)脈并留置鞘管，經(jīng)鞘管注入普通肝素200單位抗凝，沿導(dǎo)引鋼絲逆行插入球囊，連接手推式壓力表，造影后將球囊定位，置于髂動(dòng)脈以上2個(gè)椎體處，以405.30607.95 kPa壓力擴(kuò)張球囊，同時(shí)注射造影劑證實(shí)球囊完全擴(kuò)張堵閉腹主動(dòng)脈，自近心端緩慢回拉(拉傷長(zhǎng)度4 cm)，反復(fù)3次，每次球囊擴(kuò)張持續(xù)時(shí)間20 s，每次間隔15 s，撤出球囊，拔除動(dòng)脈鞘管，壓迫止血15 min后，繃帶包扎固定，4 h后解除繃帶包扎。1.2.2  I

16、L8單克隆抗體的干預(yù)治療 干預(yù)組動(dòng)物實(shí)驗(yàn)前開始通過耳緣靜脈注射IL8單克隆抗體2 mg/kg，連續(xù)3 d。1.2.3  病理學(xué)檢查 剝離腹主動(dòng)脈大體標(biāo)本（胸主動(dòng)脈至髂動(dòng)脈分叉處）。剪除血管外結(jié)締組織及脂肪，生理鹽水沖洗殘血。截取成形部位及對(duì)照組血管標(biāo)本，立即放入10%福爾馬林溶液內(nèi)浸泡固定。常規(guī)酒精脫水，石蠟包埋，制成3 m厚連續(xù)切片24 h，給予蘇木素伊紅（HE）染色。利用HE染色的切片，隨機(jī)選取5個(gè)視野，在40倍光鏡下，利用微機(jī)圖像處理測(cè)量系統(tǒng)對(duì)切片進(jìn)行圖像攝取和轉(zhuǎn)換，測(cè)量斑塊纖維帽及新生內(nèi)膜厚度（IT）、中膜厚度(MT)、內(nèi)膜面積(IA)、中膜面積(MA)，

17、計(jì)算血管狹窄程度、IT/MT比值，以及IA/MA比值。1.3  統(tǒng)計(jì)學(xué)方法 采用SPSS13.0軟件包完成統(tǒng)計(jì)處理，先進(jìn)行組間均衡性檢驗(yàn)，計(jì)量資料以x±s表示，采用2檢驗(yàn)。先行方差分析，然后再作多組均數(shù)的q檢驗(yàn)，相關(guān)資料采用相關(guān)分析法計(jì)算相關(guān)系數(shù)，并進(jìn)行多元Logistic回歸分析。2  結(jié)  果2.1  三組實(shí)驗(yàn)動(dòng)物之間體重、月齡、性別、血脂等一般情況比較，差異無(wú)統(tǒng)計(jì)學(xué)意義（P>0.05），見表1。表1  三組動(dòng)物一般情況比較(略)2.2  三組動(dòng)物術(shù)前、術(shù)后IL8水平比較 術(shù)前三組IL8水平無(wú)明顯差異；術(shù)后4 h實(shí)驗(yàn)組IL8水平較術(shù)前以及干預(yù)組和對(duì)照組明顯升高，術(shù)后1 d達(dá)到峰值，持續(xù)增高4 w。干預(yù)組與治療組無(wú)上述變化趨勢(shì)，干預(yù)組顯著低于實(shí)驗(yàn)組（P<0.01），與對(duì)照組比較，無(wú)顯著性差異(P>0.05)，見表2。表2  三組之間不同時(shí)間點(diǎn)IL8水平(略)2.3  三組動(dòng)物病理形態(tài)學(xué)改變  HE染色可見對(duì)照組動(dòng)脈內(nèi)膜光滑，內(nèi)皮下有少量結(jié)締組織，無(wú)平滑肌細(xì)胞存在，中膜平滑肌細(xì)胞成梭形，環(huán)繞管腔排列；實(shí)驗(yàn)組兔內(nèi)膜、中膜明顯增厚，管腔明顯狹窄，內(nèi)膜下平滑肌細(xì)胞明顯增生，而且排列不規(guī)則，內(nèi)皮及