芪丹通脈片聯(lián)合骨髓間充質(zhì)干細(xì)胞移植對(duì)急性心肌缺血大鼠心肌細(xì)胞凋亡及心功能的影響

1、芪丹通脈片聯(lián)合骨髓間充質(zhì)干細(xì)胞移植對(duì)急性心肌缺血大鼠心肌細(xì)胞凋亡及心功能的影響         11-05-14 15:21:00     編輯：studa20          作者：王文,王宗仁,張金洲,李軍昌,蘇海礫,王文勇,馬福成,李靜【摘要】  目的 觀察芪丹通脈片聯(lián)合骨髓間充質(zhì)干細(xì)胞(MSCs)移植對(duì)急性心肌缺血大鼠心肌細(xì)胞凋亡、心功能的影響。方法 采用密

2、度梯度離心法體外分離培養(yǎng)大鼠MSCs,擴(kuò)增至第3代后,應(yīng)用溴脫氧尿嘧啶(BrdU)標(biāo)記細(xì)胞。16只健康SD大鼠隨機(jī)分為芪丹通脈片聯(lián)合移植組與單純移植組,分別灌服芪丹通脈片浸膏干粉溶液(2 g/kg)和等體積生理鹽水,每日1次,連續(xù)14 d。結(jié)扎大鼠冠狀動(dòng)脈左前降支,制備急性心肌梗死模型,將BrdU標(biāo)記的異體MSCs以微量注射的方法移植入缺血心肌內(nèi)。移植后3 h應(yīng)用HP5500超聲診斷儀,探頭頻率12 MHz,檢測(cè)大鼠心功能,4周后復(fù)查,并用免疫熒光法檢測(cè)缺血區(qū)的微血管密度(MVD),用脫氧核苷酸轉(zhuǎn)移酶(TdT酶)介導(dǎo)的dUTP缺口末端標(biāo)記法(TUNNEL)檢測(cè)心肌細(xì)胞凋亡指數(shù)(AI)。結(jié)果 芪

3、丹通脈片聯(lián)合移植組與單純移植組大鼠心功能均有所改善,而芪丹通脈片聯(lián)合移植組改善更明顯。其AI較單純移植組明顯減少,而缺血區(qū)MVD明顯增加。結(jié)論 芪丹通脈片聯(lián)合MSCs移植能改善急性心肌缺血大鼠的心功能,減少心肌細(xì)胞凋亡,增加缺血區(qū)MVD,與MSCs單純移植組比較療效更顯著。 【關(guān)鍵詞】  芪丹通脈片；骨髓間充質(zhì)干細(xì)胞；心功能；心肌細(xì)胞凋亡；大鼠Abstract：Objective  To observe the effect of marrow mesenchymal stem cells (MSCs) transplantation combined with Qidan

4、tongmai tablet and simple MSCs transplantation on myocardial cells apoptosis and heart function in acute myocardial ischemia rats. Method Rat MSCs were isolated by density gradient method, and then cultured and amplified to the third passage labeled with BrdU. 16 SD rats were divided into two groups

5、 randomly, transplantation combined with Qidantongmai tablet group and simple transplantation group. The isochoric of suspension of Qidantongmai tablet (2 g/kg) and NS were administered to SD rats by gastrogavage respectively for 14 days. The left anterior descending coronary artery of rat was ligat

6、ed to establish animal models of acute myocardial infarction. The MSCs labeled with BrdU were implanted intramyocardially into the infarct area. After 3 hours, the heart function were detected by an echocardiographic system (HP 5500 system), equipped with a 12 MHz probe. After 4 weeks, the heart fun

7、ction were rechecked and MVD were detected by immunofluorescence, myocardial cells apoptosis were detected by TUNNEL. Results Heart function were improved in both groups, and was more significant in MSCs transplantation combined with Qidantongmai tablet group. Apoptotic index were decreased and the

8、MVD of ischemic zone were increased in transplantation combined with Qidantongmai tablet group than simple transplantation group. Conclusion MSCs transplantation combined with Qidantongmai tablet could improve heart function of acute myocardial ischemia rats, decrease apoptosis of myocardial cells,

9、increase the MVD of ischemic zone, the therapeutic effect were more significant than simple MSCs transplantation.Key words：Qidantongmai tablet；marrow mesenchymal stem cells；heart function；apoptosis of myocardial cells；rat前期研究表明,益氣活血復(fù)方芪丹通脈片含藥血清具有體外誘導(dǎo)骨髓間充質(zhì)干細(xì)胞(MSCs)向內(nèi)皮細(xì)胞分化的作用1。本實(shí)驗(yàn)旨在探討芪丹通脈片協(xié)同MSCs移植可否增加局

10、部微血管密度(MVD)及對(duì)急性心肌缺血大鼠心肌細(xì)胞凋亡、心功能的影響。1  實(shí)驗(yàn)材料 1.1  動(dòng)物2月齡SD大鼠3只(制備MSCs用),雄性,體質(zhì)量(100±20)g；健康成年SD大鼠16只,雄性,體質(zhì)量(250±20)g,中國(guó)人民解放軍第四軍醫(yī)大學(xué)動(dòng)物中心提供。1.2  藥物芪丹通脈片浸膏干粉(黃芪30 g,丹參30 g,當(dāng)歸15 g,桂枝10 g,紅花30 g,此制劑1 g相當(dāng)于原材藥2.86 g),西京醫(yī)院藥劑科提供。1.3  主要儀器與試劑小動(dòng)物呼吸機(jī)、二氧化碳孵箱(HERA cell),激光共聚焦顯微鏡,percoll分離

11、液(pharmacia公司),F12-DMEM培養(yǎng)基、進(jìn)口胎牛血清(JIBCO公司),BrdU(pharmacia公司),小鼠抗BrdU單克隆抗體,小鼠抗大鼠CD31單克隆抗體,FITC標(biāo)記的山羊抗小鼠二抗,HP5500超聲診斷儀,TUNNEL試劑盒(武漢博士德生物技術(shù)公司)。2  實(shí)驗(yàn)方法2.1  分組成年SD大鼠16只分為芪丹通脈片聯(lián)合移植組與單純移植組,造模前分別灌服等體積芪丹通脈片浸膏干粉溶劑(2 g原藥材/kg)及生理鹽水14 d。2.2  骨髓間充質(zhì)干細(xì)胞的分離、純化和培養(yǎng)將3只幼齡SD大鼠斷頸處死,浸泡在75%乙醇中5 min,取出后用無菌PBS沖洗

12、鼠體至無乙醇,在超凈臺(tái)下無菌取下股骨、脛骨置于平皿中,更換器械及超凈臺(tái),剔除周圍肌肉組織等,用PBS反復(fù)沖洗。剪去骨干兩頭的關(guān)節(jié)面,暴露骨髓腔,用7號(hào)針頭穿刺兩骨端,用5 mL含10% FBS的F12-DMEM沖出骨髓入離心管,1 500/min離心10 min。棄上清,用F12-DMEM重懸細(xì)胞,輕輕疊加到新鮮配制的密度為1.073的percoll分離液上,2 500 r/min離心30 min,收集界面層混濁液,用PBS洗滌2次。然后用F12-DMEM培養(yǎng)液(含體積分?jǐn)?shù)10%胎牛血清、100 U/mL青霉素、100 g/mL 鏈霉素)重懸細(xì)胞,按1×106/mL的密度接種,37

13、、5%飽和濕度的CO2孵箱培養(yǎng),5 d后首次更換培養(yǎng)液,棄去未貼壁細(xì)胞,34 d換液1次,接近融合的MSCs用含0.1 mmol/LEDTA的0.25%胰酶室溫消化,按13比例傳代,傳至第3代。MSCs回植前48,換用含5 mol/L 5-BrdU的F12-DMEM培養(yǎng)液培養(yǎng)。臨用前加入0.25% 胰蛋白酶消化,離心、收集細(xì)胞,調(diào)整細(xì)胞密度備用。2.3  造模2組大鼠于第15日,采用Olivette方法,結(jié)扎冠狀動(dòng)脈建立急性心肌梗死模型。50 mg/kg戊巴比妥腹腔麻醉,氣管插管,呼吸機(jī)正壓輔助呼吸；開胸暴露心臟,以4-0無損傷線結(jié)扎左冠狀動(dòng)脈前降支,造成左室前壁心肌梗死模型。2.4

14、  骨髓間充質(zhì)干細(xì)胞移植2組大鼠于左冠狀動(dòng)脈前降支結(jié)扎后1,直視下在梗死心肌周圍用微量注射器分4點(diǎn)植入標(biāo)記5-BrdU的MSCs(5×106個(gè)細(xì)胞/100 L), 每點(diǎn)25 L。關(guān)胸,維持輔助呼吸至自主呼吸恢復(fù),送回籠中飼養(yǎng)。2.5  超聲心動(dòng)圖移植后3 h及移植后4周,乙醚麻醉大鼠后固定,左側(cè)胸部剃毛,經(jīng)胸應(yīng)用HP5500超聲診斷儀,探頭頻率12 MHz。取乳頭肌水平左室短軸切面,分別于左室收縮末期(LVES)和左室舒張末期(LVED)在二維圖像上測(cè)量左室舒張末期前后徑(LVEDD)、左室收縮末期前后徑(LVESD)、收縮末期室壁厚度(WTS)、舒張末期室壁厚度(WTD)、前壁室壁增厚率(AWTF)、后壁室壁增厚率(PWTF)。所有數(shù)值均由連續(xù)3個(gè)心動(dòng)周期測(cè)量數(shù)據(jù)平均得出。根據(jù)測(cè)值計(jì)算左室短軸縮短率(FS)及室壁增厚率(WTF)。FS(%)(LVEDDLVESD)/LVEDD×100；WTF(%)(WTS-WT