間歇運(yùn)動(dòng)和粒細(xì)胞集落刺激因子促進(jìn)心梗大鼠干細(xì)胞動(dòng)員與內(nèi)源性心肌細(xì)胞增殖的激光共聚焦/流式細(xì)胞術(shù)觀察分析

史秀超，蔡夢(mèng)昕，田振軍

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間歇運(yùn)動(dòng)和粒細(xì)胞集落刺激因子促進(jìn)心梗大鼠干細(xì)胞動(dòng)員與內(nèi)源性心肌細(xì)胞增殖的激光共聚焦/流式細(xì)胞術(shù)觀察分析

史秀超1,2，蔡夢(mèng)昕1，田振軍1

目的：探討跑臺(tái)間歇運(yùn)動(dòng)結(jié)合粒細(xì)胞集落刺激因子對(duì)心肌梗死大鼠干細(xì)胞動(dòng)員的效果和在內(nèi)源性心肌細(xì)胞增殖中的作用及其對(duì)心功能的影響。方法：90只成體雄性SD大鼠隨機(jī)分為假手術(shù)組(Sham)、心梗組(MI)、心梗+間歇運(yùn)動(dòng)組(ME)、心梗+粒細(xì)胞集落刺激因子(G-CSF)組(MG)和心梗+G-CSF+間歇運(yùn)動(dòng)組(MGE)。結(jié)扎大鼠左冠狀動(dòng)脈前降支制備心肌梗死模型。ME和MGE組大鼠在心肌梗死手術(shù)結(jié)束1周后進(jìn)行3周跑臺(tái)間歇運(yùn)動(dòng)。MG和MGE組大鼠術(shù)后1 h皮下注射人源重組粒細(xì)胞集落刺激因子(rhG-CSF)，10 μg/kg/d×5 d，其他各組大鼠給予同劑量生理鹽水。免疫熒光法檢測(cè)并計(jì)算梗死邊緣區(qū)心肌細(xì)胞增殖百分率；流式細(xì)胞術(shù)檢測(cè)外周血單個(gè)核細(xì)胞中c-kit+和CD29+細(xì)胞百分率；Western Blot方法檢測(cè)心肌組織中c-kit和CD29蛋白表達(dá)水平。TTC染色檢測(cè)心肌梗死面積百分率；彩色多普勒超聲和血流動(dòng)力學(xué)方法檢測(cè)心功能。結(jié)果：與Sham組相比，MI組PCNA+心肌細(xì)胞百分率、外周血單個(gè)核細(xì)胞中c-kit+和CD29+細(xì)胞百分率、心肌組織中c-kit和CD29蛋白表達(dá)和心肌梗死面積顯著增加，心功能顯著降低；與MI組比較，ME、MG和MGE組大鼠梗死邊緣區(qū)心肌組織中PCNA+心肌細(xì)胞百分率、外周血單個(gè)核細(xì)胞中c-kit+和CD29+細(xì)胞百分率、心肌組織中c-kit和CD29蛋白表達(dá)均顯著增加，梗死面積顯著減小，心功能顯著增強(qiáng)，且MGE組變化最為顯著。結(jié)論：間歇運(yùn)動(dòng)或粒細(xì)胞集落刺激因子可顯著促進(jìn)干細(xì)胞動(dòng)員歸巢，誘導(dǎo)心肌細(xì)胞增殖，縮小梗死面積，提升心功能，且二者聯(lián)合效果優(yōu)于單一因素作用。

心肌梗死；間歇運(yùn)動(dòng)； 粒細(xì)胞集落刺激因子；干細(xì)胞動(dòng)員；心肌細(xì)胞增殖；鼠；動(dòng)物實(shí)驗(yàn)

心肌梗死(myocardial infarction，MI)發(fā)生后，心肌細(xì)胞大量壞死或凋亡，心室發(fā)生惡性重塑，導(dǎo)致心力衰竭[43]。增加有收縮功能的心肌細(xì)胞數(shù)目是基礎(chǔ)和臨床研究的熱點(diǎn)。一般認(rèn)為，成體哺乳動(dòng)物心肌細(xì)胞缺乏增殖能力。近年研究證實(shí)，成體哺乳動(dòng)物心肌細(xì)胞存在更新現(xiàn)象，其更新率隨年齡增加而降低[6,21,35]。研究表明，心肌細(xì)胞具有增殖現(xiàn)象[28]，但其機(jī)制與方法研究需要不斷豐富，尋求有效促進(jìn)內(nèi)源性心肌細(xì)胞增殖的細(xì)胞來(lái)源、方法和手段，對(duì)促進(jìn)心肌損傷修復(fù)意義重大。針對(duì)心臟損傷修復(fù)的干細(xì)胞療法已有大量研究報(bào)道[30,49]，目前認(rèn)為，可參與心肌再生的細(xì)胞來(lái)源包括部分有收縮功能的單個(gè)核心肌細(xì)胞、心肌固有的干/祖細(xì)胞(Cardiac stem/progenitor cells，CSCs/CPCs)[24,26,53]以及外周成體干細(xì)胞，如骨髓干細(xì)胞(Bone marrow stem cells,BMSCs)和誘導(dǎo)型多功能干細(xì)胞(Induced pluripotent stem cells，iPSCs)等[49,50,51]。MI發(fā)生后，梗死區(qū)微環(huán)境發(fā)生改變，可在一定程度上動(dòng)員CSCs/CPCs和外周成體干細(xì)胞，如BMSCs等趨化至損傷部位進(jìn)行增殖與分化，參與內(nèi)源性心肌修復(fù)[13，14,42,52,58]。

間歇運(yùn)動(dòng)是指高強(qiáng)度與低強(qiáng)度鍛煉或自由活動(dòng)等方式交替進(jìn)行的運(yùn)動(dòng)形式。文獻(xiàn)表明，高強(qiáng)度間歇運(yùn)動(dòng)較中強(qiáng)度有氧運(yùn)動(dòng)對(duì)心臟結(jié)構(gòu)與功能的影響更為顯著[12,17,31,56,59]。 MI患者進(jìn)行運(yùn)動(dòng)康復(fù)鍛煉可有效改善心臟惡性重塑現(xiàn)象，提升心功能和改善生活質(zhì)量[15,41,46]。動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn)，運(yùn)動(dòng)可促進(jìn)正常生理狀態(tài)下心肌細(xì)胞增殖[7,57]。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn)，8周持續(xù)有氧運(yùn)動(dòng)可促進(jìn)正常和MI大鼠心肌組織增殖細(xì)胞核抗原(proliferating cell nuclear antigen，PCNA)和細(xì)胞增殖標(biāo)記物Ki67蛋白表達(dá)[1]，有效提高心功能。粒細(xì)胞集落刺激因子(granulocyte colony-stimulating factor，G-CSF)可促進(jìn)骨髓間充質(zhì)干細(xì)胞和造血干細(xì)胞等趨化至損傷部位，參與心肌損傷后的修復(fù)[27，45]。運(yùn)動(dòng)可促進(jìn)血液循環(huán)中干細(xì)胞數(shù)目增多[8,9,22]，但間歇運(yùn)動(dòng)結(jié)合干細(xì)胞動(dòng)員劑G-CSF能否更有效促進(jìn)自體干細(xì)胞動(dòng)員和心臟內(nèi)源性心肌細(xì)胞增殖，缺乏直接證據(jù)。文獻(xiàn)表明，心?；蛐墓：笞⑸銰-CSF動(dòng)員BMSCs入血均存在時(shí)間窗[44，55]。根據(jù)G-CSF動(dòng)員BMSCs時(shí)間窗和心臟運(yùn)動(dòng)負(fù)荷產(chǎn)生效應(yīng)的最短時(shí)間，本研究采用心梗1 h后給予G-CSF動(dòng)員，手術(shù)后1周進(jìn)行3周間歇運(yùn)動(dòng)干預(yù)，采用激光共聚焦顯微術(shù)和流式細(xì)胞術(shù)及Western Blot方法，觀察與分析間歇運(yùn)動(dòng)和粒細(xì)胞集落刺激因子對(duì)心梗大鼠內(nèi)源性心肌細(xì)胞增殖和干細(xì)胞動(dòng)員的效果。

1 材料與方法

1.1 主要儀器和試劑

主要儀器：ALC V8 動(dòng)物呼吸機(jī)、BM Ⅱ型病理組織包埋機(jī)、LEICA RM 2126切片機(jī)、尼康Nikon C2 Plus激光共聚焦顯微鏡(laser scanning confocal microscope,LSCM)、PowerLab/8S生理信號(hào)采集系統(tǒng)、美國(guó)BD公司FACS Aria流式細(xì)胞儀、BioTek epoch超微量微孔板分光光度計(jì)、GE Logiq 7彩色多普勒超聲診斷儀、Bio Rad電泳儀和轉(zhuǎn)移槽等。

主要試劑：甘氨酸、牛血清白蛋白、Tween20、甲叉雙丙烯酰胺、丙烯酰胺、DAPI(羅氏公司)、重組人粒細(xì)胞集落刺激因子(recombinant human granulocyte colony-stimulating factor，rhG-CSF 山東齊魯制藥有限公司)、小鼠抗大鼠PCNA(Cell Signaling Technology公司)、2，3，5-氯化三苯基四氮唑(2,3,5-Triphenyltetrazolium chloride,TTC Amresco公司)、心肌型兔抗大鼠肌鈣蛋白T(cardiac troponin T，cTnT)、兔抗大鼠CD29和c-kit(北京博奧森公司)、FITC標(biāo)記的山羊抗兔抗體和TRITC標(biāo)記的山羊抗小鼠抗體(武漢博士德生物工程有限公司)、動(dòng)物外周血和臟器組織單個(gè)核細(xì)胞分離試劑盒(灝洋生物公司)。

1.2 動(dòng)物分組與MI模型制備

動(dòng)物分組：清潔級(jí)3月齡Sprague Dawley雄性大鼠90只，體重180～220 g，購(gòu)于西安交通大學(xué)實(shí)驗(yàn)動(dòng)物管理中心(動(dòng)物質(zhì)量合格證號(hào)：SCXK2013-003號(hào))。隨機(jī)分為假心梗組(Sham)、心梗組(MI)、心梗+間歇運(yùn)動(dòng)組(ME)、心梗+G-CSF組(MG)和心梗+G-CSF+間歇運(yùn)動(dòng)組(MGE)，每組18只。大鼠飼養(yǎng)室溫度保持20～29℃，相對(duì)濕度50%～60%，采用國(guó)家標(biāo)準(zhǔn)嚙齒類動(dòng)物干燥飼料喂養(yǎng)，自由飲食。

MI大鼠模型制備：采用左冠狀動(dòng)脈前降支結(jié)扎法[1]，同時(shí)監(jiān)測(cè)心電圖并觀察結(jié)扎點(diǎn)下方至心尖部位心肌顏色變化。以心電圖ST段弓背抬高、結(jié)扎線以下心肌發(fā)白，或出現(xiàn)病理性Q波或T波倒置為結(jié)扎成功標(biāo)志，之后逐層縫合關(guān)胸。Sham組只開胸穿線，不結(jié)扎。

1.3 間歇運(yùn)動(dòng)和G-CSF給藥方案

1.4 心功能檢測(cè)

心動(dòng)超聲檢測(cè)：實(shí)驗(yàn)造模結(jié)束后，常規(guī)腹腔麻醉并稱重，8%Na2S脫毛凈胸，取仰臥位將頭部及四肢固定在手術(shù)臺(tái)上。使用GE Logiq7彩色多普勒超聲診斷儀，i12L寬帶，術(shù)中探頭頻率為12 MHz，超聲檢測(cè)中圖像深度調(diào)至2 cm，增益固定為60 dB。將探頭置于左前胸，與前正中線呈30°左右夾角，顯示胸骨旁左室長(zhǎng)軸切面，探頭順時(shí)針旋轉(zhuǎn)90°顯示左室短軸切面圖像，于胸骨旁左室長(zhǎng)軸切面二維測(cè)量左室室壁厚度及梗死區(qū)范圍。在二維導(dǎo)引下，將取樣線置于左室健索水平，取M型超聲心動(dòng)圖，速度為200 mm/s。測(cè)量左室舒張末期內(nèi)徑(left ventricle diastolic diameter，LVDd)及左室收縮末期內(nèi)徑(left ventricle systolic diameter,LVDs)。儀器自動(dòng)計(jì)算左室短軸縮短率(left ventricular fractional shortening，LVFS)=(LVDd-LVDs)/LVDd ×100%和左室射血分?jǐn)?shù)(Left ventricle ejection fraction，LVEF)=[(LVDd3-LVDs3)/LVDd3]×100%。測(cè)量指標(biāo)均取3個(gè)連續(xù)心動(dòng)周期的平均值。

血流動(dòng)力學(xué)檢測(cè)：經(jīng)右頸總動(dòng)脈逆行插管至大鼠左心室，以多導(dǎo)生理記錄儀記錄最大左室收縮壓(Left ventricle systolic pressure，LVSP)、左室舒張末壓(left ventricle end diastolic pressure，LVEDP)、左室壓力最大上升速率(The maximum ascending velocity of left ventricle pressure，LV+dp/dtmax)和最大下降速率(The maximum descending velocity of left ventricle pressure，LV-dp/dtmax)。

1.5 取材與樣本制備

大鼠心功能測(cè)試結(jié)束后，立刻斷頭取血，肝素鈉抗凝，分離外周血單個(gè)核細(xì)胞。摘取心臟，每組隨機(jī)取6個(gè)心臟中性甲醛固定，常規(guī)石蠟包埋，連續(xù)切片(厚度5 μm)，用于免疫熒光實(shí)驗(yàn)；另取6個(gè)心臟液氮速凍，置-80℃冰箱，用于western blot 實(shí)驗(yàn)。剩余6只大鼠常規(guī)腹腔麻醉，開胸摘取心臟，5% TTC溶液主動(dòng)脈逆向灌流染色，液氮速凍，10 min后取出，手工切片(厚約2 mm)，數(shù)碼相機(jī)拍照，統(tǒng)計(jì)梗死區(qū)(乳白色)與非梗死區(qū)(紅色)面積，并計(jì)算梗死面積百分率。

1.6 外周血單個(gè)核細(xì)胞分離和流式細(xì)胞儀檢測(cè)

大鼠外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cell，PBMC)分離嚴(yán)格按照灝洋生物公司的“各種動(dòng)物外周血和臟器組織單個(gè)核細(xì)胞分離試劑盒操作說(shuō)明”進(jìn)行。在15 ml玻璃管中依次加入A和D液各2 ml，取1 ml新鮮抗凝血按1∶1比例與全血及組織稀釋液混勻疊加于D液的液面上，400倍重力離心15 min，收集離心管中由上至下第二層單個(gè)核細(xì)胞懸液，注入5 ml細(xì)胞洗滌液試管中，以500倍重力離心20 min，棄上清，沉淀的細(xì)胞用PBS重懸2次即得單個(gè)核細(xì)胞懸液，500倍重力離心20 min ，沉淀的單個(gè)核細(xì)胞用2.5%戊二醛固定30 min，PBS洗滌3次，離心棄上清，3% BSA 37℃封閉1 h，離心去上清，加入兔抗大鼠c-kit或CD29抗體，37℃孵育2 h，PBS洗滌3次，F(xiàn)ITC標(biāo)記的山羊抗兔c-kit或CD29抗體37℃孵育2 h，PBS洗滌5次。不加一抗，只加FITC標(biāo)記的山羊抗兔二抗的PBMCs作為陰性對(duì)照，F(xiàn)ACS Aria 流式細(xì)胞儀進(jìn)行檢測(cè)。

1.7 免疫熒光檢測(cè)

石蠟切片脫蠟至水，pH6.0枸櫞酸緩沖液抗原修復(fù)，切片于修復(fù)液中降至室溫，PBS清洗，濕盒中正常山羊血清37℃封閉1 h，滴加兔抗大鼠心肌型肌鈣蛋白T(cTnT，1∶50)和小鼠抗大鼠細(xì)胞增殖核抗原(PCNA，1∶100)，濕盒中4℃過(guò)夜。室溫復(fù)溫45 min，PBS清洗，滴加FITC標(biāo)記的山羊抗兔抗體(1∶50)、TRITC標(biāo)記的山羊抗小鼠抗體(1∶50)和DAPI(1 μg/ml)，濕盒中37℃孵育1 h，PBS清洗，甘油緩沖液封片。設(shè)置空白對(duì)照和陰性對(duì)照。尼康Nikon C2 Plus激光共聚焦顯微鏡觀察拍照。

1.8 Western Blot實(shí)驗(yàn)

取心梗邊緣區(qū)心肌組織提取蛋白，10% Tris-甘氨酸SDS聚丙烯酰胺凝膠電泳分離蛋白，轉(zhuǎn)膜，封閉后孵育干細(xì)胞表面抗原c-kit和CD29(濃度均為1∶500)，4℃過(guò)夜。室溫孵育二抗30 min(濃度為1∶2 000)，洗膜后ECL發(fā)光，內(nèi)參照為GAPDH。

1.9 圖像和數(shù)據(jù)采集與分析

免疫熒光顯微圖片經(jīng)Image-Pro Plus 5.1軟件采集并分析；Western Blot膠片經(jīng)掃描后采用Quantity One 4.6軟件進(jìn)行分析；GraphPad Prism 5.0軟件作圖。所有數(shù)據(jù)均用SPSS 17.0軟件進(jìn)行處理，采用One-Way ANOVA進(jìn)行統(tǒng)計(jì)學(xué)分析，組間顯著性差異水平為P<0.05和P<0.01。

2 實(shí)驗(yàn)結(jié)果

2.1 間歇運(yùn)動(dòng)和/或G-CSF可促進(jìn)心梗大鼠骨髓干細(xì)胞動(dòng)員增加

c-kit+和CD29+均為骨髓干細(xì)胞標(biāo)記物[29,53]。通過(guò)分離大鼠外周血單個(gè)核細(xì)胞，用FITC標(biāo)記c-kit和CD29抗原，流式細(xì)胞儀計(jì)數(shù)被標(biāo)記的和未標(biāo)記的細(xì)胞，計(jì)算免疫標(biāo)記的單個(gè)核細(xì)胞百分率，反映骨髓干細(xì)胞動(dòng)員效果。結(jié)果表明，與Sham組比較，MI組外周血單個(gè)核細(xì)胞中c-kit+和CD29+細(xì)胞的百分率增加顯著(P<0.01)。與MI組比較，ME組和MG組外周血c-kit+和CD29+干細(xì)胞百分率顯著增加(P<0.05)，MGE組更為顯著(P<0.01，圖1)。

圖1 本研究大鼠外周血c-kit+和CD29+細(xì)胞的流式細(xì)胞儀檢測(cè)結(jié)果與統(tǒng)計(jì)圖Figure 1.The Result of c-kit and CD29 Positive PBMCs Detected by Flow Cytometer and Statistical Graph

2.2 間歇運(yùn)動(dòng)和/或G-CSF可促進(jìn)大鼠心梗邊緣區(qū)c-kit和CD29蛋白表達(dá)增加

Western Blot檢測(cè)結(jié)果顯示，與Sham組比較，MI組心梗邊緣區(qū)心肌c-kit和CD29蛋白表達(dá)均顯著增加(P<0.01)。與MI組比較，ME組和MG組心梗邊緣區(qū)心肌c-kit和CD29蛋白表達(dá)顯著增加(P<0.05)，且MGE組增加效果顯著優(yōu)于單一因素(P<0.01，圖2)。

圖2 本研究大鼠心梗邊緣區(qū)c-kit和CD29蛋白表達(dá)結(jié)果與統(tǒng)計(jì)圖Figure 2.Expression of c-kit and CD29 Protein in Peri-infarct Area of Rat Heart and Statistical Graph

2.3 間歇運(yùn)動(dòng)和/或G-CSF干預(yù)可促進(jìn)心梗大鼠心肌細(xì)胞增殖

PCNA為定位于增殖細(xì)胞核的抗原，cTnT為定位于心肌細(xì)胞質(zhì)的特異性結(jié)構(gòu)蛋白。DAPI為DNA強(qiáng)力結(jié)合染料。PCNA+和cTnT+雙陽(yáng)性細(xì)胞可反映新生心肌細(xì)胞；新生心肌細(xì)胞百分率=新生心肌細(xì)胞數(shù)/所有心肌細(xì)胞數(shù)×100%。免疫熒光顯示，Sham組PCNA+和cTnT+雙陽(yáng)性細(xì)胞極少。MI組、ME組、MG組和MGE組均可見PCNA+和cTnT+雙陽(yáng)性細(xì)胞。與Sham組比較，MI組PCNA+和cTnT+雙陽(yáng)性細(xì)胞數(shù)顯著增加(P<0.01)；與MI組比較，ME組、MG組和MGE組心肌細(xì)胞增生百分率顯著增加，且MGE組PCNA+和cTnT+雙陽(yáng)性細(xì)胞數(shù)最多(P<0.05，P<0.01，圖3)。

2.4 間歇運(yùn)動(dòng)和/或G-CSF干預(yù)可縮小心梗面積，改善心功能

TTC染色結(jié)果顯示，與Sham組比較，MI組梗死面積顯著增加(P<0.01)；與MI組比較，ME組和MG組大鼠心梗面積顯著縮小(P<0.05)，且MGE組梗死面積縮小更加明顯(P<0.01，圖4)。

圖3 本研究心梗邊緣區(qū)大鼠心肌細(xì)胞增殖的激光共聚焦掃描顯微鏡觀察結(jié)果與統(tǒng)計(jì)圖Figure 3.The Immunofluorescent Results of Cardiomyocyte Proliferation Detected by LSCM in Peri-infarct Area of Rats and Statistical Graph

圖4 本研究心梗大鼠心臟TTC染色結(jié)果與統(tǒng)計(jì)圖Figure 4.The Results of TTC Staining of Rat Hearts with Myocardial Infarction and Statistical Graph

血流動(dòng)力學(xué)和超聲心動(dòng)檢測(cè)結(jié)果顯示，與Sham組比較，MI組大鼠LVEF、LVFS、LVSP、LV+dp/dtmax和LV-dp/dtmax顯著降低(P<0.01)，LVEDP顯著升高(P<0.01)，心功能嚴(yán)重受損；與MI組比較，ME組和MG組大鼠LVEF、LVFS、LVSP、LV+dp/dtmax和LV-dp/dtmax顯著升高(P<0.05)，LVEDP顯著下降(P<0.05)； MGE組大鼠LVEF、LVFS、LVSP、LV +dp/dtmax和LV-dp/dtmax升高更顯著(P<0.01)，LVEDP下降更顯著(P<0.01)，心梗大鼠心功能改善明顯(圖5、圖6)。

圖5 本研究心梗大鼠心動(dòng)超聲結(jié)果與統(tǒng)計(jì)圖Figure 5.Echocardiography of Myocardial Infarction Rats and Statistical Graph

圖6 本研究心梗大鼠心臟血流動(dòng)力學(xué)結(jié)果與統(tǒng)計(jì)圖Figure 6.Hemodynamic Statistical Results of Myocardial Infarction Rats and Statistical Graph

3 分析與討論

3.1 間歇運(yùn)動(dòng)和/或G-CSF均可促進(jìn)心梗大鼠動(dòng)員干細(xì)胞歸巢與分化

近年的研究發(fā)現(xiàn)，干細(xì)胞在心肌再生過(guò)程中扮演著重要角色[29，34,42]，心臟干細(xì)胞歸巢行為的發(fā)現(xiàn)為MI后心臟損傷修復(fù)的研究帶來(lái)新希望。eCSCs/CPCs作為心肌細(xì)胞增殖的最直接來(lái)源細(xì)胞，在損傷發(fā)生后可被炎性因子誘導(dǎo)激活，進(jìn)一步分化為心肌細(xì)胞并修復(fù)受損心肌組織[16,38]。研究發(fā)現(xiàn)，注入c-kit+CSCs可促進(jìn)心肌修復(fù)，降低29%的梗死面積和左室重塑，提升心功能[11]。因此，促進(jìn)心肌c-kit+細(xì)胞數(shù)量增加具有重要意義。Orlic等研究證實(shí)，BMSCs可被誘導(dǎo)分化為心肌細(xì)胞、內(nèi)皮細(xì)胞和血管平滑肌細(xì)胞，促進(jìn)心肌細(xì)胞新生，改善血供和心功能，降低個(gè)體死亡率[39,40]。此外，近期研究發(fā)現(xiàn)，干細(xì)胞同樣可通過(guò)旁分泌效應(yīng)，促使存活的心肌細(xì)胞再生[18,32]。CD29為BMSCs的主要標(biāo)記物之一，來(lái)自大鼠股骨和脛骨的BMSCs與新生大鼠心室肌細(xì)胞共培養(yǎng)可分化為心肌細(xì)胞，提升心功能[29]。因此認(rèn)為，促進(jìn)干細(xì)胞動(dòng)員和提高損傷部位干細(xì)胞水平對(duì)心肌再生具有積極作用。外源性注射G-CSF可通過(guò)SDF-1/CXCR4信號(hào)軸誘導(dǎo)BMSCs趨化，參與組織再生[37，52]。G-CSF作為強(qiáng)有力的干細(xì)胞動(dòng)員劑，可有效促進(jìn)骨髓干細(xì)胞進(jìn)入循環(huán)或直接趨化至心肌損傷部位，促進(jìn)心肌組織再生。有研究報(bào)道，運(yùn)動(dòng)可促進(jìn)CSCs和循環(huán)中干細(xì)胞的激活、動(dòng)員、歸巢和分化[14,54]，誘導(dǎo)MI患者循環(huán)內(nèi)皮祖細(xì)胞水平增加[25]。

3.2 間歇運(yùn)動(dòng)和/或G-CSF促進(jìn)心梗大鼠心肌細(xì)胞增殖

心肌梗死后有功能的心肌細(xì)胞大量丟失是造成心肌組織惡性重塑的主要原因之一。如何有效促進(jìn)內(nèi)源性心肌增殖是近年來(lái)臨床醫(yī)學(xué)和基礎(chǔ)研究的熱點(diǎn)問(wèn)題。有文獻(xiàn)報(bào)道，正常生理和病理狀態(tài)下，心肌細(xì)胞存在增殖現(xiàn)象。Kajstura等發(fā)現(xiàn)，缺血心肌中處于有絲分裂期的心肌細(xì)胞數(shù)量比正常生理狀態(tài)下增加了近10倍[20]。Bostrom等研究發(fā)現(xiàn)，游泳可促進(jìn)心肌PCNA蛋白表達(dá)，誘導(dǎo)心肌細(xì)胞發(fā)生有絲分裂，其機(jī)制可能與C/EBP beta信號(hào)有關(guān)[7]。Waring等報(bào)道，有氧運(yùn)動(dòng)可上調(diào)NRG1、FGF2和Periostin等多種細(xì)胞生長(zhǎng)因子表達(dá)，增加Brdu+和Ki67+心肌細(xì)胞數(shù)目[57]。以上研究表明，運(yùn)動(dòng)可促進(jìn)正常生理狀態(tài)下心肌細(xì)胞的增殖。本研究采用PCNA和cTnT雙陽(yáng)性表達(dá)來(lái)反映心肌細(xì)胞增殖水平。結(jié)果發(fā)現(xiàn)，MI后的第4周，梗死邊緣區(qū)心肌組織中檢測(cè)到PCNA+和cTNT+雙陽(yáng)性細(xì)胞水平顯著增加，證實(shí)了MI后心肌細(xì)胞增殖被激活。ME組和MG組PCNA+心肌細(xì)胞比MI組大鼠顯著增加，說(shuō)明間歇運(yùn)動(dòng)和G-CSF均可使梗死邊緣區(qū)心肌細(xì)胞產(chǎn)生較強(qiáng)的增殖效應(yīng)。MGE組梗死邊緣區(qū)PCNA+心肌細(xì)胞比ME組和MG組顯著，提示，間歇運(yùn)動(dòng)協(xié)同G-CSF能產(chǎn)生比單一因素干預(yù)更強(qiáng)的心肌細(xì)胞增殖效應(yīng)。

3.3 間歇運(yùn)動(dòng)和/或G-CSF縮小心肌梗死面積，提升心功能

研究報(bào)道，心肌梗死后的運(yùn)動(dòng)康復(fù)或給予G-CSF可改善心肌損傷后心功能[4,16]。本研究顯示，間歇運(yùn)動(dòng)和G-CSF均可縮小心梗面積，二者協(xié)同作用效果更為顯著，說(shuō)明間歇運(yùn)動(dòng)和G-CSF在心肌梗死后心肌組織再重塑方面發(fā)揮積極作用。本研究進(jìn)一步發(fā)現(xiàn)，心肌梗死后大鼠心臟LVEF、LVFS、LVSP、LV+dp/dtmax和LV-dp/dtmax顯著降低，LVEDP顯著升高，心功能嚴(yán)重惡化。心梗后進(jìn)行間歇運(yùn)動(dòng)或G-CSF干細(xì)胞動(dòng)員，均可以顯著升高LVEF、LVFS、LVSP、LV+dp/dtmax和LV-dp/dtmax和降低LVEDP，心功能得到有效改善，間歇運(yùn)動(dòng)和G-CSF聯(lián)合作用效果優(yōu)于單一因素。

間歇運(yùn)動(dòng)或G-CSF均可動(dòng)員心梗大鼠骨髓干細(xì)胞進(jìn)入外周血，有效增加循環(huán)血中c-kit+和CD29+單個(gè)核細(xì)胞比率和增加心肌梗死邊緣區(qū)c-kit和CD29蛋白表達(dá)，提示，進(jìn)入外周血的干細(xì)胞可能在趨化因子SDF-1作用下沿SDF-1/CXCR4信號(hào)軸向心肌梗死及其邊緣區(qū)歸巢、分裂和分化，生成新的心肌細(xì)胞或者通過(guò)干細(xì)胞旁分泌作用，促進(jìn)心肌細(xì)胞數(shù)目增加，縮小梗死面積，提升心功能。間歇運(yùn)動(dòng)聯(lián)合G-CSF干預(yù)可更有效動(dòng)員干細(xì)胞歸巢，增加梗死邊緣區(qū)新生心肌細(xì)胞比率，其心功能改善效果優(yōu)于單一因素的作用。

4 結(jié)論

間歇運(yùn)動(dòng)協(xié)同G-CSF干預(yù)可顯著促進(jìn)內(nèi)源性干細(xì)胞動(dòng)員歸巢，誘導(dǎo)心肌細(xì)胞增殖，縮小梗死面積和提升心功能，且二者聯(lián)合效果優(yōu)于單一因素的作用。

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The Observation and Analysis of Stem Cell Mobilization and Endogenous Cardiomyocyte Proliferation Promoted by Interval Exercise and Granulocyte Colony-stimulating Factor Using Confocal Microscopy and Flow Cytometry in Myocardial Infarction Rats

SHI Xiu-chao1,2,CAI Meng-xin1,TIAN Zhen-jun1

Objectives:To discuss the effects of treadmill interval exercise and granulocyte colony-stimulating factor(G-CSF) on stem cell mobilization,endogenous cardiomyocyte proliferation and cardiac function in rats with myocardial infarction(MI).Methods:90 Adult male sprague-dawley rats were randomly divided into:Sham-operated group(Sham),MI group(MI),MI with interval exercise group(ME),MI with G-CSF treatment group(MG)and MI with G-CSF treatment plus interval exercise group(MGE).The MI model of rats was established by ligation of the left anterior descending (LAD) coronary artery.Rats in ME and MGE groups were subjected to 3-week treadmill interval exercise seven days after myocardial infarction.Rats in MG and MGE groups were injected with rhG-CSF subcutaneously 1h after myocardial infarction,10 μg/kg/d for 5 days.Cardiomyocyte proliferation ratio was detected by immunofluorescence and calculated in the peri-infarct region.C-kit and CD29 positive peripheral blood mononuclear cells (PBMCs) were counted by flow cytometer.C-kit and CD29 expression in peri-infarct area was measured by western blot.Hearts were picked for TTC dyeing to decide the myocardial infarction area of each group.Hemodynamic measurement and echocardiography were performed to evaluate cardiac function.Results:Compared to Sham group,the ratio of PCNA and cTnT dual positive cardiomyocytes,c-kit+and CD29+cells ratio in the PBMCs,c-kit and CD29 expression in the peri-infarct region,myocardial infarction area were significantly increased and cardiac performance decreased significantly in MI rats;compared to MI rats in peri-infarct region,percentage of PCNA and cTnT dual positive cardiomyocytes,c-kit and CD29 positive PBMCs ratio,the expression of c-kit and CD29 protein in the peri-infarct region,cardiac performance were increased significantly,and myocardial infarction area decreased significantly in ME,MG and MGE group.The combined treatment with G-CSF and interval exercise had a better effect than either of them．Conclusions:Interval exercise or G-CSF could promote stem cell mobilization and homing,cardiomyocyte proliferation,cardiac performance and decrease myocardial infarction area significantly in MI rats.The better effects were shown with the combined treatment.

myocardialinfarction;intervalexercise;granulocytecolony-stimulatingfactor;stemcellmobilization;cardiomyocyteproliferation；rat;animalexperiment

2015-10-23；

2016-02-01

國(guó)家自然科學(xué)基金資助項(xiàng)目(31171141)。

史秀超(1973-)，男，陜西城固人，講師，在讀博士研究生，主要研究方向?yàn)檫\(yùn)動(dòng)心血管生物學(xué)，E-mail：tianzhj2015@hotmail.com；蔡夢(mèng)昕(1987-)，女，河南商丘人，在讀博士研究生，主要研究方向?yàn)檫\(yùn)動(dòng)心血管生物學(xué)；田振軍(1965-)，男，陜西綏德人，教授，博士研究生導(dǎo)師，主要研究方向?yàn)檫\(yùn)動(dòng)心血管生物學(xué)，E-mail:tianzj611@hotmail.com。

1.陜西師范大學(xué) 體育學(xué)院暨運(yùn)動(dòng)生物學(xué)研究所，陜西 西安 710119；2.渭南師范學(xué)院 化學(xué)與環(huán)境學(xué)院，陜西 渭南 714000 1.Shaanxi Normal University,Xi’an 710119,China；2.Weinan Normal University,Weinan 714000,China.

G804.7

A

10.16469/j.css.201604008