史秀超，蔡夢昕，田振軍

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

史秀超1,2，蔡夢昕1，田振軍1

目的：探討跑臺間歇運動結(jié)合粒細胞集落刺激因子對心肌梗死大鼠干細胞動員的效果和在內(nèi)源性心肌細胞增殖中的作用及其對心功能的影響。方法：90只成體雄性SD大鼠隨機分為假手術(shù)組(Sham)、心梗組(MI)、心梗+間歇運動組(ME)、心梗+粒細胞集落刺激因子(G-CSF)組(MG)和心梗+G-CSF+間歇運動組(MGE)。結(jié)扎大鼠左冠狀動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周后進行3周跑臺間歇運動。MG和MGE組大鼠術(shù)后1 h皮下注射人源重組粒細胞集落刺激因子(rhG-CSF)，10 μg/kg/d×5 d，其他各組大鼠給予同劑量生理鹽水。免疫熒光法檢測并計算梗死邊緣區(qū)心肌細胞增殖百分率；流式細胞術(shù)檢測外周血單個核細胞中c-kit+和CD29+細胞百分率；Western Blot方法檢測心肌組織中c-kit和CD29蛋白表達水平。TTC染色檢測心肌梗死面積百分率；彩色多普勒超聲和血流動力學方法檢測心功能。結(jié)果：與Sham組相比，MI組PCNA+心肌細胞百分率、外周血單個核細胞中c-kit+和CD29+細胞百分率、心肌組織中c-kit和CD29蛋白表達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+心肌細胞百分率、外周血單個核細胞中c-kit+和CD29+細胞百分率、心肌組織中c-kit和CD29蛋白表達均顯著增加，梗死面積顯著減小，心功能顯著增強，且MGE組變化最為顯著。結(jié)論：間歇運動或粒細胞集落刺激因子可顯著促進干細胞動員歸巢，誘導心肌細胞增殖，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；間歇運動； 粒細胞集落刺激因子；干細胞動員；心肌細胞增殖；鼠；動物實驗

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

間歇運動是指高強度與低強度鍛煉或自由活動等方式交替進行的運動形式。文獻表明，高強度間歇運動較中強度有氧運動對心臟結(jié)構(gòu)與功能的影響更為顯著[12,17,31,56,59]。 MI患者進行運動康復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]。動物實驗發(fā)現(xiàn)，運動可促進正常生理狀態(tài)下心肌細胞增殖[7,57]。本實驗室前期研究發(fā)現(xiàn)，8周持續(xù)有氧運動可促進正常和MI大鼠心肌組織增殖細胞核抗原(proliferating cell nuclear antigen，PCNA)和細胞增殖標記物Ki67蛋白表達[1]，有效提高心功能。粒細胞集落刺激因子(granulocyte colony-stimulating factor，G-CSF)可促進骨髓間充質(zhì)干細胞和造血干細胞等趨化至損傷部位，參與心肌損傷后的修復[27，45]。運動可促進血液循環(huán)中干細胞數(shù)目增多[8,9,22]，但間歇運動結(jié)合干細胞動員劑G-CSF能否更有效促進自體干細胞動員和心臟內(nèi)源性心肌細胞增殖，缺乏直接證據(jù)。文獻表明，心?；蛐墓：笞⑸銰-CSF動員BMSCs入血均存在時間窗[44，55]。根據(jù)G-CSF動員BMSCs時間窗和心臟運動負荷產(chǎn)生效應(yīng)的最短時間，本研究采用心梗1 h后給予G-CSF動員，手術(shù)后1周進行3周間歇運動干預(yù)，采用激光共聚焦顯微術(shù)和流式細胞術(shù)及Western Blot方法，觀察與分析間歇運動和粒細胞集落刺激因子對心梗大鼠內(nèi)源性心肌細胞增殖和干細胞動員的效果。

1 材料與方法

1.1 主要儀器和試劑

主要儀器：ALC V8 動物呼吸機、BM Ⅱ型病理組織包埋機、LEICA RM 2126切片機、尼康Nikon C2 Plus激光共聚焦顯微鏡(laser scanning confocal microscope,LSCM)、PowerLab/8S生理信號采集系統(tǒng)、美國BD公司FACS Aria流式細胞儀、BioTek epoch超微量微孔板分光光度計、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標記的山羊抗兔抗體和TRITC標記的山羊抗小鼠抗體(武漢博士德生物工程有限公司)、動物外周血和臟器組織單個核細胞分離試劑盒(灝洋生物公司)。

1.2 動物分組與MI模型制備

動物分組：清潔級3月齡Sprague Dawley雄性大鼠90只，體重180～220 g，購于西安交通大學實驗動物管理中心(動物質(zhì)量合格證號：SCXK2013-003號)。隨機分為假心梗組(Sham)、心梗組(MI)、心梗+間歇運動組(ME)、心梗+G-CSF組(MG)和心梗+G-CSF+間歇運動組(MGE)，每組18只。大鼠飼養(yǎng)室溫度保持20～29℃，相對濕度50%～60%，采用國家標準嚙齒類動物干燥飼料喂養(yǎng)，自由飲食。

MI大鼠模型制備：采用左冠狀動脈前降支結(jié)扎法[1]，同時監(jiān)測心電圖并觀察結(jié)扎點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 間歇運動和G-CSF給藥方案

1.4 心功能檢測

心動超聲檢測：實驗造模結(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，顯示胸骨旁左室長軸切面，探頭順時針旋轉(zhuǎn)90°顯示左室短軸切面圖像，于胸骨旁左室長軸切面二維測量左室室壁厚度及梗死區(qū)范圍。在二維導引下，將取樣線置于左室健索水平，取M型超聲心動圖，速度為200 mm/s。測量左室舒張末期內(nèi)徑(left ventricle diastolic diameter，LVDd)及左室收縮末期內(nèi)徑(left ventricle systolic diameter,LVDs)。儀器自動計算左室短軸縮短率(left ventricular fractional shortening，LVFS)=(LVDd-LVDs)/LVDd ×100%和左室射血分數(shù)(Left ventricle ejection fraction，LVEF)=[(LVDd3-LVDs3)/LVDd3]×100%。測量指標均取3個連續(xù)心動周期的平均值。

血流動力學檢測：經(jīng)右頸總動脈逆行插管至大鼠左心室，以多導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

大鼠心功能測試結(jié)束后，立刻斷頭取血，肝素鈉抗凝，分離外周血單個核細胞。摘取心臟，每組隨機取6個心臟中性甲醛固定，常規(guī)石蠟包埋，連續(xù)切片(厚度5 μm)，用于免疫熒光實驗；另取6個心臟液氮速凍，置-80℃冰箱，用于western blot 實驗。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)碼相機拍照，統(tǒng)計梗死區(qū)(乳白色)與非梗死區(qū)(紅色)面積，并計算梗死面積百分率。

1.6 外周血單個核細胞分離和流式細胞儀檢測

大鼠外周血單個核細胞(peripheral blood mononuclear cell，PBMC)分離嚴格按照灝洋生物公司的“各種動物外周血和臟器組織單個核細胞分離試劑盒操作說明”進行。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核細胞懸液，注入5 ml細胞洗滌液試管中，以500倍重力離心20 min，棄上清，沉淀的細胞用PBS重懸2次即得單個核細胞懸液，500倍重力離心20 min ，沉淀的單個核細胞用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標記的山羊抗兔二抗的PBMCs作為陰性對照，F(xiàn)ACS Aria 流式細胞儀進行檢測。

1.7 免疫熒光檢測

石蠟切片脫蠟至水，pH6.0枸櫞酸緩沖液抗原修復，切片于修復液中降至室溫，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)和小鼠抗大鼠細胞增殖核抗原(PCNA，1∶100)，濕盒中4℃過夜。室溫復溫45 min，PBS清洗，滴加FITC標記的山羊抗兔抗體(1∶50)、TRITC標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)置空白對照和陰性對照。尼康Nikon C2 Plus激光共聚焦顯微鏡觀察拍照。

1.8 Western Blot實驗

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，封閉后孵育干細胞表面抗原c-kit和CD29(濃度均為1∶500)，4℃過夜。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軟件進行處理，采用One-Way ANOVA進行統(tǒng)計學分析，組間顯著性差異水平為P<0.05和P<0.01。

2 實驗結(jié)果

2.1 間歇運動和/或G-CSF可促進心梗大鼠骨髓干細胞動員增加

c-kit+和CD29+均為骨髓干細胞標記物[29,53]。通過分離大鼠外周血單個核細胞，用FITC標記c-kit和CD29抗原，流式細胞儀計數(shù)被標記的和未標記的細胞，計算免疫標記的單個核細胞百分率，反映骨髓干細胞動員效果。結(jié)果表明，與Sham組比較，MI組外周血單個核細胞中c-kit+和CD29+細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+細胞的流式細胞儀檢測結(jié)果與統(tǒng)計圖Figure 1.The Result of c-kit and CD29 Positive PBMCs Detected by Flow Cytometer and Statistical Graph

2.2 間歇運動和/或G-CSF可促進大鼠心梗邊緣區(qū)c-kit和CD29蛋白表達增加

Western Blot檢測結(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蛋白表達結(jié)果與統(tǒng)計圖Figure 2.Expression of c-kit and CD29 Protein in Peri-infarct Area of Rat Heart and Statistical Graph

2.3 間歇運動和/或G-CSF干預(yù)可促進心梗大鼠心肌細胞增殖

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

2.4 間歇運動和/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)大鼠心肌細胞增殖的激光共聚焦掃描顯微鏡觀察結(jié)果與統(tǒng)計圖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)計圖Figure 4.The Results of TTC Staining of Rat Hearts with Myocardial Infarction and Statistical Graph

血流動力學和超聲心動檢測結(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超聲結(jié)果與統(tǒng)計圖Figure 5.Echocardiography of Myocardial Infarction Rats and Statistical Graph

圖6 本研究心梗大鼠心臟血流動力學結(jié)果與統(tǒng)計圖Figure 6.Hemodynamic Statistical Results of Myocardial Infarction Rats and Statistical Graph

3 分析與討論

3.1 間歇運動和/或G-CSF均可促進心梗大鼠動員干細胞歸巢與分化

近年的研究發(fā)現(xiàn)，干細胞在心肌再生過程中扮演著重要角色[29，34,42]，心臟干細胞歸巢行為的發(fā)現(xiàn)為MI后心臟損傷修復的研究帶來新希望。eCSCs/CPCs作為心肌細胞增殖的最直接來源細胞，在損傷發(fā)生后可被炎性因子誘導激活，進一步分化為心肌細胞并修復受損心肌組織[16,38]。研究發(fā)現(xiàn)，注入c-kit+CSCs可促進心肌修復，降低29%的梗死面積和左室重塑，提升心功能[11]。因此，促進心肌c-kit+細胞數(shù)量增加具有重要意義。Orlic等研究證實，BMSCs可被誘導分化為心肌細胞、內(nèi)皮細胞和血管平滑肌細胞，促進心肌細胞新生，改善血供和心功能，降低個體死亡率[39,40]。此外，近期研究發(fā)現(xiàn)，干細胞同樣可通過旁分泌效應(yīng)，促使存活的心肌細胞再生[18,32]。CD29為BMSCs的主要標記物之一，來自大鼠股骨和脛骨的BMSCs與新生大鼠心室肌細胞共培養(yǎng)可分化為心肌細胞，提升心功能[29]。因此認為，促進干細胞動員和提高損傷部位干細胞水平對心肌再生具有積極作用。外源性注射G-CSF可通過SDF-1/CXCR4信號軸誘導BMSCs趨化，參與組織再生[37，52]。G-CSF作為強有力的干細胞動員劑，可有效促進骨髓干細胞進入循環(huán)或直接趨化至心肌損傷部位，促進心肌組織再生。有研究報道，運動可促進CSCs和循環(huán)中干細胞的激活、動員、歸巢和分化[14,54]，誘導MI患者循環(huán)內(nèi)皮祖細胞水平增加[25]。

3.2 間歇運動和/或G-CSF促進心梗大鼠心肌細胞增殖

心肌梗死后有功能的心肌細胞大量丟失是造成心肌組織惡性重塑的主要原因之一。如何有效促進內(nèi)源性心肌增殖是近年來臨床醫(yī)學和基礎(chǔ)研究的熱點問題。有文獻報道，正常生理和病理狀態(tài)下，心肌細胞存在增殖現(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)，游泳可促進心肌PCNA蛋白表達，誘導心肌細胞發(fā)生有絲分裂，其機制可能與C/EBP beta信號有關(guān)[7]。Waring等報道，有氧運動可上調(diào)NRG1、FGF2和Periostin等多種細胞生長因子表達，增加Brdu+和Ki67+心肌細胞數(shù)目[57]。以上研究表明，運動可促進正常生理狀態(tài)下心肌細胞的增殖。本研究采用PCNA和cTnT雙陽性表達來反映心肌細胞增殖水平。結(jié)果發(fā)現(xiàn)，MI后的第4周，梗死邊緣區(qū)心肌組織中檢測到PCNA+和cTNT+雙陽性細胞水平顯著增加，證實了MI后心肌細胞增殖被激活。ME組和MG組PCNA+心肌細胞比MI組大鼠顯著增加，說明間歇運動和G-CSF均可使梗死邊緣區(qū)心肌細胞產(chǎn)生較強的增殖效應(yīng)。MGE組梗死邊緣區(qū)PCNA+心肌細胞比ME組和MG組顯著，提示，間歇運動協(xié)同G-CSF能產(chǎn)生比單一因素干預(yù)更強的心肌細胞增殖效應(yīng)。

3.3 間歇運動和/或G-CSF縮小心肌梗死面積，提升心功能

研究報道，心肌梗死后的運動康復或給予G-CSF可改善心肌損傷后心功能[4,16]。本研究顯示，間歇運動和G-CSF均可縮小心梗面積，二者協(xié)同作用效果更為顯著，說明間歇運動和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，心功能嚴重惡化。心梗后進行間歇運動或G-CSF干細胞動員，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，間歇運動和G-CSF聯(lián)合作用效果優(yōu)于單一因素。

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

4 結(jié)論

間歇運動協(xié)同G-CSF干預(yù)可顯著促進內(nèi)源性干細胞動員歸巢，誘導心肌細胞增殖，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。

[1]蔡夢昕,張娟娟,史秀超,等.有氧運動和G-CSF干預(yù)對心梗大鼠心肌細胞再生的影響及其機制探討[J].體育科學,2013，33(5):50-58.

[2]陳運賢,歐瑞明,鐘雪云,等.粒細胞集落刺激因子動員骨髓干細胞治療大鼠急性心肌梗塞[J].中國病理生理雜志,2002,18(1):1-3.

[3]田振軍,賀志雄,劉智煒,等.持續(xù)和間歇有氧運動對心梗大鼠心肌Myostatin及其受體表達的影響[J].體育科學,2013，33(11):66-74.

[4]ACHILLI F,MALAFRONTE C,LENATTI L,etal.Granulocyte colony-stimulating factor attenuates left ventricular remodelling after acute anterior STEMI:Results of the single-blind,randomized,placebo- controlled multicentre stem cell mobilization in acute myocardial infarction (STEM-AMI) trial[J].Eur J Heart Fail,2010,12(10):1111-1121.

[5]ANDJIC M,SPIROSKI D,ILIC S O,etal.Effects of short-term exercise training in patients following acute myocardial infarction treated with primary percutaneous coronary intervention[J].Eur J Phys Rehabil Med.2015,Nov 19.[Epub ahead of print].

[6]BERGMANN O,BHARDWAJ R D,BERNARD S,etal.Evidence for cardiomyocyte renewal in humans[J].Sci,2009,324(5923):98-102.

[7]BOSTROM P,MANN N,WU J,etal.C/EBPbeta controls exercise-induced cardiac growth and protects against pathological cardiac remodeling[J].Cell,2010,143(7):1072-1083.

[8]BREHM M,PICARD F,EBNER P,etal.Effects of exercise training on mobilization and functional activity of blood-derived progenitor cells in patients with acute myocardial infarction[J].Eur J Med Res,2009,14(9):393-405.

[9]CHENG F C,SHEU M L,SU H L,etal.The effect of exercise on mobilization of hematopoietic progenitor cells involved in the repair of sciatic nerve crush injury[J].J Neurosurg,2013,118(3):594-605.

[10]CIOLAC E G,BOCCHI E A,BORTOLOTTO L A,etal.Effects of high-intensity aerobic interval training vs.moderate exercise on hemodynamic,metabolic and neuro-humoral abnormalities of young normotensive women at high familial risk for hypertension[J].Hypertens Res,2010,33(8):836-843.

[11]DAWN B,STEIN A B,URBANEK K,etal.Cardiac stem cells delivered intravascularly traverse the vessel barrier,regenerate infarcted myocardium,and improve cardiac function[J].PNAS,2005,102(10):3766-3771.

[12]ELLIOTT A D,RAJOPADHYAYA K,BENTLEY D J,etal.Interval training versus continuous exercise in patients with coronary artery disease:A meta-analysis[J].Heart Lung Circ,2015,24(2):149-157.

[13]ELLISON G M,NADAL-GINARD B,TORELLA D.Optimizing cardiac repair and regeneration through activation of the endogenous cardiac stem cell compartment[J].J Cardiovasc Transl Res,2012,5(5):667-677.

[14]FIGUEIREDO P A,APPELL C H,DUARTE J A.Cardiac regeneration and cellular therapy:Is there a benefit of exercise?[J].Int J Sports Med,2014,35(3):181-190.

[15]FONTES-CARVALHO R,SAMPAIO F,TEIXEIRA M,etal.The role of a structured exercise training program on cardiac structure and function after acute myocardial infarction:Study protocol for a randomized controlled trial[J].Trials,2015,16:90.

[16]FUKUHARA S,TOMITA S,NAKATANI T,etal.G-CSF promotes bone marrow cells to migrate into infarcted mice heart,and differentiate into cardiomyocytes[J].Cell Transplant,2004,13(7-8):741-748．

[17]GODFREY R,THEOLOGOU T,DELLEGROTTAGLIE S,etal.The effect of high-intensity aerobic interval training on postinfarction left ventricular remodelling[J].BMJ Case Rep,2013,DOI:10.1136/bcr-2012-007668.

[18]GNECCHI M,ZHANG Z,NI A,etal.Paracrine mechanisms in adult stem cell signaling and therapy[J].Circ Res,2008,103(11):1204-1219.

[19]HARAM P M,KEMI O J,LEE S J,etal.Aerobic interval training vs.continuous moderate exercise in the metabolic syndrome of rats artificially selected for low aerobic capacity[J].Cardiovasc Res.2009,81(4):723-732.

[20]KAJSTURA J,LERI A,FINATO N,etal.Myocyte proliferation in end-stage cardiac failure in humans[J].PNAS,1998,95(15):8801-8805.

[21]KAJSTURA J,ROTA M,CAPPETTA D,etal.Cardiomyogenesis in the aging and failing human heart[J].Circulation,2012,126(15):1869-1881.

[22]KESER I,SUYANI E,AKI S Z,etal.The positive impact of regular exercise program on stem cell mobilization prior to autologous stem cell transplantation[J].Transfus Apher Sci,2013,49(2):302-306.

[23]KIM C,CHOI H E,LIM M H.Effect of high interval training in acute myocardial infarction patients with drug-eluting stent[J].Am J Phys Med Rehabil.2015,94(10 Suppl 1):879-886.

[24]LAFLAMME M A,MURRY C E.Heart regeneration[J].Nature,2011,473(7347):326-335.

[25]LAUFS U,WERNER N,LINK A,etal.Physical training increases endothelial progenitor cells,inhibits neointima formation,and enhances angiogenesis[J].Circulation,2004,109(2):220-226.

[26]LAUGWITZ K L,MORETTI A,LAM J,etal.Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages[J].Nature,2005,433(7026):647-653.

[27]LEONE A M,RUTELLA S,BONANNO G,etal.Mobilization of bone marrow-derived stem cells after myocardial infarction and left ventricular function[J].Eur Heart J,2005,26(12):1196-1204.

[28]LI M, IZPISUA B J C.Mending a Faltering Heart[J].Circ Res,2016,118(2):344-351.

[29]LI X,YU X,LIN Q,etal.Bone marrow mesenchymal stem cells differentiate into functional cardiac phenotypes by cardiac microenvironment[J].J Mol Cell Cardiol,2007,42(2):295- 303.

[30]LIU J,SLUIJTER J P,GOUMANS M J,etal.Cell therapy for myocardial regeneration[J].Curr Mol Med,2009,9(3):287-298.

[31]LU K,WANG L,WANG C,etal.Effects of high-intensity interval versus continuous moderate-intensity aerobic exercise on apoptosis,oxidative stress and metabolism of the infarcted myocardium in a rat model[J].Mol Med Rep,2015,12(2):2374-2382.

[32]MALTAIS S,TREMBLAY J P,PERRAULT L P,etal.The paracrine effect:Pivotal mechanism in cell-based cardiac repair[J].J Cardiovasc Transl Res,2010,3(6):652-662.

[33]MCGREGOR G,GAZE D,OXBOROUGH D,etal.Reverse left ventricular remodelling- effect of cardiac rehabilitation exercise training in myocardial infarction patients with preserved ejection fraction[J].Eur J Phys Rehabil Med,2015,Nov 4.[Epub ahead of print].

[34]MERCOLA M,RUIZ-LOZANO P,SCHNEIDER M D.Cardiac muscle regeneration:Lessons from development [J].Genes Dev,2011,25(4):299-309.

[35]MOLLOVA M,BERSELL K,WALSH S,etal.Cardiomyocyte proliferation contributes to heart growth in young humans[J].PNAS,2013,110(4):1446-1451.

[36]MOTOHIRO M,YUASA F,HATTORI T,etal.Cardiovascular adaptations to exercise training after uncomplicated acute myocardial infarction[J].Am J Phys Med Rehabil.2005,84(9):684-691.

[37]NIENABER C A,PETZSCH M,KLEINE H D,etal.Effects of granulocyte-colony-stimulating factor on mobilization of bone marrow-derived stem cells after myocardial infarction in humans[J].Nat Clin Pract Cardiovasc Med,2006,3(Suppl 1)：S73-S77．

[38]OH H,BRADFUTE S B,GALLARDO T D,etal.Cardiac progenitor cells from adult myocardium:Homing,differentiation,and fusion after infarction[J].PNAS,2003,100(21):12313-12318.

[39]ORLIC D,KAJSTURA J,CHIMENTI S,etal.Bone marrow stem cells regenerate infarcted myocardium[J].Pediatr Transplant,2003,7(Suppl 3):86-88.

[40]ORLIC D,KAJSTURA J,CHIMENTI S,etal.Mobilized bone marrow cells repair the infarcted heart,improving function and survival[J].PNAS,2001,98(18):10344-10349.

[41]PEIXOTO T C,BEGOT I,BOLZAN D W,etal.Early exercise-based rehabilitation improves health-related quality of life and functional capacity after acute myocardial infarction:A randomized controlled trial[J].Can J Cardiol,2015,31(3):308-313.

[42]PFEFFER M A,BRAUNWALD E.Ventricular remodeling after myocardial infarction.Experimental observations and clinical implications[J].Circulation,1990,81(4):1161- 1172.

[43]REDDY K,KHALIQ A,HENNING R J.Recent advances in the diagnosis and treatment of acute myocardial infarction[J].World J Cardiol,2015,7(5):243-276.

[44]RIPA R S,HAACK-SORENSEN M，WANG Y，etal.Bone marrow derived mesenchymal cell mobilization by granulocyte-colony stimulating factor after acute myocardial infarction:Results from the Stem Cells in Myocardial Infarction (STEMMI) trial[J].Circulation.2007,116(11 Suppl):124-130.

[45]RIPA R S,JORGENSEN E,WANG Y,etal.Stem cell mobilization induced by subcutaneous granulocyte- colony stimulating factor to improve cardiac regeneration after acute ST- elevation myocardial infarction:Result of the double-blind,randomized,placebo-controlled stem cells in myocardial infarction (STEMMI) trial[J].Circulation,2006,113(16):1983-1992.

[46]RIVAS-ESTANY E,SIXTO-FERNANDEZ S,BARRERA-SARDUY J,etal.Effects of long-term exercise training on left ventricular function and remodeling in patients with anterior wall myocardial infarction[J].Arch Cardiol Mex,2013,83(3):167-173.

[47]ROGNMO O,HETLAND E,HELGERUD J,etal.High intensity aerobic interval exercise is superior to moderate intensity exercise for increasing aerobic capacity in patients with coronary artery disease[J].Eur J Cardiovasc Prev Rehabil,2004,11(3):216-222.

[48]ROLIM N,SKARDAL K,HOYDAL M,etal.Aerobic interval training reduces inducible ventricular arrhythmias in diabetic mice after myocardial infarction[J].Basic Res Cardiol,2015,110(4):44.

[49]RUSSO V,YOUNG S,HAMILTON A,etal.Mesenchymal stem cell delivery strategies to promote cardiac regeneration following ischemic injury[J].Biomaterials,2014,35(13):3956-3974.

[50]SEGERS V F,LEE R T.Stem-cell therapy for cardiac disease[J].Nature,2008,451(7181):937-942.

[51]SINGLA D K,LONG X,GLASS C,etal.Induced pluripotent stem (iPS) cells repair and regenerate infarcted myocardium[J].Mol Pharm,2011,8(5):1573-1581.

[52]TAGHAVI S,GEORGE J C.Homing of stem cells to ischemic myocardium[J].Am J Transl Res,2013,5(4):404-411.

[53]THEISS H D,VALLASTER M,RISCHPLER C,etal.Dual stem cell therapy after myocardial infarction acts specifically by enhanced homing via the SDF-1/CXCR4 axis[J].Stem Cell Res,2011,7(3):244-255.

[54]WAHL P,BRIXIUS K,BLOCH W.Exercise-induced stem cell activation and its implication for cardiovascular and skeletal muscle regeneration[J].Minim Invasive Ther Allied Technol,2008,17(2):91-99.

[55]WANG Y,JOHNSEN H E,MORTENSEN S,etal.Changes in circulating mesenchymal stem cells,stem cell homing factor,and vascular growth factors in patients with acute ST elevation myocardial infarction treated with primary percutaneous coronary intervention[J].Heart.2006,92(6):768-774.

[56]WARBURTON D E,MCKENZIE D C,HAYKOWSKY M J,etal.Effectiveness of high-intensity interval training for the rehabilitation of patients with coronary artery disease[J].Am J Cardiol,2005,95(9):1080-1084.

[57]WARING C D,HENNING B J,SMITH A J,etal.Cardiac adaptations from 4 weeks of intensity-controlled vigorous exercise are lost after a similar period of detraining[J].Physiol Rep,2015,3(2):e12302.

[58]WEN Z,MAI Z,ZHANG H,etal.Local activation of cardiac stem cells for post-myocardial infarction cardiac repair[J].J Cell Mol Med,2012,16(11):2549-2563.

[59]WISL?FF U,ELLINGSEN ?,KEMI O J.High-intensity interval training to maximize cardiac benefits of exercise training？[J].Exe Sport Sci Rev,2009,37(3):139-146.

[60]WISL?FF U,LOENNECHEN J P,CURRIE S,etal.Aerobic exercise reduces cardiomyocyte hypertrophy and increases contractility,Ca2+sensitivity and SERCA-2 in rat after myocardial infarction[J].Cardiovasc Res,2002,54(1):162-174.

[61]WISL?FF U,ST?YLEN A,LOENNECHEN J P,etal.Superior cardiovascular effect of aerobic interval training versus moderate continuous training in heart failure patients:A randomized study[J].Circulation,2007,115(24):3086-3094.

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

國家自然科學基金資助項目(31171141)。

史秀超(1973-)，男，陜西城固人，講師，在讀博士研究生，主要研究方向為運動心血管生物學，E-mail：tianzhj2015@hotmail.com；蔡夢昕(1987-)，女，河南商丘人，在讀博士研究生，主要研究方向為運動心血管生物學；田振軍(1965-)，男，陜西綏德人，教授，博士研究生導師，主要研究方向為運動心血管生物學，E-mail:tianzj611@hotmail.com。

1.陜西師范大學 體育學院暨運動生物學研究所，陜西 西安 710119；2.渭南師范學院 化學與環(huán)境學院，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