蒋 智,贾中申,潘裕佳,,李安洁,韦 方
1贵州省人民医院心内科,贵阳 5500022贵州医科大学临床医学系,贵阳 550025
·论著·
子宫内膜干细胞来源细胞因子鸡尾酒对小鼠心肌缺血再灌注损伤的影响
蒋智1,贾中申2,潘裕佳1,2,李安洁1,韦方1
1贵州省人民医院心内科,贵阳 5500022贵州医科大学临床医学系,贵阳 550025
摘要:目的研究子宫内膜干细胞(EnSCs)来源细胞因子鸡尾酒(EdCC)对小鼠心肌缺血再灌注损伤(I/R)的影响。方法采用Millipore蛋白超滤技术从EnSCs培养液中提取EdCC,通过尾静脉注射治疗小鼠心肌I/R损伤。TTC/Evans Blue染色评估梗死面积,TUNEL染色计数凋亡细胞,Western blot法检测cleaved caspase 3。结果EdCC提取效率为每24 h (222.4±29.3) μg/106细胞,经-80℃冻存后EdCC蛋白浓度及蛋白总量逐渐降低。与I/R组比较,100 μg新鲜提取的EdCC可显著缩小心肌梗死面积(P=0.001),减少梗死周边区细胞凋亡数量(P=0.019),降低cleaved caspase 3表达(P=0.002),增加EdCC剂量不能进一步缩小梗死面积。EdCC经-80 ℃冻存90 d以上心肌保护效应逐渐降低。结论EdCC可能是通过抑制细胞凋亡保护心肌I/R损伤。经-80 ℃冻存30 d以内可保持较强保护效应。
关键词:子宫内膜干细胞;细胞因子;心肌梗死;缺血再灌注损伤;凋亡
ActaAcadMedSin,2016,38(3):253-259
急性心肌梗死已成为国人主要致死原因[1]。急诊再灌注治疗可显著缩小急性心肌梗死的梗死面积从而改善预后,但缺血再灌注损伤(ischemic reperfusion injury,I/R)会加重心肌结构破坏和功能障碍,削弱再灌注治疗的获益[2]。如何防治再灌注损伤保护缺血心肌日益成为急性心肌梗死治疗中亟待解决的问题[3]。单一细胞因子如粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)和促红细胞生成素(erythropoietin,EPO)治疗心肌梗死的临床试验大多得到阴性结果[4- 5],而干细胞移植却取得治疗有效的结论[6],大量研究提示旁分泌效应是其最主要的治疗机制[7]。子宫内膜干细胞(endometrial stem cells,EnSCs)具有间充质干细胞特性,可从月经血中分离获得,具有无创获取、增殖速度快、传代稳定、低免疫原性及无伦理道德问题等优势,适宜异体移植,是干细胞产品中极具潜力的种子细胞[8]。本课题组前期研究发现,人EnSCs移植可通过分泌多种细胞因子保护大鼠心肌细胞,减少心肌梗死面积[9]。为避免直接移植EnSCs潜在的致心律失常[10]、肿瘤形成[11]、血栓栓塞[12]等风险,本研究从EnSCs培养液中提取并浓缩EnSCs来源细胞因子鸡尾酒(EnSCs derived cytokine cocktail,EdCC),并观察了EdCC对小鼠心脏I/R损伤梗死面积和细胞凋亡的影响。
材料和方法
材料8~12周龄雄性健康昆明小鼠,体重25~30 g,购自贵州医科大学实验动物中心。胎牛血清(Hyclone,美国),DMEM/F12细胞培养液(吉诺,中国),PBS(吉诺,中国),25 cm2细胞培养瓶(Corning,美国),Millipore超滤离心管(Millipore,美国),异氟烷(玉研,中国),6- 0手术缝线(Sharpoint,美国),TTC(Sigma,美国),Evans Blue(凯信,中国),多聚甲醛(凯信,中国),TUNEL试剂盒(Invitrogen,美国),OCT包埋剂(Takura,日本),兔抗小鼠cleaved caspase3(CST,美国),ECL显色液(Pierce,美国)。
人EnSCs的培养人EnSCs细胞株购自易文赛生物技术有限公司(杭州,中国),37 ℃快速溶解后用含10%胎牛血清的DMEM/F12于37 ℃、5% CO2培养箱中培养,细胞生长至80%按1∶2或1∶3传代。
EdCC的制备和储存将8×105个EnSCs种入25 cm2培养瓶中,24 h后用PBS洗3次,加入3 ml无血清无酚红DMEM/F12培养液,于37 ℃、5% CO2培养箱中培养,24 h后吸出培养液,5000 r/min离心15 min,将上清加入Millipore超滤离心管,5000 r/min离心30 min,上层浓缩液即为EdCC,培养瓶中EnSCs消化后细胞计数。用200 μl移液器测得EdCC总体积V,然后取1 μl EdCC加99 μl无酚红DMEM/F12,BCA法进行蛋白定量测得蛋白浓度C,蛋白总量Q=C×V,EdCC提取效率E=Q μg/(细胞总数·24 h)。蛋白定量后供实验用或-80 ℃冰箱储存。
小鼠心肌I/R损伤模型小鼠用2%异氟烷持续吸入麻醉,6-0圆针丝线于肺动脉下方2 mm处用活结结扎左冠状动脉前降支开始缺血,将活结线头预留5 mm在胸腔外,5 min后经尾静脉注射药物,30 min后适当用力拉活结线头将活结松开开始再灌注[13]。I/R组注射200 μl无血清DMEM/F12,I/R+EdCC组按实验设计注射不同剂量及不同冻存时间的EdCC(用无血清DMEM/F12稀释成200 μl)。
TTC/Evans Blue染色脱颈椎处死小鼠,将原先松开的线结再次结扎,从升主动脉逆行注入2% Evans Blue染液,剪下心脏用PBS洗去多余染液,保鲜膜包裹后放入-20 ℃冰箱冷冻15 min,取出后垂直左心室长轴从心尖至心底间隔1 mm切成5~6片,放入37 ℃含2% TTC的PBS中避光染色20 min,4%多聚甲醛溶液固定20 min,压平、拍照,非蓝染区域为危险区(area at risk,AAR),白色区域为梗死区(infarct area,IA)。用image-pro plus软件分别计算各切片的左心室面积(left ventricle,LV)、AAR面积和IA面积。梗死面积(IA/AAR%)=IA总和/AAR总和×100%,危险区面积(AAR/LV%)=AAR总和/LV总和×100%。
冰冻切片脱颈椎处死小鼠,经主动脉逆行灌注10 ml冰PBS,剪下心脏用滤纸吸去多余水分,分离多余结缔组织,用10%、20%、30%蔗糖PBS溶液梯度脱水,OCT包埋,液氮速冻,7 μm切片留样。
TUNEL染色冰冻切片于常温放置解冻,晾干,4%多聚甲醛溶液固定10 min,PBS洗3次,0.2% TritonX100穿膜5 min,PBS洗3次,加混合好的TUNEL染液在37 ℃下孵育60 min,PBS洗3次,浸入含1 μg/ml Hoechst的PBS溶液中5 min,PBS洗3次,封片。用荧光显微镜观察,每个样本取5个梗死周边区高倍镜视野,用image-pro plus软件计数TUNEL阳性细胞数。
Western blot检测小鼠脱颈椎处死后立即经主动脉逆行灌注10 ml冰PBS,剪去心包、大血管、右心室游离壁等,仅保留冠脉结扎线以下左心室心肌组织,冰PBS反复冲洗。心肌组织用锡箔纸包裹,液氮速冻,砸碎后移入EP管,加RIPA蛋白裂解液及蛋白酶抑制剂,冰上裂解30 min,期间反复震荡混匀,离心后取上清用BCA法蛋白定量,准备蛋白样品,用15%梯度胶电泳后,转膜,用5 %的脱脂牛奶封闭1 h,膜与兔抗小鼠cleaved caspase 3一抗4℃孵育过夜,PBS-T洗3次,加入HRP偶联羊抗兔二抗于室温孵育2 h,PBS-T洗3次,加ECL液曝光、显影、定影。用扫描仪扫描曝光胶片,用Bandscan软件分析条带像素灰度值。
统计学处理采用SPSS 16.0统计软件,所有数据以均数±标准差表示,2组间数据比较采用独立样本t检验,3组以上数据比较采用单因素方差分析和LSD-t检验,P<0.05为差异有统计学意义。
结果
EnSCs的生长形态人EnSCs贴壁生长,大部分为纺锤形,细胞核圆形位于细胞中间(图1A),细胞间融合后形成旋涡样结构(图1B)。
EdCC的提取及保存效率每15 ml EnSCs上清液经Millipore超滤技术超滤浓缩后的体积为(200.5±11.2) μl,蛋白浓度为(5.57±0.95) μg/μl,EdCC的提取效率为每24 h (222.4±29.3)μg/106细胞。-80 ℃长期储存可导致EdCC蛋白浓度及总量逐渐降低(图2)。
EdCC减轻心肌I/R损伤将小鼠分为I/R组、I/R+EdCC 50 μg组、I/R+EdCC 100 μg组和I/R+EdCC 200 μg组(注射新鲜提取EdCC),再灌注24 h行心脏TTC/Evans Blue染色,结果显示,各组小鼠危险区面积差异无统计学意义(P均>0.05);I/R+EdCC 100 μg组[(34.1±8.6)% 比(48.5±7.9)%,P<0.01;(34.1±8.6)% 比(43.4±8.7)%,P<0.05]和I/R+EdCC 200 μg组[(34.3±10.0)% 比(48.5±7.9)%,P<0.01;(34.3±10.0)% 比(43.4±8.7)%,P<0.05]梗死面积较I/R组和I/R+EdCC 50 μg组明显缩小,I/R+EdCC 100 μg组与I/R+EdCC 200 μg组间差异无统计学意义(P>0.05)(图3)。
EnSCs:子宫内膜干细胞
EnSCs:endometrial stem cells
A.融合度30%;B.融合度100%
A.EnSCs with 30% confluent;B. EnSCs with 100% confluent
图 1倒置显微镜下EnSCs的生长形态
Fig 1Phase-contrast microscopic view of EnSCs
EdCC:EnSCs来源细胞因子鸡尾酒
EdCC:EnSCs derived cytokine cocktail
图 2EdCC经-80℃冻存后蛋白浓度及蛋白总量逐渐降低(n=4)
Fig 2The protein concentration and total quantity of EdCC decreased following-80℃ storage(n=4)
EdCC减少细胞凋亡将小鼠分为I/R组和I/R+EdCC组(注射新鲜提取100 μg EdCC),再灌注3 h取心脏冰冻切片行TUNEL染色,留蛋白用Western blot检测cleaved caspase 3,结果显示,I/R+EdCC组梗死周边区凋亡细胞数量较I/R组明显减少[(16.2±4.9)个/高倍镜视野比(23.8±4.9)个/高倍镜视野,P<0.05],cleaved caspase 3表达水平明显降低(0.150±0.027比0.400±0.052,P<0.01)(图4)。
-80 ℃储存降低EdCC心肌保护效应将小鼠分为I/R组、I/R+EdCC 1 d、I/R+EdCC 30 d、I/R+EdCC 90 d组和I/R+EdCC 180 d组(注射相应冻存时间EdCC 100 μg),再灌注24 h行心脏TTC/Evans Blue染色,结果显示,各组间危险区面积差异无统计学意义(P均>0.05);I/R+EdCC 1 d组[(36.5±7.7)%比(47.0±6.4)%,P<0.01]和I/R+EdCC 30 d组[(39.1±8.9)%比(47.0±6.4)%,P<0.05]的梗死面积明显小于I/R组,其余各组间差异均无统计学意义(P均>0.05)(图5)。
讨论
EnSCs具有间充质干细胞特性,是目前极具潜力的干细胞产品的种子细胞,但直接移植间充质干细胞存在多种风险:(1)干细胞因缺乏Connexin 43表达[14- 15]或无法与正常心肌形成节律偶联[16- 17],将在移植局部形成传导阻滞区导致折返性心律失常,也可能通过降低心肌细胞传导速度形成致心律失常基质[10];(2)体外短期培养的间充质干细胞可能出现染色体畸形,移植后导致肿瘤形成[11];(3)细胞表面的组织因子引起经血管途径注射后血栓栓塞[12]。近来研究提示,干细胞的旁分泌效应是治疗心肌梗死的重要机制[7]。故本研究采用EdCC注射代替EnSCs移植,以期能避免直接细胞移植导致的致心律失常、肿瘤形成和血栓栓塞风险。
I/R:缺血再灌注损伤;AAR/LV:危险区面积;IA/AAR:梗死面积;与I/R组比较,aP<0.01;与I/R+EdCC 50 μg组比较,bP<0.05
I/R:ischemic reperfusion injury;AAR/LV:risk area;IA/AAR:infarct area;aP<0.01 compared with I/R group;bP<0.05 compared with I/R+EdCC 50 μg group
A.心脏TTC/Evans Blue染色;B.定量分析危险区面积;C.定量分析梗死面积
A.representative images of TTC/Evans Blue staining of mouse heart;B.quantification of AAR/LV%;C.quantification of IA/AAR%
图 3EdCC剂量对梗死面积的影响
Fig 3Influence of EdCC dosage on infarct size
与I/R组比较,aP<0.05,bP<0.01
aP<0.05,bP<0.01 compared with I/R group
A.心脏冰冻切片TUNEL染色,蓝色荧光为细胞核,绿色荧光为TUNEL阳性细胞核(箭头);B. 定量分析梗死周边区凋亡细胞数量(n=6);C. Western blot检测cleaved caspase 3结果;D.定量分析cleaved caspase 3表达水平(n=3)
A.representative images of TUNEL positive nuclei in the infarct border zone,blue fluorescence denotes nuclei and green fluorescence denotes apoptotic nuclei (arrow);B.quantification of the apoptotic nuclei(n=6);C. Western blotting of cleaved caspase 3;D.quantification of the ratio of cleaved caspase 3 and β-actin(n=3)
图 4EdCC减少细胞凋亡,抑制caspase 3活化
Fig 4EdCC reduced cell apoptosis and inhibited activation of caspase 3
与I/R组比较,aP<0.05,bP<0.01
aP<0.05,bP<0.01 compared with I/R group
A.心脏TTC/Evans Blue染色;B.定量分析危险区面积;C.定量分析梗死面积
A.representative images of TTC/Evans Blue staining of mouse heart;B.quantification of AAR/LV%;C.quantification of IA/AAR%
图 5-80℃冻存对EdCC心肌保护效应的影响
Fig 5Influence of-80 ℃ storage on the myocardial protective effect of EdCC
当前急性心肌梗死最重要的治疗措施是在时间窗内尽早开通闭塞血管使心肌恢复血供[3]。随着急性心肌梗死发病率的增加、急诊再灌注治疗的广泛应用,如何保护缺血心肌、减轻心肌I/R损伤越来越受到重视。本研究在心肌缺血30 min即松开活结使前降支恢复血流,即急诊再灌注治疗,研究设计接近真实临床情况。结果发现,在心肌缺血5 min时经尾静脉注射EdCC可使梗死面积显著缩小,说明EdCC可在静脉注射后快速介导心肌保护作用。已知EnSCs经心肌移植可激活细胞外信号调节激酶1/2、信号传导与转录激活因子3和丝氨酸/苏氨酸激酶促存活信号通路减少细胞凋亡[9],推测EdCC的心肌保护效应可能是通过上述信号通路介导。此外,microRNA可能也参与了该保护效应[18]。研究发现,I/R损伤引起心肌细胞凋亡约占所有心肌细胞死亡的80%[19],caspase 3活化形成cleaved caspase 3是细胞凋亡的主要执行者[20],抑制凋亡可以显著缩小梗死面积[21]。本研究采用TUNEL染色发现,EdCC可显著减少梗死周边区凋亡细胞数量。Western blot检测结果显示,EdCC可明显抑制心肌组织cleaved caspase 3表达,从细胞和蛋白水平证明了EdCC的抗凋亡作用。本研究还显示,EdCC随冻存时间延长心肌保护效应逐渐降低,推测可能是因为蛋白质聚集、构象改变导致生物学活性丧失[22],如能优化冻融过程或制备成冻干粉可进一步提高EdCC储存效率。
目前干细胞旁分泌效应的研究主要存在以下问题:(1)多种因子的协同作用;(2)心肌梗死不同时期发挥治疗效应的因子不同;(3)因子浓度不同生物学效应不同;(4)移植微环境影响旁分泌谱[23]。这些问题对深入理解旁分泌效应造成巨大的障碍,需联合基因、蛋白组学技术和质谱分析才能进一步探索并验证具体的有效治疗成分[24]。随着我国《干细胞临床研究管理办法(试行)》的发布,备受争议的干细胞治疗迎来新一轮曙光以及挑战。本研究结果支持干细胞治疗策略从细胞移植向细胞因子注射转化的新方案,在建立EnSCs细胞库的同时可建立商品化EdCC以供临床研究及应用。
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基金项目:国家自然科学基金(81460050)、贵州省科技厅科学技术基金(黔科合J字[2014]2112号)和贵州省卫生厅科学技术基金(gzwkj2013- 1- 006) Supported by the National Natural Sciences Foundation of China (81460050),the Science and Technology Foundation of Guizhou Provincial Science and Technology Department (黔科合J字[2014]2112号),and the Science and Technology Foundation of Guizhou Provincial Health Department (gzwkj2013- 1- 006)
通信作者:韦方电话:0851- 85937194,电子邮件:weifanggzsy@qq.com
中图分类号:R54
文献标志码:A
文章编号:1000- 503X(2016)03- 0253- 07
DOI:10.3881/j.issn.1000- 503X.2016.03.002
Corresponding author:WEI FangTel:0851- 85937194,E-mail:weifanggzsy@qq.com
(收稿日期:2015- 12- 25)
Effect of Endometrial Stem Cell-derived Cytokine Cocktail on a Mouse Model of Myocardial Reperfusion Injury
JIANG Zhi1,JIA Zhong-shen2,PAN Yu-jia1,2,LI An-jie1,WEI Fang1
1Department of Cardiology,Guizhou Provincial People’s Hospital,Guizhou 550002,China2Department of Clinical Medicine,Guizhou Medical University,Guizhou 550025,China
ABSTRACT:ObjectiveTo study the effect of endometrial stem cells (EnSCs) derived cytokine cocktail (EdCC) on myocardial ischemic reperfusion injury (I/R) in a mouse model. MethodsEdCC was concentrated from the culture medium of EnSCs with Millipore ultra-filtration technology and was administrated to a myocardial I/R mouse models through tail vein injection. The infarct area was determined by TTC/Evans Blue staining. The apoptotic cells were counted by TUNEL assay and the protein level of cleaved caspase 3 was evaluated by Western blotting. ResultsThe EdCC extraction efficiency was (222.4±29.3) μg/106 cells in every 24 h,but the protein gradually degraded under-80 ℃ storage. As compared with I/R group,100 μg fresh EdCC decreased infarct area (P=0.001),reduced apoptotic nuclei in the infarct border (P=0.019),and inhibited cleaved caspase 3 expression (P=0.002). Increasing EdCC dosage did not further reduce the infarct area. The myocardial protective effect of EdCC diminished after 90 days’ storage under-80 ℃. ConclusionEdCC reduces myocardial I/R injury through protecting cardiomyocytes from apoptosis within 30 days storage under-80 ℃.
Key words:endometrial stem cells;cytokine;myocardial infarction;ischemic reperfusion injury;apoptosis