CN108486046B - 一种抗缺氧损伤的干细胞制剂及其制备方法与在制备治疗急性心梗药物中的应用 - Google Patents
一种抗缺氧损伤的干细胞制剂及其制备方法与在制备治疗急性心梗药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种抗缺氧损伤的干细胞制剂及其制备方法与在制备治疗急性心梗药物中的应用,所述抗缺氧损伤的干细胞制剂的制备方法为,将干细胞与Fstl1过表达慢病毒液在培养基中混合,得到抗缺氧损伤的干细胞制剂。本发明提供的Fstl1修饰的MSCs具有优异的抗缺氧损伤能力,可提高移植存活率,经心肌直接注射可有效提高梗死后心功能。
Description
技术领域
本发明属于细胞药物技术,具体涉及一种抗缺氧损伤的干细胞制剂及其制备方法与在制备治疗急性心梗药物中的应用。
背景技术
心肌梗死是一种严重危害人类健康的心血管疾病,随着我国人民生活水平的不断提高,缺血性心肌梗死的发病率也在不断上升。缺血性心肌梗死会导致心肌细胞坏死和瘢痕形成,进而影响心脏功能。目前药物或器械治疗大多只能缓解症状,但却不能逆转心脏组织损伤。心脏移植虽能彻底改善心脏状态,但因供体来源稀缺、免疫排斥以及昂贵的治疗费用等因素,在临床上很难广泛应用。
干细胞移植治疗心肌梗死,因其各方面的优势有望成为治疗心肌梗死的重要治疗手段。间充质干细胞(Mesenchymal stem cells,MSCs)是一种来源丰富且容易获得的原代成体干细胞,主要存在于骨髓、脂肪、脐带、胎盘和羊水等组织中。近年来,MSCs因其卓越的组织修复能力,常作为细胞移植的种子细胞广泛应用于再生医学领域。多项研究证实MSCs主要依赖旁分泌发挥作用。目前困扰干细胞移植的瓶颈问题系干细胞移植存活率低,因此,如何最大限度提高干细胞存活率是该领域的研究热点。心肌梗死局部恶劣的缺氧微环境是造成移植干细胞存活率低的主要原因,因此提高干细胞的抗缺氧损伤能力是未来的努力方向。
卵泡抑素样蛋白1(Follistatin like 1,Fstl1)最初从小鼠成骨细胞系MC3T3-E1中克隆获得,是一种分泌型胞外糖蛋白,并不参与细胞外基质结构,而是在细胞外水平通过改变细胞微环境调节细胞生理活动。Fstl1是维持心脏稳态与病理重塑的重要内源性活性因子,在心脏广泛表达。作为慢性收缩性心衰左室重塑的标志物,Fstl1可降低压力超负荷导致的病理性心脏肥大;但是Fstl1是否可以提高干细胞的抗缺氧损伤能力,进而提高移植细胞的驻留尚不明确。
发明内容
本发明公开了一种抗缺氧损伤的干细胞制剂及其制备方法与在制备治疗急性心梗药物中的应用。
本发明采用如下技术方案:
一种抗缺氧损伤的干细胞制剂的制备方法,包括以下步骤,将干细胞与Fstl1过表达慢病毒液在培养基中混合,10~15小时后换液,得到抗缺氧损伤的干细胞制剂。
本发明还公开了一种预防或治疗心梗药物的制备方法,包括以下步骤,将干细胞与Fstl1过表达慢病毒液在培养基中混合,10~15小时后换液,得到抗缺氧损伤的干细胞制剂;将抗缺氧损伤的干细胞制剂与分散介质,比如缓冲液、生理盐水混合,得到预防或治疗心梗药物;具体的给药方式可采取直接心肌注射或各种途径的经血注射等。
优选的,在聚凝胺(polybrene)存在下,将干细胞与Fstl1过表达慢病毒液在培养基中混合,以增加病毒感染效率,polybrene的终浓度为8 μg/ml。
上述技术方案中,干细胞为小鼠骨髓间充质干细胞;按照感染复数(multiplicityof infection,MOI)= 10计算Fstl1过表达慢病毒液的用量。Fstl1过表达慢病毒液中,Fstl1过表达慢病毒滴度为107~108 TU/ml;培养基一般为DMEM/F12培养基。
优选的,选择2-3周龄的雄性C57BL/6J小鼠,颈椎脱臼法处死小鼠,将小鼠浸泡在75%酒精中10min;无菌条件下取小鼠双下肢浸泡于DMEM/F12无血清基础培养基中,去除股骨及胫骨表面肌肉;用1ml注射器吸取添加青/链霉素的DMEM/F12无血清基础培养基并冲洗骨髓腔;待骨髓全部冲出后离心弃上清,加入新鲜的小鼠骨髓来源MSCs专用培养基反复吹打至单细胞悬液,将细胞悬液接种于培养皿中;72h后换液并去除未贴壁细胞,以后每两天换液一次,待培养皿中的集落互相融合且细胞密度达70%左右时进行传代,此时标记为P1代;待MSCs传至P5代以后,将干细胞与Fstl1过表达慢病毒液在培养基中混合以制备抗缺氧损伤的干细胞制剂;优选按照3 X 105/cm2的密度将细胞悬液接种于培养皿中。
优选的,将干细胞与Fstl1过表达慢病毒液在培养基中混合,12h后换液,得到抗缺氧损伤的干细胞制剂;更优选的,换液后培养一段时间以获得更多抗氧损伤干细胞,培养时间可以为72h或者更长。
本发明还公开了根据上述抗缺氧损伤的干细胞制剂的制备方法制备的抗缺氧损伤的干细胞制剂;Fstl1过表达慢病毒在提高干细胞移植存活率中的应用或者Fstl1过表达慢病毒在制备抗缺氧损伤干细胞制剂中的应用。
本发明制备的抗缺氧损伤的干细胞制剂通过倒置荧光显微镜下观察mCherry荧光判断细胞感染效率,可以发现大部分细胞均显示强烈mCherry荧光,提示慢病毒感染成功。
作为一种来源丰富且容易获得的原代成体干细胞, MSCs因其卓越的组织修复能力,常作为细胞移植的种子细胞广泛应用于再生医学领域。但是现有MSCs存在明显的抗缺氧损伤能力弱的问题,导致实际干细胞修复效果远低于理论设计。本发明利用Fstl1改造干细胞后可明显提高干细胞抗缺氧损伤能力,从而提高其移植存活率,所以本发明还公开了Fstl1在提高干细胞移植存活率中的应用。
现有技术研究了Fstl1对心肌梗死的作用,但是没有文献涉及利用Fstl1改造干细胞,也没有关于Fstl1改造干细胞达到何种效果的报道;本发明提供Fstl1修饰的MSCs具有优异的抗缺氧损伤能力,可提高移植存活率,因此本发明还公开了Fstl1在制备抗缺氧损伤干细胞制剂中的应用;本发明还公开了上述抗缺氧损伤的干细胞制剂在制备抗缺氧损伤干细胞体系或者提高干细胞移植存活率中的应用。同时本发明公开了抗缺氧损伤的干细胞制剂在制备预防或治疗心梗药物或保健品中的应用。本发明的抗缺氧损伤的干细胞制剂具有抵抗心肌梗死局部恶劣的缺氧微环境能力,可成为调控心脏稳态与病理重塑的重要手段。
附图说明
图1为小鼠骨髓来源MSCs的形态鉴定;
图2为小鼠骨髓来源MSCs的流式鉴定;
图3为持续缺氧刺激引起MSCs的Fstl1表达显著下降;
图4为MSCs-mCherry和MSCs-Fstl1均强烈表达mCherry荧光;
图5为MSCs-mCherry和MSCs-Fstl1的流式鉴定;
图6为MSCs-Fstl1高表达且高分泌Fstl1;
图7为MSCs-Fstl1更有效抵抗缺氧引起的细胞凋亡;
图8为MSCs-Fstl1在缺氧环境下的细胞增殖能力更强;
图9为MSCs-Fstl1在常氧和缺氧环境下的细胞活力均更佳;
图10为MSCs-Fstl1细胞移植显著改善心梗后心功能(M型超声图);
图11为MSCs-Fstl1细胞移植显著促进EF、FS、LVID;d和LVPW;d指标恢复;
图12为MSCs-Fstl1细胞移植缩小心梗面积(Masson染色示意图);
图13为MSCs-Fstl1细胞移植有效缩小心梗面积(统计结果);
图14为以mCherry自身荧光和免疫荧光共定位评估移植细胞在缺氧心肌中的驻留;
图15为以DiI标记移植细胞来评价移植细胞在缺氧心肌中的驻留。
具体实施方式
以下所列实施例,仅为帮助本领域技术人员更全面理解本发明,但不以任何方式限制本发明。本发明中,换液是指将病毒感染液100%换为正常完全细胞培养基。
实施例一 抗缺氧损伤的干细胞制剂的制备及性能检测
所使用的主要材料和来源分别如下:
C57BL/6J小鼠(昭衍新药研究中心,此实验由苏州大学伦理委员会批准);DMEM/F12培养基(Gibco,美国);小鼠骨髓来源MSCs专用培养基(赛业生物,中国);胰蛋白酶(Sigma,美国);超净工作台(安泰,中国);二氧化碳培养箱(Thermo,美国);台式离心机(Thermo,美国);流式细胞仪(Millipore,美国);倒置荧光显微镜(ZEISS,德国);Nanodrop2000超微量分光光度计(Thermo,美国);实时荧光定量PCR仪(ABI ,美国);全波段多功能酶标仪(BIO-TEK,美国);反转录试剂盒(Takara,日本); FITC-CD29抗体(Biolegend,美国);APC-CD44抗体(Biolegend,美国); FITC-CD90抗体(Biolegend,美国);PE-CD45抗体(Biolegend,美国);APC-CD117抗体(Biolegend,美国);Fstl1 ELISA检测试剂盒(R&D,美国);Click-iT Plus EdU Alexa Fluor 647流式检测试剂盒(Life,美国);Annexin V-FITC试剂盒(BD,美国);CCK-8试剂盒(Dojindo,日本);Fstl1过表达慢病毒(LV-Fstl1)及对照(LV-mCherry)(吉凯,中国)
1.1 小鼠骨髓来源MSCs的获得
选择2-3周龄的雄性C57BL/6J小鼠,颈椎脱臼法处死小鼠,将小鼠浸泡在75%酒精中10min消毒;无菌条件下取小鼠双下肢浸泡于DMEM/F12无血清基础培养基中,用剪刀和镊子去除股骨及胫骨表面肌肉;用1ml注射器吸取添加青/链霉素的DMEM/F12无血清基础培养基并缓慢冲洗骨髓腔;待骨髓全部冲出后离心弃上清,加入新鲜的小鼠骨髓来源MSCs专用培养基反复吹打至单细胞悬液,按照3 X 105/cm2的密度将细胞悬液接种于培养皿中;72h后换液并去除未贴壁细胞,以后每两天换液一次,待培养皿中的集落互相融合且细胞密度达70%左右时进行传代,此时标记为P1代。图1为P6代MSCs,可以看出光镜下小鼠骨髓来源MSCs分布均匀,形态均一,表现为成纤维细胞样或扁平状,少数呈梭形,有长短不一、粗细不均的突起。
1.2 流式细胞术鉴定小鼠骨髓来源MSCs
细胞汇合度达到80%时,常规胰酶消化,调节细胞浓度至5 X 106 cells/ml;分别加入FITC-CD29抗体、APC-CD44抗体、FITC-CD90抗体、PE-CD45抗体和APC-CD117抗体,4℃孵育30 min;PBS洗涤,流式细胞术检测细胞表面标志。图2为流式细胞术检测结果图,小鼠骨髓来源MSCs表达CD29、CD44和CD90,不表达CD45和CD117。
1.3 持续缺氧刺激引起MSCs的Fstl1表达显著下降
当MSCs汇合度达到70%时,行持续缺氧刺激(1%O2),分别于0h、24h和48h收集细胞并提取总RNA、反转和qRT-PCR。每个样品设4个复孔,反应体系为10μl,以GAPDH作为内参。所用引物序列如下:Fstl1: 5-TTATGATGGGCACTGCAA-3和5-ACTGCCTTTAGAGAACCAG-3;GAPDH:5-TGCCCAGAACATCATCCCT-3和5-GGTCCTCAGTGTAGCCCAAG-3。图3显示,持续缺氧刺激引起Fstl1在MSCs中的表达显著下调,提示Fstl1在缺氧损伤保护中可能具有重要作用。###P <0.001(0h vs 24h);***P < 0.001(0h vs 48h)。
1.4 抗缺氧损伤干细胞制剂的获得
步骤1中,待MSCs汇合度达到50%时,按照感染复数(multiplicity of infection,MOI)= 10换算病毒颗粒数量;分别吸取Fstl1过表达慢病毒(LV-Fstl1)液(吉凯公司)及相应空载对照病毒液(LV-mCherry),并加入polybrene(8μg/ml)以增加病毒感染效率;37℃、5%CO2孵箱感染过夜;12h后换除病毒液并加入正常培养基;72h后倒置荧光显微镜下观察mCherry荧光强度判断细胞感染效率,qRT-PCR和ELISA检测Fstl1高表达和分泌情况,流式鉴定MSCs表明标志。以上即可得到抗缺氧损伤的干细胞制剂,称为MSCs-Fstl1,相应对照称为MSCs-mCherry。
图4为MSCs-mCherry和MSCs-Fstl1的荧光图,MSCs-mCherry和MSCs-Fstl1组细胞均可见强烈的mCherry荧光,仍然保留典型的MSCs外观形态,证明慢病毒感染成功;图5为MSCs-mCherry和MSCs-Fstl1流式细胞术检测结果图,MSCs-mCherry和MSCs-Fstl1均表达CD29和CD44,不表达CD45和CD117。
1.5 qRT-PCR鉴定MSCs-Fstl1细胞的Fstl1转录水平
按常规提取MSCs-mCherry和MSCs-Fstl1总RNA,Fstl1转录水平测定同实施例1.3。如附图6A显示MSCs-Fstl1细胞的Fstl1转录水平提高为对照MSCs-mCherry组的9.18倍,***P < 0.001。
1.6 ELISA鉴定MSCs-Fstl1细胞的Fstl1分泌水平
用Fstl1捕获抗体包被96孔微孔板制成固相载体,依次加入标本(MSCs-mCherry或MSCs-Fstl1上清)或标准品、生物素化的Fstl1检测抗体、HRP标记的亲和素,经过彻底洗涤后用底物显色。颜色深浅和样品的Fstl1含量呈正相关。用酶标仪在450nm波长下测定吸光度(OD值),根据标准曲线计算样品中Fstl1浓度。
附图6B显示MSCs-Fstl1细胞上清的Fstl1浓度为对照组的12.44倍,证明MSCs-Fstl1可以实现Fstl1的高分泌,***P < 0.001。
1.7 Annexin V标记检测MSCs-Fstl1抵抗缺氧诱导的细胞凋亡的能力
将MSCs-mCherry和MSCs-Fstl1按8 X 104/孔接种于12孔板中培养,贴壁过夜后换为新鲜培养基,持续缺氧刺激(1%O2)48h,用不含EDTA的0.25%胰酶消化并收集细胞,PBS洗涤2次,加入100μl Annexin V标记缓冲液重悬细胞,细胞浓度约为106个/ml。加入5μlAnnexin V-FITC混匀,室温避光反应15min,流式细胞仪检测。
附图7的Annexin V染色实验显示缺氧刺激下,MSCs-Fstl1组的Annexin V+细胞比例显著低于MSCs-mCherry组。
1.8 EdU标记检测MSCs-Fstl1缺氧环境下的增殖能力
将MSCs-mCherry和MSCs-Fstl1按一定密度接种于12孔板中培养,待贴壁后行持续缺氧刺激(1%O2)48h,添加EdU(10μM)37℃孵育1h,胰酶消化并收集细胞,PBS洗涤2次,固定,荧光渗入反应,流式细胞仪检测。
附图8的EdU渗入实验结果显示常氧和缺氧条件下,MSCs-Fstl1组的增殖能力均高于MSCs-mCherry组。
1.9 CCK-8实验检测MSCs-Fstl1在缺氧环境下的细胞活力
将MSCs-mCherry和MSCs-Fstl1按一定密度铺板于96孔板中。待贴壁后行持续缺氧刺激(1%O2)48h,每孔更换100μl新鲜培养基和10μl CCK-8反应液,在0.5~2h间每0.5h用酶标仪测定在450nm处的吸光值。
附图9的CCK-8实验结果显示常氧和缺氧条件下MSCs-Fstl1的细胞活力分别为MSCs-mCherry的1.24倍和2.19倍,*P < 0.05,**P < 0.01,***P < 0.001。
实施例二 抗缺氧损伤的干细胞制剂有效改善心梗后心功能且驻留效果更佳
所使用的主要材料和来源分别如下:
C57BL/6J小鼠(昭衍新药研究中心,此实验由苏州大学伦理委员会批准);小动物呼吸机(奥尔科特生物,上海);手术器械(六六视觉,苏州);缝合针线(金环医疗,上海);小动物超声影像系统(Visual Sonics Vevo 2100);倒置荧光显微镜(ZEISS,德国);Masson染色试剂盒(Sigma,美国);mCherry抗体(Abcam,美国);FITC标记的山羊抗兔IgG二抗(SentaCruz,美国);CM-DiI(Invitrogen,美国)
2.1 小鼠心肌梗死模型的建立
选用25g左右的C57BL/6J雄性小鼠为实验对象,采用左冠状动脉前降支(leftanterior descending artery,LAD) 结扎法制作心梗模型。腹腔注射麻醉后,经口气管插管,接空气呼吸机,呼吸频率110次/min,潮气量3ml,吸呼比1:1.3。右侧卧位,左胸纵切口切开外层皮肤,剥离胸大肌,第三、四肋间横切口开胸,暴露心脏,用镊子撕开心包膜。借助手术显微镜可见左冠状动脉大致走行。在左心耳下缘约1~2mm处,将LAD 连同少量心肌组织一起结扎,进针深度约1mm,宽度控制在3mm以内。逐层关胸。假手术组(sham)仅穿过LAD下方不打结,其余同模型组;结扎后肉眼可见结扎处至心尖变白,7天后取左心室组织进行心脏组织染色,可看到明显的纤维化,证明心梗模型建立成功。
2.2 心肌注射抗缺氧损伤的干细胞制剂
将抗缺氧损伤的干细胞制剂MSCs-Fstl1与分散介质PBS混合,得到预防或治疗心梗的药物。按照上述步骤1的方法LAD结扎后,选择结扎位点附近的左下和右下两个位点注射药物,每只小鼠MSCs用量为5 X 105个/20μl,每个位点10μl,以PBS作为阴性对照。选择合适的角度,以避免注射入左心室腔内。心肌颜色轻微变浅就表明溶液进入了梗死心室壁。
2.3 心脏超声检测心梗后心功能
心梗术后7天行小鼠麻醉术(方法同前),脱毛后左侧卧位,将心脏超声诊断仪探头置于心前壁,于乳头肌水平取左室二维短轴观,同时记录M型扫描,连续3个心动周期测量左室射血分数(ejection fraction,EF)、缩短分数(fractional shortening,FS)、左室舒张末期内径(left ventricular internal diameter at end-diastole,LVID;d)和左室舒张末期后壁厚(left ventricular posterior wall thickness at end-diastole,LVPW;d)。
参见附图10、图11,心梗/移植术后7天,单纯注射PBS组小鼠心脏功能完全符合典型的心肌梗死后心脏超声特点,EF和FS显著降低,LVID;d显著增高,提示心梗后心室重构明显。MSCs-mCherry组的心功能各指标均明显优于单纯注射PBS组。MSCs-Fstl1组改善心梗后心功能效果最佳,与其余各组相比差异显著。*P < 0.05,**P < 0.01,***P < 0.001。
2.4 Masson染色评估心肌梗死面积
心梗术后7天处死小鼠,取左心室组织进行常规组织切片和Masson染色。以普通光学显微镜观察并拍照、采用图像分析软件Image J分析各部分面积。心梗面积的计算参照公式如下:
心梗面积(%)=实际心梗面积/实际心脏横切面面积;
参见附图12、图13,以Masson染色观察各组心梗术后7天梗死面积,发现MSCs-mCherry组和MSCs-Fstl1组的心梗面积分别为PBS组的74.75%和52.06%;MSCs-Fstl1组的心梗面积最低,为MSCs-mCherry组的69.64%;*P < 0.05。附图12显示的是每组样本中的代表性图片,标尺显示1mm。
2.5 mCherry免疫荧光评估MSCs-Fstl1驻留
细胞移植和心梗术后1天处死小鼠,取左心室组织进行冰冻切片,按常规步骤进行mCherry免疫荧光染色,添加FITC标记的山羊抗兔IgG二抗,用含DAPI的抗荧光衰减封片剂封片,以荧光显微镜同时观察并拍照mCherry自身信号(红),FITC信号(绿)和DAPI信号(蓝)。
参见附图14,荧光显微镜下观察各组荧光,发现MSCs-Fstl1组的细胞驻留显著高于MSCs-mCherry对照组。照片显示的是每组样本中的代表性图片,标尺显示50μm。
2.6 CM-DiI标记MSCs-Fstl1评估其在缺氧心脏的驻留
消化并收集MSCs-mCherry和MSCs-Fstl1,CM-DiI染色工作液(1μg/ml)重悬各组MSCs,37˚C孵育5min,4˚C孵育15min,PBS洗涤2次,行心梗手术和局部心肌注射。移植术后3天处死小鼠,取注射位点附近组织进行常规切片,荧光显微镜观察局部细胞移植区域CM-DiI信号。
参见附图15,直接在荧光显微镜下观察各组CM-DiI荧光,发现MSCs-Fstl1组的CM-DiI信号区域显著高于MSCs-mCherry对照组,提示MSCs-Fstl1在缺氧心肌中的驻留优于MSCs-mCherry,标尺显示200μm。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
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<110> 苏州大学
<120> 一种抗缺氧损伤的干细胞制剂及其制备方法与在制备治疗急性心梗药物中的应用
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Claims (5)
1.Fstl1在制备提高干细胞移植存活率的制剂中的应用,其特征在于,所述干细胞为小鼠骨髓间充质干细胞。
2.Fstl1过表达慢病毒在制备提高干细胞移植存活率的制剂中的应用,其特征在于,所述干细胞为小鼠骨髓间充质干细胞。
3.抗缺氧损伤的干细胞制剂在制备高移植存活率干细胞中的应用,其特征在于,所述干细胞为小鼠骨髓间充质干细胞,所述抗缺氧损伤的干细胞制剂的制备方法包括以下步骤:将干细胞与Fstl1过表达慢病毒液在培养基中混合,10~15小时后换液,得到抗缺氧损伤的干细胞制剂;按照MOI = 10计算Fstl1过表达慢病毒液的用量;选择2-3周龄的雄性C57BL/6J小鼠,颈椎脱臼法处死小鼠,将小鼠浸泡在75%酒精中消毒;无菌条件下取小鼠双下肢浸泡于DMEM/F12无血清基础培养基中,去除股骨及胫骨表面肌肉;用注射器吸取添加青/链霉素的DMEM/F12无血清基础培养基并冲洗骨髓腔;待骨髓全部冲出后离心弃上清,加入新鲜的小鼠骨髓来源MSCs专用培养基反复吹打至单细胞悬液,将细胞悬液接种于培养皿中;72h后换液并去除未贴壁细胞,以后每两天换液一次,待培养皿中的集落互相融合且细胞密度达70%时进行传代,此时标记为P1代;待MSCs传至P5代以后,将干细胞与Fstl1过表达慢病毒液在培养基中混合以制备抗缺氧损伤的干细胞制剂。
4.根据权利要求3所述的应用,其特征在于,在聚凝胺存在下,将干细胞与Fstl1过表达慢病毒液在培养基中混合。
5.根据权利要求3所述的应用,其特征在于,将干细胞与Fstl1过表达慢病毒液在DMEM/F12培养基中混合;12小时后换液。
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