CN116270742A - MitoQ和MSCs的细胞混合制剂及其应用 - Google Patents
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Abstract
本发明涉及宠物临床间充质干细胞治疗领域,公开了一种MitoQ和MSCs的细胞混合制剂及其应用。本发明细胞混合制剂为MSCs、MitoQ和生理盐水的液体混合制剂,其在制备用于治疗动物糖尿病药物中的应用。本发明能够增强间充质干细胞的治疗效果,延长细胞在体内留存时间,从而对糖尿病起到更好的治疗效果,还能够减轻糖尿病导致的相关器官损伤,如肝脏、胰腺等。本发明制备及使用方法简便,可以减少细胞移植次数,并有利于节省设备和人力成本,是临床用于治疗犬等宠物的糖尿病的可行途径。
Description
技术领域
本发明涉及细胞制剂技术领域,具体涉及一种MitoQ和MSCs的细胞混合制剂及其应用。
背景技术
间充质干细胞(Mesenchymal stem cells,MSCs)是一类来源于中胚层、存在于多种组织(包括骨髓、脂肪、胎盘、脐带等)中的多能干细胞,具有干细胞的所有共性,即自我更新和多向分化能力,且增殖能力强、免疫原性小、极具临床应用价值。在血液系统、心血管系统、神经系统,内分泌代谢系统等多方面的疾病治疗中发挥极大的潜力。在多个研究中,均证明间充质干细胞对于糖尿病治疗具有显著治疗效果,能够有效控制血糖水平,其治疗途径主要包括:调节代谢、减轻炎症、对抗自身免疫、促进胰岛再生、缓解胰岛素抵抗、促进胰岛素分泌等。
目前,已经证明不同移植途径的MSCs对于机体的治疗效果存在差异。在宠物相关的临床研究中,一般采取静脉输注或者滴注治疗的方式,生物发光成像系统被广泛用于跟踪MSCs的体内生物分布,发现细胞移植后多数被留存于毛细血管中,导致细胞存活率大大降低,并在注射后数小时被消除。除此之外,细胞移植过程中如果存在较大的细胞团块或者凝集,会导致毛细血管中血液流通速度降低,从而导致血管中形成局部血栓。细胞移植过程中的这些缺点使得其无法广泛的应用于临床。
慢性炎症状态下,机体细胞容易受到高血糖的明显损害,同时增加破坏机体自身免疫功能的风险。在研究中发现,氧化应激与糖尿病的发生有很大关系,线粒体活性氧产物增加是糖尿病的各种致病通路关键始动因子。
发明内容
本发明为了克服间充质干细胞在临床应用中留存时间短,促进间充质干细胞对犬糖尿病的治疗效果,并延长细胞在体内的治疗时间,提供一种MitoQ和MSCs的细胞混合制剂及其应用。
为了实现上述目的,本发明采用的技术方案为:
MitoQ和MSCs的细胞混合制剂在制备用于治疗动物糖尿病药物中的应用。
MitoQ和MSCs的细胞混合制剂,所述细胞混合制剂为MSCs、MitoQ和生理盐水的液体混合制剂。
所述的MitoQ的浓度为1μmol/L。
所述细胞制剂的细胞含量为107-109个细胞/mL。
与现有技术相比,本发明具有以下有点:
1)本发明MitoQ可以显著提高细胞在体外的活力,并且促进对小鼠及犬糖尿病模型的治疗效果,使得细胞治疗时间有所延长。
2)本发明能够实现间充质干细胞在犬机体内对糖尿病的治疗,能够减轻糖尿病导致的相关器官损伤,如肝脏、胰腺等。
3)本发明制备及使用方法简便,可以减少细胞移植次数,并有利于节省设备和人力成本,是临床用于治疗犬等宠物的糖尿病的可行途径。
附图说明
图1为MitoQ预处理MSCs细胞形态、增殖情况比对图;其中a.Giemsa染色、b.细胞生长曲线、c.EdU染色及定量结果。
图2为MitoQ预处理MSCs的抗氧化能力检测;其中a.qRT-PCR检测氧化应激指标、b.WesternBloting实验检测P53蛋白表达量、c.细胞ROS荧光染色。
图3为MitoQ预处理MSCs移植治疗小鼠糖尿病模型相关指标。其中a.小鼠治疗期间血糖变化、b.小鼠治疗期间体重变化、c.小鼠细胞移植1周后葡萄糖耐量检测、d.小鼠细胞移植3周后葡萄糖耐量检测、e.小鼠胰腺HE染色及Insulin免疫荧光染色。
图4为MitoQ预处理MSCs移植治疗犬糖尿病模型相关指标;其中a.犬治疗期间体重变化、b.犬治疗期间血糖变化、c.犬细胞移植1周后葡萄糖耐量检测、d.犬细胞移植3周后葡萄糖耐量检测、e.犬胰腺HE染色及Insulin免疫荧光染色。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图和实施例对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
米托蒽醌甲磺酸盐(MitoQ)作为一种线粒体靶向抗氧化剂,由三苯基磷阳离子结合,包含具有抗氧化作用的醌基,共价结合到亲脂性的三苯基磷阳离子上。MitoQ被吸附到线粒体内膜,其连接链使活化的CoQ抗氧化成分渗透到膜的核心。在研究中表明,糖尿病小鼠模型,经口服MitoQ治疗后,其表现出来的肾小管和肾小球功能缺陷有所减弱,肾间质性纤维化也显著减少。Escri等人研究结果认为线粒体靶向抗氧化剂(如线粒体(MitoQ)能调节T2DM患者的氧化应激、炎症和白细胞内皮相互作用。Xiao L等人研究结果表明,MitoQ对糖尿病小鼠具有保护作用,能够恢复高血糖诱导的NF-E2相关因子(Nrf2)表达、活动和移位等,通过Nrf2/PINK1通路改善糖尿病肾病中由线粒体引起的管状损伤。
本发明MitoQ和MSCs的细胞混合制剂在制备用于治疗动物糖尿病药物中的应用。
本发明MitoQ和MSCs的细胞混合制剂为MSCs、MitoQ和生理盐水的液体混合制剂,细胞的含量为107-109个细胞/mL。
本发明MitoQ和MSCs的细胞混合制剂中MitoQ的浓度为1μmol/L,并进行体外在细胞层面的验证。
本发明的MitoQ可以显著提高细胞在体外的活力,并且促进对小鼠及犬糖尿病模型的治疗效果,使得细胞治疗时间有所延长。
间充质干细胞来源于陕西省干细胞工程技术中心,为犬脂肪间充质干细胞;MitoQ来源于Sigma。
下面通过实验对本发明做进一步详细描述:
1、间充质干细胞的形态、增殖和生长速度检测。
复苏犬脂肪间充质干细胞,使用添加10mL/100mL血清替代物的α-MEM培养基在37℃,5% CO2培养箱中正常培养传代;
待长满后进行传代准备,其中半数细胞使用正常α-MEM培养基培养,另外半数细胞使用1μmol/L MitoQ的培养基进行培养48h。
对6孔板中贴壁培养的间充质干细胞进行Giemsa染色,结果如图1a所示,ADMSCs和MitoQ-ADMSCs均呈立体的纤维状形态,ADMSCs在经MitoQ处理后形态未发生明显改变,但是同一视野下,可见MitoQ-ADMSCs细胞数量远多于ADMSCs;
除此之外,为进一步验证MitoQ是否能够起到促进细胞增殖的效果,进行了EdU细胞增殖实验,结果如图1c所示,经MitoQ处理后,ADMSCs的增殖速度明显提升,且细胞活力也有所提高。
为进一步验证细胞生长速度的变化,对24孔板中贴壁培养的ADMSCs进行消化,初始浓度0.5×104个/ml,并进行为期7天的细胞生长曲线绘制,后绘制以时间为横坐标。细胞数量为纵坐标的生长曲线,如图1b所示。
2、为验证MitoQ是否能够提高ADMSCs的抗氧化能力,延缓细胞的衰老的凋亡,进行了qRT-PCR实验、WesternBloting实验及ROS荧光染色实验。
根据制造商的说明,使用TRIzol试剂从ADMSCs和MitoQ-ADMSCs中提取总RNA,并使用逆转录酶试剂试剂盒(赛默飞世尔科技(中国)有限公司)合成cDNA。实时荧光定量聚合酶链反应(qRTPCR),结果如图2a所示,MitoQ对ADMSCs进行预处理后可以使得SOD1、SOD2、GSH-Px抗氧化物表达水平有所提高,并且使得氧化物MDA水平有所下降,可见,RNA水平上,MitoQ能够有效提高抗氧化相关基因的表达,降低氧化相关基因的表达。
为进一步验证MitoQ对细胞凋亡的影响,选用P53进行了WesternBloting实验。
首先使用含有蛋白酶抑制剂的RIPA裂解缓冲液(Beyotime,上海,中国)裂解ADMSCs和MitoQ-ADMSCs,然后用5%SDS上样缓冲液对每个样品的蛋白质进行变性。将制备的样品加载到10%SDS-聚丙烯酰胺凝胶电泳上,然后电转移到活化的聚偏二氟乙烯膜上。将膜用5%牛血清白蛋白在含有1%吐温20(TBS-T)的Tris缓冲盐水(TBS)中封闭,并与特异性抗体(p53)在4℃孵育过夜。然后,洗涤膜,并在室温下与抗小鼠辣根过氧化物酶孵育1小时,如图2b所示,ADMSCs中P53的蛋白表达量在经MitoQ预处理后显著下降,证明了MitoQ能够对ADMSCs起到减少凋亡的作用。
除此之外,为了探究MitoQ是否能够减少ADMSCs中的活性氧水平,进行了ROS荧光染色实验;按照1:1000用无血清培养液稀释DCFH-DA,使终浓度为10μmol/L。细胞收集后悬浮于稀释好的DCFH-DA中,细胞浓度为1×106/ml,37℃细胞培养箱内孵育20分钟。每隔3-5分钟吹打混匀1次。用无血清细胞培养液洗涤细胞三次,以充分去除未进入细胞内的DCFH-DA。荧光显微镜下观察、拍照,如图2c所示;同一视野中MitoQ-ADMSCs荧光量显著低于ADMSCs,表明经MitoQ处理后细胞中活性氧产生减少,能够延缓细胞衰老。
3、对小鼠适应性饲喂1周,对所有小鼠进行随机分组:正常对照组;糖尿病模型组;ADMSCs单独治疗组;MitoQ联合ADMSCs治疗组,每组12只小鼠。正常对照组全程使用普通饲料饲喂,其他三组均选用高糖高脂饲料进行为期3周的饲喂,后对其进行为期16h的禁食并进行链脲佐菌素(Streptozotin,STZ)注射,其中小鼠腹腔注射剂量为40mg/kg,1次/周,连续3周。
注射1周后,对动物血糖水平进行为期3天的连续监测,3次空腹血糖测量值均高于7.6mmol/L,随机血糖测量值高于11.1mmol/L,视为糖尿病动物模型成功建立。后进行细胞移植治疗,MSCs单独治疗组注射剂量为:小鼠尾静脉注射2×106个;MitoQ联合MSCs治疗组注射剂量为:MitoQ预处理MSCs剂量为1μmol/L,小鼠尾静脉注射2×106个。正常对照组和糖尿病组则注射同等剂量的0.9%NaCl,连续治疗3周。在此过程中,持续监测动物模型生理状态、采食量、血糖水平等变化。在治疗1周和3周时,分别进行葡萄糖耐量的检测。治疗结束两周后,禁食16h,麻醉、采血、处死、采集组织样本备用,如图3a、b所示各组小鼠在实验开始时体重、血糖水平较为统一,而糖尿病模型组、ADMSCs单独治疗组、MitoQ-ADMSCs联合治疗组小鼠在连续3周的高糖高脂饲喂后,体重和血糖水平出现明显变化,均呈现出升高的趋势,而后续配以3周的STZ注射后,可见小鼠出现明显的体重下降及血糖持续升高的趋势,到第7周时可以确定进行连续3天的空腹血糖测定,血糖均高于7.6mmol/L,确定小鼠糖尿病模型成功制作。之后针对ADMSCs组和MitoQ-ADMSCs进行细胞移植治疗,经3周治疗后可见小鼠体重呈现出升高趋势,而血糖则呈现出明显的下降趋势,两个治疗组之间的对比可以发现,MitoQ-ADMSCs移植后的治疗效果更佳显著,这在治疗后的第1周和第3周葡萄糖耐量实验中可以见得,如图3c、d所示,MitoQ-ADMSCs细胞移植后,小鼠糖耐量实验的血糖变化和糖尿病初始阶段相似,呈现出非常显著的治疗效果。
小鼠胰腺使用4%多聚甲醛固定、脱水、石蜡包埋,制成4μm切片,并进行系列染色,包括H&E染色及Insulin免疫荧光染色,如图3e所示,糖尿病模型组小鼠的胰腺组织呈现出胰岛碎片化的状态,且可见存在的瘀血状态较为严重,而ADMSCs则在一定程度上恢复了胰腺组织的损伤,而MitoQ-ADMSCs进一步增强了这一治疗效果。
4、对犬适应性饲喂1周,对所有犬进行随机分组:正常对照组;糖尿病模型组;ADMSCs单独治疗组;MitoQ联合ADMSCs治疗组,每组包括3只犬。正常对照组全程使用普通狗粮饲喂,其他三组选用普通狗粮配合馒头等高糖食物进行为期3周的饲喂,后对其进行为期16h的禁食并进行链脲佐菌素(Streptozotin,STZ)注射,犬前肢静脉注射剂量为20mg/kg,1次/天,连续3天,1周后检测结果欠佳,之后继续进行1次/天,连续3天注射。
1周后,对动物血糖水平进行为期3天的连续监测,3次,随机血糖测量值高于11.1mmol/L,视为糖尿病动物模型成功建立。后进行细胞移植治疗,MSCs单独治疗组注射剂量为:犬前肢静脉注射1×107个;MitoQ联合MSCs治疗组注射剂量为:MitoQ预处理MSCs剂量为1μmol/L,犬前肢静脉注射1×107个。正常对照组和糖尿病组则注射同等剂量的0.9%NaCl,连续治疗3周。在此过程中,持续监测动物模型生理状态、采食量、血糖水平等变化。并于治疗的第1周和第3周进行葡萄糖耐量的检测。治疗结束两周后,禁食16h,麻醉、采血、处死、采集组织样本备用,如图4a-b所示,经2次间断STZ注射后,犬随机血糖急剧升高,并在3天监测时间内维持恒定,糖尿病模型基本成立,后经3天移植治疗后,血糖水平显著下降,体重水平有所上升,同时MitoQ-ADMSCs的治疗效果显著优于ADMSCs单独治疗。在治疗第1周的葡萄糖耐量实验中,可见血糖变化趋势有所恢复,且在第3周时,血糖在120min的观察时间内更加平稳,基本恢复到糖尿病初期水平,如图c、d所示。
犬胰腺使用4%多聚甲醛固定、脱水、石蜡包埋,制成4μm切片,并进行H&E染色和Insulin免疫荧光染色,如图4e所示,经细胞移植治疗后,胰腺组织损伤有所恢复,且胰岛素分泌有所提升,MitoQ则进一步提高了修复功能。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (4)
1.MitoQ和MSCs的细胞混合制剂在制备用于治疗动物糖尿病药物中的应用。
2.MitoQ和MSCs的细胞混合制剂,其特征在于:所述细胞混合制剂为MSCs、MitoQ和生理盐水的液体混合制剂。
3.根据权利要求2所述的MitoQ和MSCs的细胞混合制剂,其特征在于:所述的MitoQ的浓度为1μmol/L。
4.根据权利要求2或3所述的MitoQ和MSCs的细胞混合制剂,其特征在于:所述细胞制剂的细胞含量为107-109个细胞/mL。
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