CN115317519A - 脂肪间充质干细胞在制备治疗猫脂肪肝药物中的用途 - Google Patents
脂肪间充质干细胞在制备治疗猫脂肪肝药物中的用途 Download PDFInfo
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Abstract
本发明公开了脂肪间充质干细胞在制备治疗猫脂肪肝药物中的用途,属于制药领域。申请人通过制作FHL动物模型并静脉移植了ADSCs及其培养液冻干粉剂,结果发现,移植组能显著模型组动物的死亡率,病理组织学染色还表明,ADSCs及MSCM移植能显著减少肝脏病变,降低肝细胞中的脂质含量,对猫脂肪肝的治疗具有积极作用。因此,ADSCs对于猫脂肪肝具有很好的治疗效果,具有开发相关药物的潜力。
Description
技术领域
本发明涉及脂肪间充质干细胞的制药新用途,尤其是制备治疗猫脂肪肝药物的新用途,属于制药领域。
背景技术
1.ADSCs简介及生物学特性
干细胞是未成熟的组织前体细胞,具有自我更新、形成克隆细胞群以及多潜能分化等的能力,干细胞疗法用于机体急慢性损伤后,再生修复受损的组织器官并重建其功能。根据细胞来源、发育阶段以及分化潜能,干细胞可分为胚胎干细胞、诱导多能干细胞以及成体干细胞三种类型。胚胎干细胞虽然具有发育的全能性及扩增的无限性,但其“非定位性”分化难以控制,容易导致畸胎瘤,同时细胞获取来源也存在伦理争议,阻碍了在临床疾病治疗上的应用。诱导多能干细胞技术尚在起步阶段,诱导效率低下,难以获得状态、质量稳定的干细胞制品。相较而言,成体干细胞包括造血干细胞(HSCs)、间充质干细胞(MSCs)、神经干细胞等(NSCs),在体内广泛存在,取材方便,不涉及伦理问题且致瘤性低,是最具有临床应用前景的细胞类型。
HSCs是血液系统中的成体干细胞,它是目前研究历史最长且最为深入的一类成体干细胞,但异基因移植时免疫排斥需配型,且应用局限于治疗恶性血液病和肿瘤。NSCs存在于神经系统中,能够分化为神经元、星型胶质细胞及少突胶质细胞,对于开发治疗神经退行性疾病及脊髓损伤的疗法至关重要。与人类医学相比,HSCs和NSCs在兽医学中起的作用较小,主要是由于缺乏对应的移植和恢复条件,同时代价较为昂贵。相比之下,MSCs异体移植免疫原性较低,且近年来多种来源的MSCs分离培养及大规模制备技术逐渐成熟,利用MSCs来治疗各种急慢性损伤有望成为兽医再生医学的临床常规方法,是兽医临床转化医学理想的“种子细胞”。
迄今为止,已经成功地从各种组织,包括骨髓、脂肪、胎盘、脐带、牙髓等分离出了MSCs,目前最常用的MSCs来源是骨髓和脂肪组织,相较于其他组织,它们能提供更多的MSCs。脂肪间充质干细胞(ADSCs)与骨髓间充质干细胞(BMSCs)所表达的表面蛋白标志物基本相似,一样具有高度增殖和多向分化的能力,但ADSCs的增殖速率显著大于BMSCs,而且动物体脂肪储备丰富,取材微创,术后恢复迅速,并发症少,因此,对于临床应用而言,脂肪组织是首选的MSCs来源。
ADSCs的特性根据分离脂肪的位置不同而有所差异,而从腹股沟皮下分离的ADSCs可塑性最高。一般利用外科手术切取或吸脂术获取动物腹股沟皮下脂肪,运用酶消化法或组织贴块法接种培养细胞,通过不断换液除去未贴壁细胞而获得较纯的ADSCs。体外培养ADSCs形态上与成纤维细胞相似,主要表现为长梭形、纺锤形,常呈“旋涡状”排列,传代后,细胞逐渐纯化,3-4d可传代一次。ADSCs事实上并不是完全同质的细胞群体,是包含前脂肪细胞、血管内皮细胞、平滑肌细胞、成纤维细胞等的不均匀混合物。目前尚未发现特异性的ADSCs表面抗原,它既有间质细胞的表面抗原,又有内皮细胞、上皮细胞、肌肉细胞的表面抗原,而且ADSCs的体外表型呈动态改变,随着传代次数的增加,部分标志物的表达增多或减少甚至丧失,例如CD34是ADSCs短期培养和原代培养鉴定的潜在标志物,但长期培养的ADSCs不表达CD34。因此,目前通常联合多种表面标志物对ADSCs进行鉴定,根据国际细胞治疗协会间充质和组织干细胞委员会(ISCT)的提出的标准,体外标准培养条件下MSCs具有塑料粘附性,MSCs不表达造血干细胞的表面抗原,如CD34、CD45、CD14等,95%阳性表达CD73、CD105和CD90等,在体外标准条件下能诱导分化为成骨细胞、脂肪细胞和软骨细胞,联合检测可作为分选和鉴定ADSCs的依据。
2.ADSCs的治疗潜力及医学进展
尽管最初认为MSCs主要通过分化和代偿损伤组织来发挥其损伤修复作用,但大量的实验研究表明,MSCs的治疗特性主要依赖其免疫调节功能实现,MSCs可通过旁分泌作用、细胞外囊泡(ECV)的分泌、细胞凋亡介导的免疫调节、线粒体转移等与免疫系统相互作用,发挥损伤修复的功能。
MSCs的主要作用机制依赖于旁分泌信号的传导,通过调节各种免疫调节因子的分泌例如转化生长因子(TGF-β)、吲哚胺2,3—二氧酶(IDO)、前列腺素E2(PGE2)、白细胞介素10(IL-10)和肿瘤坏死因子α(TNF-α)等,调节机体免疫细胞的功能,主要是诱导单核巨噬细胞从促炎(M1)转变为抗炎(M2)型,抑制树突状细胞(DC)、T细胞、B细胞的增殖和成熟,并诱导调节性T细胞(Tregs)的产生,抑制自然杀伤细胞(NK)的功能。
MSCs的旁分泌作用不仅限于可溶性因子的分泌,还可通过ECV转移各种分子,ECV是由质膜通过向外或向内出芽而产生的囊泡,包括外泌体、微囊泡和凋亡小体。ECV可通过表面配体与靶细胞的受体结合来激活或抑制靶细胞内的转导通路,比如ECV表达跨膜Notch配体DII4,通过与表达在内皮细胞和神经细胞上的Notch受体相互作用而分别刺激血管再生和轴突生长。此外,还可通过传递胞内蛋白、mRNA、miRNAs、线粒体等生物活性物质来影响受体细胞的功能。
细胞凋亡也可能在MSCs的免疫调节作用中起重要作用。凋亡的MSCs刺激诱导单核巨噬细胞调节表型,进行胞吞作用吞噬清除,同时通过诱导Treg细胞来调节适应性免疫系统。MSCs似乎还能够通过隧穿纳米管进行细胞器的细胞间转移,可通过向体细胞的线粒体主动转移挽救具有非功能性线粒体的哺乳动物细胞中的有氧呼吸。由于线粒体转移与各种生理和病理活动有关,因此线粒体转移对于许多病理状况的未来治疗可能是潜在有用的。
除了复杂的免疫调节机制外,MSCs的另一特点是其具有定向迁移到受损组织的能力——MSCs“归巢”,这一过程与白细胞向炎症组织的归巢机制十分相似。当机体组织器官缺血缺氧或者出现损伤时,通过释放炎症因子(如IL-1、TNF-α)和趋化因子(如基质细胞衍生因子1)等多种成分入血活化MSCs,同时可以在损伤部位周围形成配体的浓度梯度,从而趋化MSCs沿浓度梯度向着损伤部位定向迁移。而后MSCs像白细胞一样向血管内皮黏附,跟随血液循环到达损伤部位周围后跨内皮游出至靶组织。在干细胞疗法中,MSCs的局部移植是理想的治疗方法,但在很多情况下,由于移植技术和潜在侵袭性的限制,往往不能直接移植于损伤部位,因此归巢是MSCs安全有效的应用于临床的关键。通常选择侵入性小且操作方便的静脉移植途径。静脉移植后MSCs的归巢主要受以下三种因素的影响。一是MSCs细胞体积大,平均大小为30μm,而肺毛细血管的平均直径仅为14μm,导致MSCs在肺中的机械滞留,有肺栓塞的风险;二是MSCs与肺内皮细胞相互作用,整联蛋白过度表达和激活导致MSCs归巢能力降低,归巢的细胞数量大大减少;三是细胞生存能力降低。因此,实际上来说。MSCs主要依赖免疫调节来发挥其治疗作用。
迄今为止,ADSCs已被用于治疗不同动物的多种疾病,大部分是研究性质的。再生兽药的最初重点是骨科疾病,但现在正在迅速扩展到其他领域,例如口腔和消化道疾病、肝、肾、心脏、呼吸、神经、肌肉、皮肤、嗅觉和生殖系统疾病。干细胞治疗最常用于犬和马,治疗各种器官系统的各种疾病,在猫中用于治疗肾、呼吸系统和炎性疾病。
肌肉骨骼系统疾病,包括肌腱、韧带和关节疾病。肌腱和韧带的创伤性和应力性损伤自然会随着瘢痕组织的形成而愈合,但形成的瘢痕组织与健康组织相比功有能缺陷,纤维化破坏了肌腱或韧带的生理结构以及功能,降低其韧性,引起运动功能不可逆性受损,容易再次受伤。关节部位软骨细胞相对较少且无血管经过,因此软骨组织的自我修复能力有限。肌肉骨骼损伤的常规治疗一般是应用抗炎皮质醇结合外科手术,以减轻疼痛和炎症,往往导致动物运动性能大幅度降低和慢性跛行。因此,理想的治疗方法应旨在再生正常的肌腱基质和提高软骨组织的再生修复能力。
脊髓损伤。脊髓损伤是人和动物最常见的神经肌肉损伤之一,是导致死亡和残疾的重要原因,常见的有脊柱骨折、椎间盘脱出或突出等,原发性损伤尚无有效的治疗方法,脊髓损伤后及时的阻断自由基氧化作用、减少炎症反应,减少钙离子的内流入细胞,最大程度上减少继发性脊髓损伤(出血、水肿等)是目前治疗、改善预后和降低致畸率的最有效处理方法。Jung等通过手术建立30只比格犬重度脊髓损伤模型,10只鞘内注射自体间质干细胞,10只注射异体MSCs,10只作为模型对照,治疗一周后结果表明,不论自体或异体来源的MSCs对恢复犬下肢活动能力均有明显的提高。Penha等使用自体BMSCs治疗4只脊髓损伤的犬,移植10d后,动物膜反射逐渐恢复,浅表和深部疼痛反应减轻,持续移植18个月后,动物运动能力明显提高,说明MSCs移植对脊髓损伤是一种有效的治疗方法。
肾脏疾病。由于摄入饮食和药物等的不合理,犬猫易发各种急慢性肾病,约1/3的猫和1/10的狗会患上慢性肾病,而目前肾移植是唯一可以恢复肾功能的治疗方法。将25只患有慢性肾病的猫分成3组,分别静脉注射2×106、4×106、6×106剂量的异体MSCs两周一次,实验结果表明第一组猫无明显不良反应,血清肌酐浓度小幅度下降,蛋白尿轻度改善,第二、三组动物出现不同程度呕吐、呼吸频率加快等不良反应,血清肌酐、尿常规也未见改善。临床病例也证实,MSCs在猫中确实有降低肌酐、尿素,改善肾衰的功能。Yasser等利用缺血再灌注建立犬急性肾损伤模型,直接在肾皮质注射BMSCs或ADSCs,通过超声检查、组织病理学以及炎症因子的检测,说明MSCs对犬急性肾损伤具有保护作用,且ADSCs显示出优于BMSCs的治疗效果。
心脏疾病。心脏病主要多发于老年犬猫,有较高的发病率和死亡率,大部分手术难度大,一般以保守治疗为主(减轻心脏负担、增强心肌收缩力、对症治疗等)。Memon等将自体BMSCs直接注射入犬心脏受损部位,4周后结果显示患犬左心室扩张减小和射血分数增加,治疗组心功能较对照组有明显的改善,表明BMSCs能促进心肌生成和血管再生,增强心脏功能并改善心肌重构,是临床治疗缺血性心脏病的新的治疗方案。
皮肤病。MSCs可以激活细胞间接触和旁分泌,促进成纤维细胞的增殖,从而加速伤口的愈合。Kim等在健康的比格犬背部制作全层皮肤损伤模型,皮内注射同种异体BMSCs,发现治疗组在伤口愈合、胶原合成、细胞增殖和血管生成方面均优于对照组,且治疗组的炎性细胞因子和创口愈合相关因子表达量减少,说明其不仅可以用于治疗皮肤损伤,还可应用于治疗炎性和纤维化皮肤病。
猫的MSCs的研究主要集中在猫口炎、慢性肾病、慢性肠病、哮喘几方面。猫口炎是发生在猫口腔黏膜表层或深层组织的炎症,该病时间长、难治愈、易复发,一般认为该病是由于口腔中抗原刺激引起不恰当的免疫反应引起的。常规的治疗手段包括全口拔牙、抗生素治疗和皮质类固醇给药,给药时间长,且可能复发。干细胞疗法可以帮助纠正可能与口炎有关的免疫系统异常。对7只已进行全口拔牙治疗的猫进行为期6个月的静脉注射ADSCs,其中4只猫对治疗表现出良好反馈,有明显的临床改善,持续进行干细胞移植,在18-20个月时,有两只猫表现逐步改善和完全治愈的迹象。目前为止共有超过40只猫接受了干细胞治疗,总的治愈率为75%,且均未出现不良副作用。对于猫的慢性肠病、哮喘等研究的开展,主要是由于MSCs的免疫调节作用,但实验结果患猫指标仅见轻微下降,与安慰组相比指标变化小,可以尝试作为辅助疗法,不能完全依赖干细胞治愈。
目前,MSCs在肝脏中的研究应用主要集中在疑难及终末期肝病,例如自身免疫性肝炎、急慢性肝衰竭、肝硬化、肝癌等,原因主要是现阶段能够长期有效的治疗晚期肝病的方法只有肝脏移植,而肝脏移植又受限于捐献者短缺及代价昂贵而难以普及,且术后容易出现并发症包括急慢性排异反应、胆道并发症、腹腔感染等。多数终末期肝病患者只能采取姑息治疗以缓解病痛和维持生命,在这种情况下,我们亟须新的有效的治疗方法来改善预后和延长患者的生存时间、提高生存质量。
大量的实验研究表明,MSCs移植在终末期肝病的治疗中发挥着积极作用,可延缓患者病情,改善肝功能,且无明显的不良反应,具有极大的临床应用潜力,但MSCs的具体作用机制尚不十分明确。目前可以确定的是MSCs移植后在肝脏损伤微环境的影响下首先定植、增殖,并分化成肝细胞,从而修复肝脏;还有可能通过直接与肝细胞融合,进而启动肝细胞的增殖过程;同时MSCs可以分泌不同水平的细胞因子相互影响,激活肝内的肝干细胞,促进肝细胞再生;分泌的免疫活性因子发挥免疫调节的作用,减轻肝脏的炎症反应和损伤;对于肝硬化的的恢复可通过高表达基质金属蛋白酶降解细胞外基质,直接降解肝内过量沉积的细胞外基质,而减轻肝纤维化。
总而言之,许多积极的结果表明,ADSCs治疗在各种动物疾病上应用前景广阔,有望成为兽医临床治疗的一种现实可行的选择。
3.猫脂肪肝简介及治疗现状
猫脂肪肝又称猫肝脏脂肪沉积综合征(FHL),是猫特有的、也是猫最常见的一种肝脏疾病,是由于各种病因导致的猫原发或继发性肝脏脂肪代谢异常,使甘油三酯在肝脏细胞中大量聚积,导致肝功能继发性损伤和胆汁淤积。该病不分年龄和品种,但雌性猫、老年猫、肥胖猫多发。肥胖和应激是引起该病的主要因素,厌食通常是疾病发生的直接因素。当肥胖猫持续厌食5-7d时,就有患脂肪肝的风险。由于发病初期一般仅出现厌食症状,不易引起饲主重视,往往错过最佳治疗时间,若不进行积极有效的救治,死亡率在90%以上。如早期就诊,并配合合理治疗和特殊护理,治愈率可达80%-85%。随着宠物市场的不断发展,以及对宠物猫以胖为美的审美驱使下,FHL的发病率逐渐提高,已经成为宠物猫健康的一大杀手,如何有效、安全的治疗猫脂肪肝,提高患病动物的生存率,已是摆在我们面前亟待解决的问题。
引起FHL的原因多种多样,一般以原发性和继发性区分。原发性主要与动物应激如饥饿、环境改变、更换日粮以及惊吓等引起的食欲废绝有关。此外,长期的营养不均衡,如营养过剩(长期摄入高脂肪和高碳水化合物的食物)和营养缺乏(蛋白质、维生素、精氨酸、蛋氨酸等缺乏)、肝脏慢性毒素损伤、动物运动不足、妊娠等也会引起动物摄食减少而引起原发性脂肪肝。
早期典型的临床症状表现为猫病理性厌食、抗拒喂食,随后动物逐渐消瘦、体重下降(通常会超过体重的25%)、便秘、被毛粗乱、出现慢性脱水以及间歇性呕吐。病程继续发展,动物表现精神沉郁、四肢乏力、肌肉萎缩、可视黏膜、皮肤、内耳和齿龈黄染,进一步恶化后,猫的凝血功能异常,皮肤上可见淤青,出现皮肤脆皮综合征,极易出现皮肤瘢痕。少数病猫会出现肝性脑病,表现出抽搐、昏迷等神经异常的症状。
临床上猫脂肪肝的诊断主要包括一般检查、血液学检查、影像学检查以及肝脏组织病理学检查。一般检查主要根据患猫精神状况、体态、体温、脱水情况、黏膜黄染程度、腹部触诊肝脏肿胀程度(30%动物显示肝肿大)等临床症状进行初步诊断。血液学检查可见患猫有轻度的非再生障碍性贫血,并且血凝能力下降,应激性白细胞总数增加。生化指标常显示碱性磷酸酶(ALP)升高,谷丙转氨酶(ALT)升高,谷草转氨酶(AST)、总胆固醇(CHOL)、甘油三酯(TG)均升高。血气分析30%左右患猫会出现低钾血症,少数还可能出现低血磷、低血镁。腹部超声可见肝脏弥漫性回声增强现象,同时还会出现门静脉增粗以及胆管的不规则增粗。肝脏穿刺活检是确诊FHL最准确的办法,但也是最危险的诊断方法。剖检时可见肝肿大,边缘钝圆、表面平整、略带鼓胀感且易碎,整体呈斑驳或弥漫的黄白色,触感油腻。组织病理学可见肝细胞广泛空泡化,表现为大空泡型、小空泡型或混合型脂沉积,并有胆汁阻塞,可有小的炎性灶或坏死灶。
猫脂肪肝治疗的关键点在于及时尽早纠正机体液体和电解质紊乱。大部分患猫就诊时已出现严重脱水,需要及时补液,若问诊动物有明显呕吐,一般伴随低血钾,选择静脉滴注林格氏液。根据血气分析结果,注意额外补充镁、磷。重症猫脂肪肝动物乳酸代谢障碍,体内乳酸浓度异常升高,应避免使用乳酸林格氏液。为支持动物机体能量供应,应添加ATP+coA+VC,促进机体代谢恢复。葡萄糖是猫脂肪肝治疗禁忌,因为葡萄糖会减少脂肪酸氧化,促进肝脏脂质蓄积,进一步导致脂肪肝恶化,还可因渗透性利尿造成更加严重的脱水和电解质紊乱。
营养支持让猫尽快摄入食物,并恢复自主进食是脂肪肝能否痊愈的核心。根据动物的状态可选择强饲、食欲刺激、鼻饲管、食道饲管、胃饲管等方法。临床上为防止产生厌食综合征以及减少应激,最常用的是安置鼻饲管、食道饲管或胃饲管,改善动物体况,通过饲管给予全价高蛋白日粮,研究表明,饲料中丰富的蛋白质能显著减少脂肪在肝脏的蓄积,应避免饲料中碳水化合物过量。同时在饲料中添加复合维生素、牛磺酸、L-肉毒碱等,促进机体氧化代谢和脂质转运。日粮应少量多次给予,总量满足机体基础能量消耗,不宜饲喂过多而导致高血糖和胃肠负担。
对症治疗,通过静脉滴注VK治疗和预防动物凝血障碍;使用胃复安来抑制疾病和安置饲管引起的呕吐;茵栀黄、熊去氧胆酸保肝、利胆、退黄;抗生素防止继发感染;动物精神好转后,科特壮刺激食欲,加快动物自主采食。治疗过程中要密切关注动物状态,调整治疗方法和药物,注意治疗原发病和防止并发症如贫血、肝性脑病、糖尿病、胰腺炎、再饲喂综合征等的出现,改善疾病预后。
经过综合治疗,动物的生化指标一般在1-2周时有所改善,需要3-6周才能完全康复,康复的时间与进行营养支持的时间正相关,积极饲喂的动物康复时间短、预后良好。目前关于其他治疗药物的探索多是研究具有护肝利胆作用的中药或提取物如绞股蓝、水飞蓟素等在治疗中的作用,大多都具有良好的效果,但其与临床使用的茵栀黄作用相近,对提高动物的生存率和缩短病程的效果并不十分显著。
发明内容
人们视干细胞为“种子细胞”或“万能细胞”。在基础和临床医学研究领域中,干细胞疗法已成为治疗多种疾病的首选方法。其中,ADSCs具有干细胞的生物特性,针对修复组织缺陷和损伤,它有独特的优势。FHL是临床上猫发病率及死亡率极高的一种内科疾病,且其治疗需要长期的特殊护理,代价昂贵。
鉴于此,本发明研究ADSCs在猫脂肪肝中的治疗效果,旨在为猫脂肪肝的临床治疗提供新思路、新方法,缩短治疗周期,同时提高患病动物的生存率和治愈率,同时,进一步了解和探索ADSCs的功能,为研发干细胞药物提供理论依据。
首先,分离培养猫ADSCs并对其进行了鉴定和原体检测,接着制作FHL模型并静脉移植了ADSCs及其培养液冻干粉剂,结果发现,移植组能显著模型组动物的死亡率,病理组织学染色表明,ADSCs及MSCM移植能显著减少肝脏病变,降低肝细胞中的脂质含量,对猫脂肪肝的治疗具有积极作用。
以上结果表明,ADSCs对于猫脂肪肝具有很好的治疗效果,具有开发相关药物的潜力。
更详尽的方案参见具体实施例。
附图说明
图1:不同时期猫ADSCs的形态学观察。(A)猫原代ADSCs培养24h,ADSCs已经贴壁伸展,培养液中可见大量悬浮的脂滴和不贴壁细胞;(B)原代ADSCs首次换液后,细胞呈明显的纺锤状,培养液澄清无悬浮物;(C)P1代ADSCs;(D)P3代ADSCs。P1、P3代ADSCs呈“鱼鳞样”或“旋涡状”均匀排列生长。
图2:P3代ADSCs细胞增殖曲线。
图3:猫ADSCs的诱导分化鉴定。
图4:细胞表面标志物鉴定。
图5:细胞病原体检测。
图6:治疗期各组小鼠的生存曲线。
图7:治疗期各组小鼠的病理组织学染色结果。
具体实施方式
下面通过具体实施例对本发明进行详细说明。
1、材料和仪器
1.1实验动物
25只健康成年家猫,年龄3-6岁,雌雄不限,体重5.5-6.5kg,实验前接种猫三联(荷兰英特威)和狂犬疫苗(美国辉瑞)。
1.2主要试剂与溶液
Ⅰ型胶原酶(9001-12-1,Biofroxx公司)
PBS缓冲液(MA0010,Meilunbio公司)
低糖DMEM培养基(SH30021.01)、0.25%胰蛋白酶(SH30042.01)均购自HyClone公司
表皮生长因子(96-AF-100,PeproTech公司)
胎牛血清(GIBCO公司)
青链霉素混合液(P1400)、间充质干细胞无血清培养基(N6010)、油红O染液(细胞专用)(G1262)、茜素红染液(G3281)、油红O染液(G1260)、GoldviewⅡ型核酸染色剂(G8142)均购自Solarbio公司
海藻糖(T100012,阿拉丁公司)
CCK8细胞增殖毒性检测试剂盒(BS350A,Biosharp公司)
CD34抗体(ab81289,Abcam公司)
CD45抗体(NBP2-34287,Novus公司)
CD90抗体(A2126)、CD105抗体(A5639)均购自ABclonal公司
DAPI(C1002,上海碧云天生物技术有限公司)
成脂诱导分化培养基(CAXMX-90021)、成软骨诱导分化培养基(CAXMX-90031)均购自Cyagen Biosciences公司
DL2000 DNA Marker(272023AX,北京艾德莱生物科技有限公司)
Trizol(9109)、反转录试剂盒(toyobo fsk-101)均购自TaKaRa公司
氯仿、无水乙醇均购自国药集团化学试剂有限公司
酚磺乙胺注射液(湖北中佳公司)
盐酸肾上腺素注射液(中国远大医药有限公司)
硫酸阿托品注射液(天津金耀公司)
速眠新Ⅱ注射液(吉林省方正动物药业有限责任公司)
3.3主要仪器设备
优普系列超纯水器(UPT-11-20T,四川优普超纯有限公司)
凝胶成像系统(NEWBIO INDUSRTY公司)
雪花制冰机(XUEKE,武汉科昊佳生物科技有限公司)
电子天平(AX124ZH/E,奥豪斯仪器有限公司)
紫外可见光分光光度计(UV-3100PC,上海美谱达仪器有限公司)
普通PCR仪(A200,杭州朗基科学仪器有限公司)
电泳仪及电泳槽(DYY-6C型,北京市六一仪器厂)
台式高速冷冻离心机(5404,德国Eppendorf公司)
-20℃冰箱(DW-YL270,中科美菱公司)
-80℃超低温冰箱(DW-86L626,青岛海尔特种电器有限公司)
超净工作台(HDL APPARATUS,哈尔滨市东联公司)
立式自动电热压力蒸汽灭菌器(LX-B50L型,合肥华泰医疗设备有限公司)
高通量组织研磨器(SCIENTZ-48,宁波新芝生物科技股份有限公司)
涡旋震荡仪(MX-S,SCILOGEX公司)
电热恒温培养箱(DNP-9162型,上海精宏实验设备有限公司)
数显恒温水浴锅(HH-6,国华电器有限公司)
冷冻干燥机(Labconco)、激光共聚焦显微镜(Zeiss LSM800)由华中农业大学农业微生物国家重点实验室公共平台提供
2.实验方法
2.1猫ADSCs的分离及培养
2.1.1猫ADSCs的分离
运用酶消化法分离猫ADSCs。
①通过无菌术,取猫腹股沟皮下脂肪约拇指指甲盖大小,放于含有2%双抗PBS的15mL离心管内;
②取到的脂肪在无菌超净台内依次用75%酒精浸泡3次,每次3s,PBS洗2次,最后用双抗PBS洗涤两次;
③然后剪碎约为1mm3大小的组织块,剪碎后转移至15mL离心管中,加入两倍体积的浓度为1mg/mL的Ⅰ型胶原酶溶液,于37℃水浴锅中消化60min;
④之后加入等体积的完全培养液(含10%FBS、10ng/mL EGF、1%双抗的低糖DMEM)终止消化;
⑤消化液用200目细胞筛过滤,滤液以3000g离心5min,弃去上清液;
⑥沉淀用完全培养基重悬,混匀制成细胞悬液,接种至25cm2细胞培养瓶中。将细胞置于37℃、5%CO2培养箱中培养48h后换液,以后每3d换一次液,直到细胞长至80%-90%融合,可进行传代。
2.1.2猫ADSCs的培养
细胞换液及传代:
①先用吸管吸去培养瓶内的培养液,加入PBS多次洗涤,每次洗涤轻轻晃动培养瓶,使液面覆盖培养瓶底,直至PBS澄清后弃去,再加入5mL完全培养基,置于5%CO2、37℃饱和湿度箱中培养,48h后换液;
②待细胞达到80%-90%融合,弃去培养基,PBS洗涤1-2次,加入1mL0.25%胰蛋白酶,轻轻晃动使消化液盖满瓶底;
③弃去胰酶,至于镜下观察,至大多细胞形状变圆,边缘发亮,根据细胞状态不同,消化时间在15s-1min;
④用完全培养基终止消化,小心吹打,避免出现气泡,避免长时间多次吹打,形成均一的单细胞悬液,平均接种到5个25cm2细胞培养瓶中,轻轻摇匀,平稳置于5%CO2、37℃饱和湿度箱中培养,隔天换液。
细胞计数:
①用吸管轻轻反复吹打细胞悬液使细胞重悬均匀后,吸取细胞悬液少许,向另一离心管中滴入细胞悬液9滴,再滴入0.4%台盼蓝染色液1滴混匀。向细胞计数板边缘轻轻滴入1-2滴已染色的细胞悬液,使之充满计数板和盖片间的空隙中;
②置于倒置显微镜下计数死细胞与活细胞数目,计算细胞活力(死细胞能被台盼蓝染上色,镜下呈深蓝色,活细胞不被染色,镜下无色透明)和细胞密度;
③计数四个大方格内的总细胞数及着色细胞数,计算细胞存活率。
细胞冻存:
①取对数生长期的细胞,用胰蛋白酶把单层生长的细胞消化下来,完全培养基终止消化,收集入离心管中;
②于水平离心机内离心1900g,5min;
③去除旧的培养液,加入适量配制好的冻存液(10%DMSO+40%血清+50%完全培养基),用吸管轻轻吹打使细胞均匀,计数,调节冻存液中细胞的最终密度为5×106-1×107/mL;
④将细胞分装入冻存管中,每管1-1.5mL;
⑤在冻存管上标明细胞的名称,冻存时间及操作者;
⑥将装有细胞的冻存管平稳放入4℃冰箱中30-60min,随后放入-20℃冰箱30-60min,然后放入-80℃冰箱中过夜,次日液氮转移冻存管,放入液氮罐内并登记位置。细胞复苏:
①从液氮罐中取出冻存管,浸入37℃温水中,并快速晃动令其尽快融化,时间控制在2min之内;
②完全融化后取出冻存管,在超净台中打开盖子,用吸管吸出细胞悬液,加到5mL的离心管并添加等体积的完全培养基,轻轻吹打混匀;
③于水平离心机内离心,1 900g,5min;
④弃去上清液,加入完全培养基重悬沉淀,细胞计数,调整细胞密度,接种培养瓶,轻轻摇匀,平稳置入5%CO2、37℃培养箱静置培养;
⑤次日更换一次培养基,继续培养。
细胞增殖曲线的绘制:
①在96孔板中接种细胞悬液(100ul/孔),每孔约2000个细胞。设置不含细胞的完全培养基对照孔;
②细胞培养每隔12h,待测细胞孔和其对照更换新鲜培养基,之后分别加入10ulCCK-8溶液,在细胞培养箱内继续孵育2h;
③用酶标仪测定在450nm处的吸光值,以培养时间为横坐标,绝对OD值为纵坐标,绘制ADSCs的生长曲线。
2.2猫ADSCs的鉴定
2.2.1诱导分化鉴定
成脂诱导分化鉴定:
以生长良好的P3代细胞为实验细胞。细胞经消化后接种于12孔培养板,24h后完全贴壁。更换成脂诱导分化培养基A液培养2d,之后换成脂诱导培养基B液培养1d,如此交替培养21d后,进行油红O染色。诱导过程中拍照记录观察细胞形态。
油红O染色:
①细胞用PBS浸洗3次,每次3min;
②用ORO Fixative固定15min,PBS浸洗玻片3次,每次3min;
③加入新配制的ORO Stain,浸染15min;
④60%异丙醇漂洗20-30s至背景无色,流水冲洗,蒸馏水稍微清洗;
⑤Mayer苏木素复染核2min,自来水洗(返蓝)1-3min;
⑥ORO Buffer 1min,流水冲洗,水溶性封片剂封片。显微镜下观察并拍照。
成软骨诱导分化鉴定:
以生长良好的第3代细胞为实验细胞。细胞经消化后接种于12孔培养板,24h后完全贴壁。更换成软骨诱导分化培养基,每两天换液,如此培养21d后,进行茜素红染色。诱导过程中拍照记录观察细胞形态。
茜素红S染色:
①细胞用PBS浸洗3次,每次3min;
②用4%多聚甲醛固定15min,弃去固定液,用ddH2O洗3次;
③将水完全吸干净后,慢慢加入茜素红S染色液,染色25min;
④弃去染液,用ddH2O洗3-5次;
⑤每孔加入适量ddH2O防止孔内干燥,显微镜下观察并拍照。
2.2.2细胞表面标志物鉴定
细胞爬片的制作:
①胰酶消化细胞后计数重悬细胞于完全培养基中;
②先在每个孔里滴10μL完全培养基,目的是使玻片与培养瓶靠培养基的张力吸附在一起,然后放置爬片,防止加细胞悬液时玻片漂起,造成双层细胞贴片。整个过程注意无菌操作;
③根据需要选择合适的细胞密度接种入培养板内即可。
免疫荧光染色:
①在培养板中将已爬好细胞的玻片用PBS浸洗3次,每次3min;
②用4%的多聚甲醛固定爬片15min,PBS浸洗玻片3次,每次3min;
③PBS浸洗玻片3次,每次3min,吸水纸吸干PBS,在玻片上滴加3%正常山羊血清(PBS配制),室温封闭30min;
④吸水纸吸掉封闭液,不洗,每张玻片滴加足够量的稀释好的一抗并放入湿盒,37℃孵育4h;
⑤加荧光二抗:PBS浸洗爬片3次,每次3min,吸水纸吸干爬片上多余液体后滴加稀释好的荧光二抗,湿盒中37℃孵育1h,TBST浸洗切片3次,每次3min;PBS洗切片3次,每次3min。注意:从加荧光二抗起,后面所有操作步骤都尽量在较暗处进行;
⑥复染核:滴加DAPI避光孵育5min,对标本进行染核;
⑦用吸水纸吸干爬片上的液体,用含抗荧光淬灭剂的封片液封片,然后在荧光显微镜下观察采集图像。
2.3猫ADSCs的病原体检测
2.3.1细胞DNA提取
①用细胞刮将细胞刮下并收集在标记的EP管中;
②添加500μL组织裂解液和10μl Proteinase K到每一管,涡旋混匀。55℃水浴过夜;
③每管加入250μL5 M NaCl,上下晃动30-50次。12000g,离心10min;
④弃沉淀,再离心,弃沉淀;
⑤每管加入700μL无水乙醇,上下晃动30-50次。12000g,离心10min;
⑥弃上清,每管加入700μL70%乙醇。12000g,离心5min;
⑦弃上清,尽量弃干净。烘箱内烘干;
⑧各用20μL双蒸水稀释,涡旋后测浓度。
2.3.2细胞总RNA提取及反转录
RNA提取:
①从培养箱取出细胞,PBS清洗3次,每孔加入1mL Trizol裂解,静置5min;
②加入0.2mL氯仿,用力震荡15sec,静置2min;
③4℃,12000g,离心15min,取上清(注意不要取到中间蛋白层);
④加入0.5mL异丙醇,将管中的液体轻轻混匀,室温静置10min;
⑤4℃,12000g,离心10min,弃上清;
⑥加入1mL 75%乙醇(DEPC水配制),轻轻洗涤沉淀。;
⑦4℃,7500g,离心5min,弃上清;
⑧沉淀自然晾至将干未干,加入适当的DEPC水溶解;
⑨取少许检测纯度(OD260/280)及RNA完整性(电泳),于-80℃保存备用。
cDNA的合成:
以提取的RNA为模板,进行反转录(两步法)。反转录(Reverse Transcription,RT)的体系(20μL)为:
第一步:
反应条件为:42℃2min,之后迅速置于冰上。
第二步:
反应条件为:37℃15min,85℃5sec,4℃保存。合成好的cDNA置于-20℃保存备用。
2.3.3病毒引物合成
根据NCBI提供的碱基序列,通过文献查找及Primer 5.0设计引物,由生工生物工程(上海)股份有限公司合成。
病毒引物序列
2.3.4聚合酶链式反应(PCR)扩增
将提取出的细胞DNA、cDNA作为模板,Taq DNApolymerase分别扩增RV、FHV-I、FCV、FPV。PCR反应体系(10μL)为:
2.3.5DNA凝胶电泳和成像
称取0.48g琼脂糖粉末于烧杯中,加入40ml 1×TAE Buffer,配制1.2%琼脂糖凝胶用于电泳。向10μL反应后的PCR体系里加入2μL 6×loading buffer,混匀后点到凝胶孔中,以4μL DNA标准分子量DL2000 Marker作为参照,15V/cm电泳,电泳结束后,在凝胶成像系统中观察,拍照保存图片。
2.4猫ADSCs培养液冻干粉的制备
2.4.1培养液(MSCM)的收集
①将培养好的待收集细胞放入显微镜观察,其密度达到80%左右为宜;
②将培养瓶中的完全培养基倾倒,用PBS冲洗培养瓶底部,确保培养瓶被冲洗干净,没有完全培养基的残留;
③向培养瓶中加入无血清培养基,置于37℃,5%二氧化碳恒温培养箱中培养48h;
④取出培养瓶,显微镜下观察细胞状态。将无血清的培养基收集到无菌烧杯中;
⑤在无菌烧杯中把细胞上清液混匀,分装到离心管中,用离心机进行离心(3000g/min,4℃),去除细胞上清液中的细胞残渣;
⑥离心后将细胞上清液重新收集到无菌烧杯中,用注射器吸取通过0.22μm滤膜进行无菌过滤,获得纯净的细胞上清液。
2.4.2MSCM冻干处理
①收集新鲜的MSCM;
②按照体积称量海藻糖粉末(8%),溶于收集的MSCM中进行配制;
③将混合物倒入15cm直径的玻璃平皿中,每个平皿大约倒入5mL左右,平皿上方用保鲜膜进行包裹,防止冰霜落入平皿中影响MSCM的纯度,保鲜膜上扎数个透气孔放入-80℃冰箱,冷冻2h;
④2h后取出玻璃平皿,将冷冻后的MSCM移至5mL西林瓶中,同样用保鲜膜覆盖住西林瓶口,保鲜膜上扎数个透气孔;
⑤打开冻干机,冻干过夜。
2.5FHL模型的制作及检测
2.5.1FHL模型的制作
选择肥胖的成年猫,通过完全禁食20d,诱导建立FHL模型,诱导造模过程中,动物独立笼位饲养,不禁水。造模后肝脏活检确定FHL模型的建立。
2.5.2肝脏活检手术
①术前15min称体重,按剂量皮下注射硫酸阿托品、盐酸酚磺乙胺。肌肉注射速眠新II全身麻醉;
②上腹部剃毛,消毒(75%酒精-聚维酮碘-75%酒精);
③剑状软骨下沿腹白线开口,依次打开皮肤-肌肉-腹膜,暴露腹腔,找到肝脏;
④沿肝脏边缘剪切1cm3肝实质,注意避开大的血管;
⑤部分肝切除后,迅速用浸有盐酸肾上腺素的纱布按压断面2min;
⑥去除纱布后,观察有无出血,出血则继续按压,未出血即可缝合;
⑦腹膜、肌肉层连续缝合,皮肤结节缝合;
⑧切口碘伏消毒。
2.5.3病理组织学检查
肝脏样本4%多聚甲醛固定48h,用以后续制备石蜡切片及冰冻切片。
石蜡切片的制作:
①取材:不超过3mm。
②流水冲洗1h。
③固定:
⑴脱水:乙醇梯度脱水Ⅰ→Ⅱ→Ⅲ(90%→100%→100%),1h/次,共3h;
⑵透明:二甲苯脱脂Ⅰ→Ⅱ→Ⅲ(100%),1h/次,共3h;
⑶浸蜡:恒温箱中55℃~65℃浸石蜡Ⅰ→Ⅱ→Ⅲ,1h/次,共3h。
④包埋:应用包埋机。
⑤切片:
⑴切片:应用切片机,切片厚度在3μm以下;
⑵展片:首先在冷水中尽量将片子展开,然后在热水(40℃到50℃)中展开;
⑶捞片:注意引出切片与载玻片之间的水分,使切片和载玻片充分贴合;
⑷烘片:在烘片机上烘干。
HE染色:
①熔蜡:恒温箱中56℃~60℃放置45min;
②脱蜡:二甲苯Ⅰ→Ⅱ→Ⅲ(100%),10min/次,共30min;
③脱水:乙醇Ⅰ→Ⅱ→Ⅲ(100%),10min/次,共30min;
④苏木精染色5min(做预实验确定时间);
⑤流水冲洗8-15min;
⑥伊红染色8min(根据染液的效果调整时间);
⑦酒精简单漂洗;
⑧乙醇Ⅰ→Ⅱ→Ⅲ(70%→90%→100%),5min/次,共15min;
⑨二甲苯Ⅰ→Ⅱ→Ⅲ(100%),5min/次,共15min。
⑩封片:中性树脂封片。注意不要进入气泡,盖上盖玻片后不要随意移动,防止拉坏组织,晾干后添加标签,光镜下观察。
冰冻切片的制作:
①4%多聚甲醛固定48h;
②30%蔗糖脱水48h;
③冰冻切片机内-25℃OCT包埋;
④应用切片机,厚度7μm;
切片-80℃保存。
油红O染色:
①称取油红O 0.5g,50%乙醇100ml。将油红O溶于乙醇内,且不断搅拌至完全溶解即可。配制好的染液过滤,避光保存待用;
②切片干燥后入50%乙醇稍洗;
③油红O乙醇染液作用8min;
④50%乙醇分化,自来水终止分化;
⑤苏木素复染核,自来水返蓝,甘油明胶封片。
2.6猫ADSCs和MSCM移植
①准备P3代猫ADSCs,随机选择,PBS清洗两遍;
②加入1-2mL胰酶使其均匀覆盖瓶底,弃掉;
③镜下观察,细胞收缩至边缘变亮,形状变钝即可用完全培养基对其进行结束消化,收集入离心管中;
④1900g,5min,常温状态下进行离心;
⑤弃上清,PBS重悬,重复离心重悬;
⑥细胞计数,按照2×106cells/kg为移植标准,收集细胞悬液入玻璃注射器中,在肝脏活检后24h、72h进行静脉移植。MSCM移植的剂量对应细胞移植量,移植培养移植剂量细胞的DMEM;
⑦疏通留置针,通过留置针缓慢匀速注射,以1mL/min为标准进行缓慢推注,生理盐水封口。
3、结果与分析
3.1猫ADSCs的鉴定
3.1.1细胞形态学观察
运用酶消化法体外分离获得猫原代ADSCs。培养12h时,原代ADSCs基本贴壁,此时大多数仍然是单核细胞,而且培养液中有大量悬浮脂肪滴。培养24h时,单核细胞已经伸展呈纺锤状,具有均匀的成纤维细胞样形态。培养48h时,PBS多次洗涤换液,通过去除不贴壁的细胞以达到猫原代ADSCs初步纯化的目的。进一步培养,根据分离到原代ADSCs数量的不同,细胞生长至100%汇合的时间也不同。分离的原代ADSCs数量稀少时,细胞生长速度较慢且呈集落式生长,以几个贴壁细胞为中心向外辐射生长,集落中心处细胞致密,当集落中心生长至80%-90%汇合时,可消化传代至P1代。未及时传代培养的话,集落中心细胞会形成多个密集的细胞层并开始从瓶底脱落。分离的原代ADSCs数量多且在培养瓶中分布均匀时,细胞生长迅速,70-80%汇合时可见呈“鱼鳞样”或“旋涡状”排列生长,可消化传代至P1代。传代后,去除不能增殖的细胞进一步纯化ADSCs。(图1)
3.1.2细胞增殖曲线
理论上讲,细胞代数越靠前,干细胞的干性也越强,随着细胞传代次数的增加,细胞会逐渐的老化,增殖能力和分化潜能不断下降。但是由于干细胞在体外培养的过程中,干细胞有从组织微环境到体外培养环境的一个调整和适应过程,一部分不适应的细胞会被淘汰,所以在前两代,干细胞基因组有着不稳定的因素,可能不宜临床应用。同时研究发现,ADSCs在传代至P5代的过程中,生物学特性及遗传特性基本稳定,无明显变化,表明ADSCs在P5代以内可以安全使用。综合考量,此次FHL治疗实验选择P3代细胞为实验细胞。P3代ADSCs呈现典型的“S”样曲线生长,在接种36h后进入对数生长期,接种120h后,进入平台期。(图2)
3.1.3诱导分化鉴定
成软骨诱导分化培养14d时,细胞表面有少量颗粒状钙盐沉积,细胞未发生明显变化(图3A);培养21d时,大量钙盐逐渐融合成致密的矿化结节覆盖细胞表面,细胞间隙增大(图3B);此时,茜素红S染色矿化结节呈深红色,成软骨诱导分化阳性(图3C)。
成脂诱导分化培养14d时,细胞开始失去梭形变圆,可见圆形折光性强的脂肪小滴(图3D);培养21d时,细胞呈圆形且体积增大,胞质中脂滴融合成大脂滴(图3E);此时,油红O染色胞质脂滴呈鲜红色,成脂诱导分化阳性(图3F)。
3.1.4细胞表面标志物鉴定
免疫荧光染色结果显示,CD34、CD45呈阴性表达,同时高表达CD90、CD105,符合ISCT对于ADSCs的鉴定标准。(图4)
3.2猫ADSCs的病原体检测
为保证ADSCs移植的安全性,避免医源性感染,我们随机选择P1、P3、P5代细胞,对猫几种常见的高死亡率的病毒进行了检测,包括FPV(猫细小病毒)、FHV-1(猫疱疹病毒)、FCV(猫杯状病毒)、RV(狂犬病毒),结果细胞未携带这几种病毒。(图5)
3.3动物实验结果
3.3.1治疗期生存曲线
恢复饮食并开始治疗的短期内,FHL+PBS组陆续有动物死亡,5d内死亡率达50%;FHL+ADSCs组和FHL+MSCM组在治疗期间未出现死亡。FHL+PBS组的生存率与FHL+ADSCs组和FHL+MSCM组相比均有显著性,Logrank=0.0173(图6)。动物出现死亡的主要原因可能是恢复饮食改变了长期禁食造模时的体内稳态,引起再饲喂综合征而导致动物急性死亡,因此,生存曲线的结果可能指向ADSCs和MSCM对于机体稳态的适应性调节具有积极作用。
3.3.2病理组织学染色结果
Control组肝细胞排列整齐,肝索明显,油红O染色胞质微量着色;FHL组肝细胞内可见出现大的空泡,细胞核被挤压于一侧,呈明显的“戒指状”,肝索紊乱,油红O染色可见弥散分布大量红染的大小不等的脂滴。表明猫脂肪肝模型建立成功。
FHL+PBS组肝细胞弥散性空泡化,胞质稀薄,细胞较大;油红O染色可见大量小脂滴弥散分布;FHL+ADSCs组和FHL+MSCM组肝索明显,肝细胞未见明显异常。油红O染色FHL+ADSCs组肝细胞内脂肪含量极少;FHL+MSCM组肝细胞内可见团状脂滴,胞核明显。
根据肝脏病变程度及肝细胞内脂滴含量可得出,恢复正常饮食后,机体可以代偿修复同时逐渐恢复正常脂质代谢,FHL+PBS组肝脏病变较FHL组轻,且脂滴含量有所下降。FHL+ADSCs组和FHL+MSCM组的肝细胞基本恢复正常状态,脂滴含量明显少于FHL+PBS组,说明ADSCs及MSCM在猫脂肪肝的恢复过程中起到了积极的促进作用,且ADSCs移植的效果优于MSCM。(图7)。
Claims (2)
1.脂肪间充质干细胞在制备治疗猫脂肪肝药物中的用途。
2.如权利要求1所述的用途,其特征在于:所述药物为冻干粉。
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