CN110772534A - 多系分化持续应激细胞在制备抗炎药物中的应用 - Google Patents
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Abstract
本发明公开了多系分化持续应激细胞在制备抗炎药物中的应用。多系分化持续应激细胞可以从骨髓、脂肪、脐带、真皮等多种间充质组织中获得。本发明研究表明多系分化持续应激细胞能够有效治疗各种炎症,如关节炎、肌腱炎、鼻炎、肺炎、肾炎、结肠炎、妇科炎症、前列腺炎、蜂窝织炎、溃疡、荨麻疹、结核、伤寒、自身免疫性疾病(如脊柱炎、红斑狼疮、淋巴性甲状腺炎等)在内的多种疾病和症状,并且作用时间长久,具有极大的临床应用价值。
Description
技术领域
本发明属于干细胞生物学和生物医药领域,具体涉及多系分化持续应激细胞在制备抗炎药物中的应用
背景技术
炎症是指具有血管系统的活体组织对损伤因子的防御性反应,如体表的外伤感染和各器官的大部分常见病和多发病(如关节炎、肺炎、肝炎、肾炎、自身免疫性炎症等),典型表现为红、肿、热、痛,是十分常见而又重要的基本病理过程,而且随着疾病的发展炎症可导致严重的功能障碍,如关节炎引起的关节软骨破裂,类风湿性关节炎引起的关节畸形,输卵管炎引起的输卵管堵塞,慢性肾炎引起的肾功能衰竭等,给患者带来了沉重的心理和经济负担。以骨关节炎为例,据世界卫生组织调查研究显示,目前全世界骨关节炎患者已超过4亿。我国的骨关节炎患者超过人口总数的10%,多达1亿以上,膝关节炎致残率全球第二,且发病率随年龄增加而增高,防控形势严峻。然而,目前对炎症,尤其是慢性炎症的治疗仍缺乏治愈性治疗手段。现有的针对慢性炎症的治疗手段主要包括一些症状缓解药物,如非甾体类抗炎药、类固醇激素、对乙酰氨基酚等,但这些药物的疗效有限,并不能阻止疾病的进展,而且长期使用后会导致严重的副作用,因此,寻找和开发新的抗炎药物具有重要的医学意义和深远的社会意义。
发明内容
多系分化持续应激细胞是2010年在成人骨髓和真皮组织中新发现的一种成体干细胞,具备如下特性:1)特异性表达阶段特异性胚胎抗原3(stage specific embryonicantigen-3,SSEA3)和CDl05;2)有应激耐受力;3)单细胞能形成细胞球,具备很强的自我更新能力;4)能分化为内、中、外三个胚层的细胞,但在裸鼠体内不形成畸胎瘤。本发明研究发现,多系分化持续应激细胞能够有效治疗各种慢性炎症。注射多系分化持续应激细胞可以显著治疗多种慢性炎症,并且作用时间长久,对包括关节炎、肌腱炎、鼻炎、肺炎、肾炎、结肠炎、妇科炎症、前列腺炎、蜂窝织炎、溃疡、荨麻疹、结核、伤寒、自身免疫性疾病(如脊柱炎、红斑狼疮、淋巴性甲状腺炎等)等在内的多种疾病和症状都有显著地治疗效果,具有极大的临床应用价值。
本发明的目的是提供一种多系分化持续应激细胞在制备抗炎药物中的应用,为临床治疗炎症的药物提供一种新的选择。
为了解决上述技术问题,本发明具体技术方案如下:
本发明提供一种多系分化持续应激细胞在制备抗炎药物中的应用。优选的,所述抗炎药物为治疗慢性炎症的药物。
所述慢性炎症包括如下因素引起的炎症:
(1)物理因素如温度(高热、低温)、射线(放射线、紫外线)等引起的炎症;
(2)外源性化学物质如强酸、强碱等腐蚀性物质及松节油、芥子气引起的炎症,以及内源性化学毒物如坏死组织的分解产物及在某些病理条件下堆积于体内的代谢产物如尿素等引起的炎症;
(3)切割、撞击、挤压等机械性因素引起的炎症;
(4)细菌、病毒、立克次体、支原体、真菌、螺旋体和寄生虫等通过在体内繁殖,产生、释放毒素直接导致细胞和组织损伤,或通过其抗原性诱发免疫反应导致的炎症;
(5)各型变态反应均能造成组织和细胞损伤而导致炎症:I型变态反应如过敏性鼻炎、荨麻疹;II型变态反应如抗基底膜性肾小球肾炎;Ⅲ型变态反在如免疫复合物性肾小球肾炎和IV型变态反应如结核、伤寒等;此外还有某些自身免疫性疾病如淋巴性甲状腺炎、溃疡性结肠炎、脊柱炎、红斑狼疮等。
进一步,本发明所述慢性炎症选自包括关节炎、肌腱炎、鼻炎、肺炎、肾炎、结肠炎、妇科炎症、前列腺炎、蜂窝织炎、溃疡、荨麻疹、结核、伤寒、淋巴性甲状腺炎、脊柱炎、红斑狼疮中的一种或几种。
进一步,本发明所述多系分化持续应激细胞来源于骨髓、脂肪、脐带、真皮等间充质组织,可以悬浮成球生长也可以贴壁生长,表达SSEA-3,Oct3/4,Sox2,CD105,CD90,不表达CD34,CD45,CD11b。本发明一个具体的实施例使用的多系分化持续应激细胞来源于骨髓。
本发明所述多系分化持续应激细胞可采用如文献(Kuroda,et al.,Uniquemultipotent cells in adult human mesenchymalcell populations.PNAS,2010,107:8639-8643.)所述的方法制备得到。
本发明一个具体的多系分化持续应激细胞制备方法如下:
参考文献(Kuroda,et al.,PNAS,2010,107:8639-8643.)公开的方法制备得到骨髓细胞,利用多系分化持续应激细胞表达SSEA-3+/CD105+/CD45-的特性采用流式细胞分选技术对骨髓细胞进行分选,对分选出的多系分化持续应激细胞进行悬浮培养,使用a-MEM+0.9%MethoCult H4100+20%FBS培养基,培养1d后细胞开始聚集,单个细胞聚集成团形成细胞集落,之后细胞集落又分裂生长,形成干细胞球,3-5d细胞数目逐渐增多,细胞球逐渐增大,呈现大而透亮的形态。多系分化持续应激细胞传代培养后的成球时间要比第1代细胞明显缩短,从第2代开始,一般2d即可形成细胞球。采用多能干细胞标记物SSEA-3、Nanog、Oct4和Sox2对多系分化持续应激细胞做鉴定。
采用本发明所述方法制备得到的多系分化持续应激细胞传代稳定性好,研究表明传代60代之后细胞性状仍保持稳定,体现为1)仍能形成细胞球悬浮生长;2)和传代5代之内的MUSE细胞具有相同的镇痛效果;3)蛋白质谱分析显示和传代5代的MUSE细胞相比,在细胞因子、趋化因子和受体等多方面蛋白表达都没有统计差异。本发明所述的多系分化持续应激细胞稳定的传代特性,对要求提供大量、标准化的细胞产品来制备抗炎药物极其有利。
进一步,本发明所述多系分化持续应激细胞以细胞悬液的形式给药,所述细胞悬液按如下方法制备:将培养的多系分化持续应激细胞用0.25%trypsin-EDTA进行消化制成单细胞悬液,生理盐水洗涤后,再用生理盐水悬浮,即为多系分化持续应激细胞悬液。
进一步,本发明所述多系分化持续应激细胞悬液中细胞浓度为1~10×107个/ml。
进一步,本发明所述多系分化持续应激细胞悬液的药物用量是以多系分化持续应激细胞个数计数为5~10×106个/kg体重,局部注射(鞘内注射、关节腔内注射、皮下注射,肌肉注射等)1次;或5~10×107个/kg体重,静脉输注1次。
本发明所述的药物,进一步还可以含有药学上可接受的辅料。
本发明利用多种慢性炎症的小鼠模型,首次证明通过局部注射或静脉注射的方式移植多系分化持续应激细胞可以长时间的显著抑制多种慢性炎症模型小鼠的病程发展,减少炎症因子产生,抑制痛觉过敏。
附图说明
图1为人多系分化持续应激细胞(MUSE细胞)的制备和鉴定结果。(其中,图A:是收集培养到第3代的人骨髓细胞,利用多系分化持续应激细胞SSEA-3+/CD105+的特性采用流式细胞分选技术进行分选的结果。图B是分选出的多系分化持续应激细胞在悬浮培养下可由分选得到的单细胞形成细胞球。图C是将多系分化持续应激细胞的悬浮细胞球消化成单细胞后,进行贴壁培养时的细胞形态)。
图2为关节腔内注射人Muse细胞对骨关节炎模型小鼠的膝关节功能指数评分的影响。
图3为关节腔内注射人Muse细胞对骨关节炎模型小鼠的膝关节肿胀度的影响。
图4为静脉注射人Muse细胞对前列腺炎模型小鼠的前列腺炎症细胞浸润的影响。
图5为人MUSE细胞与人骨髓间充质干细胞(BMSC)分泌抗炎因子的比较。
具体实施方式
以下通过实施例说明本发明的具体步骤,但不受实施例限制。
在本发明中使用的术语,除非另有说明,一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明,应理解,这些实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。
在以下实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法。
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此。
实施例1:多系分化持续应激细胞的制备和鉴定
由专业医生在捐献者的髂前上棘后上方行骨髓穿刺术,抽取1ml骨髓。将骨髓与磷酸缓冲盐溶液按1∶1混合加入到淋巴细胞分离液上层,离心后液体分为四层,最上层约占液柱的1/2,为淡红色的血浆层;第三层约占液柱的1/4,为澄清的淋巴细胞分离液层;在两层的交界处为高约5mm的乳白色云雾状层,为单核细胞层(此层为所要提取的细胞层);最底层紧附试管壁,呈深红色,主要为密度较大的红细胞层。取第二层的细胞,使用含15%FBS的DMEM/F12完全培养基,于37℃、5%CO2。的恒温培养箱中培养24h后,可见有散在单个细胞贴壁;2-3d后贴壁细胞开始分裂增殖,由最初的圆形逐渐伸展开,细胞两极朝向不规律,形态不规则,周围有混杂细胞;4-7d后细胞呈集落样生长,以梭形居多;1w后细胞扁平,多突起,胞体互相融合。至第3代后,细胞形态基本单一,呈梭形,流式细胞仪检测约有95%的细胞为CD90+/CD45-/CD11b-。收集培养到第3代的细胞,利用Muse细胞SSEA-3+/CD105+的特性采用流式细胞分选技术进行分选,对分选出的Muse细胞进行悬浮培养,使用a-MEM+0.9%MethoCultH4100+20%FBS培养基,1d后细胞开始聚集,单个细胞聚集成团形成细胞集落,之后细胞集落又分裂生长,形成干细胞球,3-5d细胞数目逐渐增多,细胞球逐渐增大,呈现大而透亮的形态。Muse细胞传代培养后的成球时间要比第1代细胞明显缩短,从第2代开始,一般2d即可形成细胞球。采用多能干细胞标记物SSEA-3、Nanog、Oct4和Sox2对Muse细胞做鉴定。
结果见图1,其中图A是收集培养到第3代的人骨髓细胞,利用多系分化持续应激细胞SSEA-3+/CD105+的特性采用流式细胞分选技术进行分选的结果。该结果表明在培养的骨髓细胞中有大约0.89%的细胞是多系分化持续应激细胞。图B是分选出的多系分化持续应激细胞在悬浮培养下可由分选得到的单细胞形成细胞球。该结果表明在多系分化持续应激细胞可以在体外大量扩增。图C是将多系分化持续应激细胞的悬浮细胞球消化成单细胞后,进行贴壁培养时的细胞形态。该结果表明多系分化持续应激细胞也可以进行贴壁培养,且细胞形态一致。可用来做进一步的实验。
实施例2:多系分化持续应激细胞的抗炎活性检测
1)小鼠炎症模型:
①胶原诱导关节炎模型:无菌条件下,用0.05mol/L冰醋酸充分溶解牛II型胶原(CII),质量浓度为4mg/mL。置4℃冰箱过夜后,与弗氏佐剂(CFA)等体积混合,振荡乳化,取乳化液滴入水中不扩散表示乳化充分,制成CII乳剂(CII终浓度为2mg/mL)。乳化全程冰浴中操作。小鼠在手术平台上用异氟烷气体持续麻醉,剃毛消毒后,在小鼠尾根部、背部两点皮内注射CII乳剂,总量100μL。2周后同法100μL加强注射。
②非细菌性前列腺炎模型:将0.1g的角叉菜胶溶入10mL生理盐水中,混匀配制成1%的角叉菜胶生理盐水溶液。小鼠在手术平台上用异氟烷气体持续麻醉,剃毛消毒后,在下腹正中剪开2cm左右的切口,逐层打开腹壁,找到膀胱,用器械轻轻挑起膀胱,在其后方找到前列腺,用无菌微量调节注射器将50μL的1%无菌角叉菜胶溶液分别注射到小鼠前列腺左右腹叶上;对照组每只注入50μL灭菌的生理盐水溶液。将前列腺放回体内,缝合肌肉、皮肤,消毒伤口,将大鼠回笼中饲养。在伤口处涂抹适量抗生素以防止伤口感染化脓,并每天观察小鼠活动状态、进食量。
2)小鼠行为检测:所有行为学实验采取“双盲”的实验设计方案。
①关节炎指数(AI)评分:采用AI评分法进行小鼠关节足肿胀评价。评分标准:0分,无关节肿胀;1分,小趾关节稍红肿;2分,趾关节和足趾肿胀;3分,踝关节以下的足爪肿胀,严重红肿;4分,包括踝关节在内全部足爪严重红肿,关节活动障碍。四肢的病变程度累积积分为AI,每只小鼠的最高分为16分。若评分为6-8分则表示为严重关节炎。
②足肿胀度测试利用肢体肿胀测量仪,精确测量关节炎小鼠后足肿胀情况。
结果如图2,3所示。图2为关节腔内注射人Muse细胞对骨关节炎模型小鼠的膝关节功能指数评分的影响。在关节炎模型造模2周后移植MUSE细胞,结果表明模型组在单次关节腔内注射人Muse细胞后可以显著降低小鼠的关节炎指数。图3为关节腔内注射人Muse细胞对骨关节炎模型小鼠的膝关节肿胀度的影响。在关节炎模型造模2周后移植MUSE细胞,结果表明模型组在单次关节腔内注射人Muse细胞后可以显著降低小鼠的后肢关节肿胀度。图2和3的结果提示多系分化持续应激细胞对关节炎模型小鼠的炎症水肿有较好的治疗效果。
3)组织病理学检查
小鼠用4%甲醛溶液全身灌注后取前列腺组织。放置进装有4%的多聚甲醛中,4℃条件固定过夜。然后将前列腺组织放入由0.1M PB所配制而成的10%、20%和30%的蔗糖溶液里进行脱水后进行冰冻切片,苏木精-伊红染色,光学显微镜下观察。
结果如图4所示,与注射生理盐水的模型组(A)相比,静脉注射人Muse细胞的模型组(B)中炎症细胞在前列腺组织中的浸润显著降低,提示移植Muse细胞可以抑制炎症病程的发展。
4)ELISA法检测
利用ELISA检测试剂盒,以人BMSC细胞作为对照,考察培养的人MUSE细胞抗炎因子TGF-β和IL-10的合成和分泌情况。
结果如图5A和5B所示,与人BMSC细胞相比,人Muse细胞在细胞内的TGF-β含量较少,有统计学差异(A);但两种细胞在培养上清中的TGF-β含量无统计学差异(B),说明人MUSE细胞和人BMSC细胞在抗炎因子TGF-β分泌的能力上无显著差别。图5C和5D显示,与人BMSC细胞相比,人Muse细胞在细胞内(C)和培养上清(D)中的IL-10的含量都显著增高且有统计学差异,说明MUSE细胞在抗炎因子IL-10的分泌上明显强于人BMSC细胞。这一结果提示与人BMSC细胞相比,人Muse细胞具备更强的抗炎活性。
综上所述,关节腔内注射或静脉注射的方式移植Muse细胞对关节炎和前列腺炎症模型小鼠都具有很好的治疗效果。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (9)
1.多系分化持续应激细胞在制备抗炎药物中的应用。
2.如权利要求1所述的应用,其特征在于所述抗炎药物为治疗慢性炎症的药物。
3.如权利要求2所述的应用,其特征在于所述慢性炎症包括如下因素引起的炎症:
(1)物理因素引起的炎症;
(2)外源性化学物质以及内源性化学毒物引起的炎症;
(3)机械性因素引起的炎症;
(4)由细菌、病毒、立克次体、支原体、真菌、螺旋体和寄生虫通过繁殖,产生毒素直接导致细胞和组织损伤,或通过其抗原性诱发免疫反应导致的炎症;
(5)变态反应或自身免疫性疾病引起的炎症。
4.如权利要求3所述的应用,其特征在于所述慢性炎症选自关节炎、肌腱炎、鼻炎、肺炎、肾炎、结肠炎、妇科炎症、前列腺炎、蜂窝织炎、溃疡、荨麻疹、结核、伤寒、淋巴性甲状腺炎、脊柱炎、红斑狼疮中的一种或几种。
5.如权利要求1所述的应用,其特征在于所述多系分化持续应激细胞来源于骨髓、脂肪、脐带、真皮等多种间充质组织。
6.一种抗炎药物,其特征在于包含多系分化持续应激细胞的细胞悬液。
7.如权利要求4所述的药物,其特征在于所述多系分化持续应激细胞的细胞浓度为1~10×107个/ml。
8.如权利要求4所述的药物,其特征在于还含有药学上可接受的辅料。
9.如权利要求4所述的药物,其特征在于所述药物通过皮下注射,肌肉注射,腹腔注射,关节腔内注射,静脉注射,鞘内注射,直肠给药,阴道给药,鼻腔给药,透皮给药,结膜下给药方式给药。
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