CN115518077A - 一种治疗炎症性肠病的细胞药物 - Google Patents
一种治疗炎症性肠病的细胞药物 Download PDFInfo
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Abstract
本发明涉及一种治疗炎症性肠病的细胞药物,通过用琥珀酸盐处理经炎症因子刺激后的间充质干细胞得到。本发明通过使用琥珀酸盐体外预处理间充质干细胞,提升MSCs胞内HIF‑1α的蛋白水平,增强细胞的糖酵解能力,提高免疫抑制因子的表达,发挥干细胞免疫调控功能,从而实现低剂量MSCs治疗炎症性肠病的目的。
Description
技术领域
本发明涉及生物制药技术领域,尤其涉及一种治疗炎症性肠病的细胞药物。
背景技术
炎性肠病又称炎症性肠病(IBD),为累及回肠、直肠、结肠的一种特发性肠道炎症性疾病。临床表现为腹泻、腹痛、粘液血便,甚至出现各种全身并发症如视物模糊、关节疼痛、皮疹等。炎症性肠病包括溃疡性结肠炎(UC)和克罗恩病(CD)。溃疡性结肠炎是结肠黏膜层和黏膜下层连续性炎症,疾病通常先累及直肠,而后逐渐向全结肠蔓延。克罗恩病可累及全消化道,为非连续性全层炎症,最常累及部位为末端回肠、结肠和肛周。
炎症性肠病的病因和发病机制尚未完全明确,目前已知肠道黏膜免疫系统异常激活所导致的高炎症反应在IBD发病中起重要作用,可能是由于多因素相互作用所致,主要包括环境、遗传、感染和免疫因素。目前的治疗选择包括抗炎药(如美沙拉嗪)、皮质类固醇、免疫调节剂(硫嘌呤、甲氨蝶呤、环孢素等)和生物制剂(抗肿瘤坏死因子-α)。但是以上治疗方法常伴有异常的毒副反应,且不能实现对于疾病进展的长期遏制。
间充质干细胞(mesenchymal stem cells,MSCs)是广泛存在于机体大部分组织中的多能干细胞,与个体生长发育、组织稳态维持和器官损伤修复密切相关。MSCs容易从成人组织中分离得到,包括脂肪组织和骨髓等,并且由于它们已被证明具有免疫规避性,现已成为包括IBD在内的许多疾病的临床可行疗法。MSCs上高表达的趋化因子受体诱导细胞向炎症部位迁移,随后通过分泌抗炎介质进行免疫调节来抑制炎症。但在临床试验中发现,使用间充质干细胞治疗炎症性肠病时,如果注射的细胞数量过少,就无法达成炎症性肠病治疗的预期目标,而随着注入干细胞数量的增多,血栓风险大幅度增加,此外细胞毒性也成倍增强。
发明内容
为解决上述技术问题,本发明提供了一种治疗炎症性肠病的细胞药物,通过使用琥珀酸盐体外预处理间充质干细胞,提升MSCs胞内HIF-1α的蛋白水平,增强细胞的糖酵解能力,提高免疫抑制因子的表达,发挥干细胞免疫调控功能,从而实现低剂量MSCs治疗炎症性肠病的目的。
本发明的第一个目的是提供一种治疗炎症性肠病的细胞药物,所述细胞药物通过用琥珀酸盐处理经炎症因子刺激后的间充质干细胞得到。
进一步地,所述炎症因子包括IFN-γ(干扰素-γ)和TNF-α(肿瘤坏死因子-α)。
进一步地,所述间充质干细胞来源于骨髓、脂肪或脐带。
所述的间充质干细胞是一种多能干细胞,它具有干细胞的所有共性,即自我更新和多向分化能力。骨髓间充质干细胞(BMSCs)是一类起源于中胚层的成体干细胞,也具有自我更新及多向分化潜能,可分化为多种间质组织,如骨骼、软骨、脂肪、骨髓造血组织等。为排除伦理问题,本发明使用的是经伦理审查批准的人骨髓间充质干细胞,包括商业化的人骨髓间充质干细胞(如Prochymal、Temcell HS Inj、Neuronata-R、Stemirac、Stempeucel和Cellgram-AMI等)。
特别指出,本发明所使用的人多能干细胞均不能够发育成完整个体,且都是已通过伦理审查而建立的多能干细胞。
进一步地,所述细胞药物由以下步骤制备得到:将间充质干细胞在增殖培养基中进行传代培养,选取20代以内的间充质干细胞,加入炎症因子和琥珀酸盐进行孵育处理,得到所述细胞药物。
进一步地,所述增殖培养基为含有胎牛血清和bFGF的DMEM低糖培养基。
进一步地,在增殖培养基中,胎牛血清的质量百分比为5-15%,bFGF的浓度为5-15ng/mL。
进一步地,所述炎症因子在培养基中的浓度为10-100ng/mL。
进一步地,所述琥珀酸盐在培养基中的浓度为10mM-50mM。
进一步地,所述炎症性肠病为溃疡性结肠炎和克罗恩病。
进一步地,所述细胞药物的剂型为注射剂。
进一步地,通过静脉注射进行给药。
进一步地,所述细胞药物的给药剂量为1-2×105个间充质干细胞/kg。当然,本领域技术人员可以知晓,可以根据需要提高给药剂量。
进一步地,上述药物通过提升HIF-1α(缺氧诱导因子-1α)蛋白水平,提高免疫抑制因子的表达,发挥干细胞免疫调控功能。
进一步地,免疫抑制因子包括HO1、COX2和iNOS。
借由上述方案,本发明至少具有以下优点:
本发明通过使用琥珀酸盐体外预处理间充质干细胞,相比于同等数量未经预处理的间充质干细胞可表现出更高效的治疗效应,从而为炎症性肠病的细胞治疗提供了一种新颖的增益策略。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚地理解,下面根据本发明的具体实施例并结合附图,对本发明做进一步详细的说明。
图1为MSCs经IFN-γ和TNF-α刺激联合琥珀酸盐处理24h后胞内HIF-1α蛋白、糖酵解基因和免疫抑制因子的表达情况;
图2为MSCs经琥珀酸盐预处理24h后增强对结肠炎治疗效果的表征。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
本发明涉及的具体实验操作如下:
1、MSCs分离提取与体外扩增
1.1间充质干细胞的分离提取
(1)C57BL/6J品系小鼠经水合氯醛麻醉后,颈椎脱位处死小鼠,将其全身浸泡于75%酒精约10min,而后去除股骨和胫骨的全部肌肉组织。
(2)眼科剪减去股骨和胫骨的两端股垢后,使用含无菌PBS的注射器反复冲洗骨内骨髓组织,待骨色由红色变为白色后,将收集的全部骨髓细胞经70μm细胞筛网过滤后,400×g离心5min。
(3)离心完成后,使用MSCs增殖培养基(DMEM低糖培养基+10%胎牛血清+10ng/mLbFGF)进行重悬,而后将细胞种植于多个培养皿中进行培养。其中,DMEM低糖培养基指培养基中葡萄糖含量≤1100mg/L。
(4)根据不同类型细胞差异化的胰酶消化时间,预分离出MSCs群体。在胰蛋白酶加入至细胞后不久,吸取出快速脱落的细胞,进而离心富集。400×g离心5min。
(5)依据上一步方法培养细胞一段时间内所进行多次的胰酶消化和离心富集,可大体去除混杂其中的免疫细胞。
(6)通过极限稀释法,将预提纯的细胞群体种植于96孔板中,确保每孔只存有一个细胞。培养数天,观察并筛选出能够长出细胞克隆的板孔,此孔中的细胞即为MSCs。
1.2间充质干细胞的扩增培养
(1)使用胰蛋白酶对孔板中的MSCs克隆进行消化传代,使用间充质干细胞增殖培养基进行离心后的细胞重悬,而后将细胞种植于培养瓶中进行扩增培养。
(2)每两天更换一次新鲜培养基直至细胞融合度达到80~90%。
(3)使用胰蛋白酶对间充质干细胞进行传代,而后将子代细胞种植于新的培养瓶中继续培养扩增。其中,只选取20代之内的MSCs细胞群体用于本实验。
2、结肠炎动物模型建立
2.1实验动物
实验所用的8~10周龄且体重约为25g雄性C57BL/6J品系小鼠均购自于北京维通利华实验动物技术有限公司,并严格遵循SPF级屏障系统标准进行饲养。本发明中所涉及的动物实验操作均经过苏州大学动物实验伦理委员会批准。
2.2结肠炎模型建立
实验开始当天,不同的C57BL/6J品系小鼠被随机分组并分别进行正常水饮食和含4%葡聚糖硫酸钠(DSS,分子量:36,000-50,000)水饮食,每两天更换小鼠饮水,共计喂养7天。根据不同的实验目的给予疾病小鼠不同的治疗处理方式,每天记录结肠炎小鼠的体重,并同时观察各小鼠便血情况、粪便形态以及小鼠活动状态等。
2.3结肠炎模型治疗
在小鼠结肠炎模型诱导第2天,给予小鼠尾静脉注射琥珀酸盐预处理的1×105个MSCs进行疾病的治疗,模型对照小鼠给予尾静脉PBS注射。
2.4结肠炎疾病活动指数
A.体重丢失(0~4分)
0分:未发生体重丢失;
1分:丢失初始体重的10%以下;
2分:丢失初始体重的10%~15%;
3分:丢失初始体重的15%~20%;
4分:丢失初始体重的20%以上。
B.腹泻程度(0~2分)
0分:没有腹泻;
1分:轻度腹泻;
2分:中度至重度腹泻。
C.直肠出血程度(0~2分)
0分:无出血;
1分:轻度出血;
2分:中度至重度出血。
D.机体活动(0~2分)
0分:正常
1分:轻度抑郁;
2分:中度至重度抑郁。
2.5结肠炎组织学评分
A.肠壁增厚程度(0~3分)
0分:无增厚;
1分:粘膜增厚;
2分:粘膜与粘膜下层增厚;
3分:穿透肠壁。
B.隐窝损伤程度(0~3分)
0分:无损伤;
1分:杯状细胞丢失;
2分:只有表面上皮细胞完整;
3分:整个隐窝和上皮细胞丢失。
C.炎症细胞浸润情况(0~2分)
0分:无炎症细胞浸润;
1分:轻度至中度浸润;
2分:重度浸润。
3、RNA抽提与基因表达检测
3.1细胞总RNA抽提
(1)PBS洗涤孔板中的细胞2遍后,加入l mL预冷的Trizol试剂,反复吹打至细胞完全裂解。而后将液体转移至1.5mL EP管中。
(2)按照1:5的氯仿:Trizol比例加入200μL氯仿,涡旋器上充分混匀后,室温静置5min。4℃,12,000×g离心15min。
(3)离心完毕后,液体混合液出现分层。小心吸出最上层液体至新的1.5mL EP管,加入1mL异丙醇,涡旋器上充分混匀后,室温静置10min。4℃,12,000×g离心10min,弃上清。
(4)此时可见管底出现白色沉淀,加入1mL经DEPC水配制的75%乙醇溶液,上下颠倒混匀,以使沉淀脱落漂浮于乙醇溶液中。4℃,12,000×g离心5min。重复洗涤离心一次。
(5)弃上清,吸水纸吸取回流液体,而后放置于通风橱中,室温干燥至无液体残留于EP管内。
(6)加入20μL DEPC水至RNA完全溶解,使用分光光度计检测所得RNA的浓度和纯度。
3.2总RNA反转录成cDNA
吸取1ng总RNA,使用PrimeScriptTMRT Master Mix试剂盒将总mRNA反转录成cDNA。
具体反应体系如下:
反应条件:37℃,15min;85℃,5s;4℃保持。
3.3实时荧光定量PCR
以cDNA为模板,按照SYBRTMSelect Master Mix说明书要求进行实时荧光定量PCR反应。以β-actin为内参,检测各基因在细胞内的相对表达水平。
具体反应体系如下:
反应条件:
表1特异性实时荧光定量PCR引物序列表
4、流式细胞术
(1)使用胰蛋白酶将不同处理得到的MSCs从6孔板中消化转移至96孔圆底板中。
(2)离心弃上清后,每孔加入200μL固定/破膜溶液重悬细胞。室温避光孵育60min。
(3)600×g离心5min,弃上清。每孔加入200μL破膜洗涤液。
(4)600×g离心5min,弃上清。每孔加入100μL含HIF-1α的抗体稀释液(内含2%正常大鼠血清)。室温避光孵育30min。
(5)每孔加入100μL破膜洗涤液。600×g离心5min,弃上清。
(7)每孔加入100μL破膜洗涤液。600×g离心5min,弃上清。使用PBS重悬细胞后,使用流式细胞术检测各样本中的细胞在APC通道内的平均荧光强度(MFI)。
5、病理组织学
5.1结肠炎小鼠肠组织样本制备
实验处理结束后,收集各组结肠炎小鼠大肠组织并量取长度。剖开肠腔,去除其内的粪便和黏液。PBS涮洗两遍后,浸泡于4%多聚甲醛溶液固定48小时,而后进行相应的病理学技术处理与检测。
5.2肠组织石蜡切片制备
将经过4%多聚甲醛溶液固定后的肠组织进行脱水、透明、浸蜡和包埋等流程处理后,制作成组织蜡块,为下步H&E染色做好准备。
表2石蜡包埋组织制作流程表
5.3H&E染色
(1)二甲苯脱蜡10min,重复3次。
(2)无水乙醇4min。
(3)95%乙醇1min。
(4)85%乙醇1min。
(5)75%乙醇1min。
(6)自来水冲洗1min。
(7)苏木素染色5min。
(8)自来水冲洗复蓝。
(9)伊红染色2min。
(10)75%乙醇脱水10s。
(11)85%乙醇脱水10s。
(12)95%乙醇脱水1min。
(13)无水乙醇脱水4min。
(14)二甲苯透明3min。
(15)中心树胶封固。
6、酶联免疫吸附测定法
(1)麻醉实验小鼠,摘取一侧眼球后,收集小鼠全血。待血液完全凝结,2400r.p.m.离心30min后,小心吸取上层血清,注意避免吸到红色血细胞。
(2)将50μL血清样本和50μL试剂盒中样品分析缓冲液依次加入样品孔中混匀,用封板膜(透明)封住反应孔,而后将ELISA板放置于水平摇床上混匀,室温孵育120min。其中将只加有TMB溶液和终止液的实验孔设定为空白孔。
(3)孵育结束后,甩出ELISA板中的液体,使用洗涤液洗板5次,且最后一次置于厚吸水纸上拍干。
(4)加入生物素化抗体100μL/孔(空白孔除外),用封板膜(透明)封住反应孔,而后将ELISA板放置于水平摇床上混匀,室温孵育60min。
(5)孵育结束后,甩出ELISA板中的液体,使用洗涤液洗板5次,且最后一次置于厚吸水纸上拍干。
(6)加入辣根过氧化物酶标记的Streptavidin 100μL/孔(空白孔除外)。用封板膜(白色)封住反应孔,而后将ELISA板放置于水平摇床上混匀,室温避光孵育20min。
(7)孵育结束后,甩出ELISA板中的液体,使用洗涤液洗板5次,且最后一次置于厚吸水纸上拍干。
(8)加入显色剂TMB溶液100μL/孔(包括空白孔),用封板膜(白色)封住反应孔,而后将ELISA板放置于水平摇床上混匀,室温避光孵育20min。
(9)加入终止液50μL/孔(包括空白孔),混匀后立即使用酶标仪测量A450处的样品吸光度值。
7、统计学分析
使用Graphpad Prism 8软件进行数据结果处理和作图。所有数据均表示为均数±标准差。对于多组比较使用one-way ANOVA检验来检测多组数据间的统计学差异。其中P小于0.05表示为具有统计学差异。
实施例1琥珀酸盐通过编程HIF-1α介导的糖酵解程序调控MSCs的免疫抑制功能
图1中,(A-C)为检测MSCs经IFN-γ(10ng/mL)和TNF-α(10ng/mL)刺激联合琥珀酸盐(20mM)处理24h后胞内HIF-1α蛋白、糖酵解基因和免疫抑制因子的表达水平。从结果得知,使用炎症因子组合IFN-γ和TNF-α作为赋能MSCs免疫调控功能的手段并通过流式细胞术检测胞内HIF-1α水平得知炎症因子激活会导致MSCs胞内HIF-1α水平大幅升高,由此诱导下游糖酵解基因的表达,促使免疫抑制因子HO1、COX2和iNOS的表达上调。而使用琥珀酸盐可进一步提升炎症因子诱导的HIF-1α蛋白表达水平,致使下游糖酵解基因表达进一步上调,最终导致免疫抑制因子HO1、COX2和iNOS表达进一步上调。
实施例2琥珀酸盐增强MSCs的疾病治疗效益
为研究琥珀酸盐是否可以增强MSCs的疾病治疗效应,本发明在DSS诱导的结肠炎模型第2天将不同预处理的MSCs通过尾静脉输注至小鼠体内,其中只接受PBS处理的小鼠作为疾病治疗的同型对照。每日监测各组小鼠体重变化、腹泻情况、直肠出血情况以及机体活动状态,具体实验流程如图2A所示。(B)为每日记录小鼠体重,绘制疾病期各组小鼠的体重变化曲线。(C)为根据结肠炎小鼠体重丢失情况、腹泻程度、直肠出血程度以及机体活动状况综合评算出机体的疾病活动指数。(D)为安乐死小鼠后解剖分离出各组小鼠的结肠组织并比较得出的肠长度。(E)为根据肠壁增厚情况、隐窝损伤情况和炎症细胞浸润情况综合评算出各组结肠组织的组织学评分。比例尺:250μm。(F)为使用酶联免疫吸附测定法分析各结肠炎小鼠血清IL-6水平。
从结果得知,单次静脉输注1×105个MSCs(使用DMSO预处理)未能缓解结肠炎所引发的体重减轻、腹泻和便血症状,也未能提高小鼠的活跃程度、降低疾病活动指数和改善结肠缩短情况(图2B-D)。在输注DMSO预处理24h的MSCs的结肠炎小鼠中,其肠壁增厚情况、隐窝损伤情况和炎症细胞浸润也都未得到改善,结肠炎小鼠的组织学评分未出现下降(图2E)。另外,指示结肠炎是否发生恶性转归的重要血清标志物IL-6也未曾下降(图2F)。然而,输注同等数量琥珀酸盐预处理24h的MSCs却可以显著缓解结肠炎小鼠以上的疾病指征。综合以上动物实验结果,表明了使用琥珀酸盐预处理MSCs可以提升干细胞的疾病治疗效能,从而实现低剂量MSCs治疗炎症性肠病的目的。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种治疗炎症性肠病的细胞药物,其特征在于:所述细胞药物通过用琥珀酸盐处理经炎症因子刺激后的间充质干细胞得到。
2.根据权利要求1所述的细胞药物,其特征在于:所述炎症因子包括IFN-γ和TNF-α。
3.根据权利要求1所述的细胞药物,其特征在于:所述间充质干细胞来源于骨髓、脂肪或脐带。
4.根据权利要求1所述的细胞药物,其特征在于,所述细胞药物由以下步骤制备得到:将间充质干细胞在增殖培养基中进行传代培养,选取20代以内的间充质干细胞,加入炎症因子和琥珀酸盐进行孵育,得到所述细胞药物。
5.根据权利要求4所述的细胞药物,其特征在于:所述增殖培养基为含有胎牛血清和bFGF的DMEM低糖培养基。
6.根据权利要求4所述的细胞药物,其特征在于:所述炎症因子的浓度为10-100ng/mL。
7.根据权利要求4所述的细胞药物,其特征在于:所述琥珀酸盐的浓度为10mM-50mM。
8.根据权利要求1所述的细胞药物,其特征在于:所述炎症性肠病为溃疡性结肠炎和克罗恩病。
9.根据权利要求1所述的细胞药物,其特征在于:所述细胞药物的剂型为注射剂。
10.根据权利要求1所述的细胞药物,其特征在于:所述细胞药物的给药剂量为1-2×105个间充质干细胞/kg。
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