CN101269088A - 用于预防与治疗慢性移植物抗宿主病的干细胞制剂 - Google Patents
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Abstract
本发明属于干细胞与免疫学领域,涉及用于预防与治疗慢性移植物抗宿主病的干细胞制剂。本发明所述的用于预防与治疗慢性移植物抗宿主病的干细胞制剂,为间质干细胞与造血干细胞的联合移植的干细胞制剂;所述的干细胞制剂经静脉输入人体受体,间质干细胞的输入剂量根据受体而调整,造血干细胞的输入剂量同常规骨髓移植方案。将本发明所述的干细胞制剂应用于骨髓移植后的病人,可预防骨髓移植(BMT)后慢性移植物抗宿主病(cGVHD)的出现,不仅明显降低GVHD的发生率,而且一旦出现GVHD时可明显降低其严重程度,从而为综合解决造血干细胞移植后的问题提供了新的途径。
Description
技术领域
本发明属于干细胞与免疫学领域,涉及一种新的干细胞制剂,尤其是涉及用于预防与治疗慢性移植物抗宿主病的干细胞制剂。
背景技术
骨髓移植是目前治疗许多恶性血液疾病和自身免疫性疾病的其中一种有效的方法,由于异基因骨髓移植日益普遍,使异基因骨髓移植后移植物抗宿主病(graft versus host disease,GVHD)的发生率和致死率不断升高,GVHD的发生主要是由于供、受者之间除主要组织相容性抗原一致外,还存在免疫遗传学的差异,供者淋巴细胞(主要是T淋巴细胞)对受者产生的免疫攻击反应所造成的,严重影响了患者的生活质量,并成为导致移植后非复发相关死亡的主要原因。为了降低GVHD,通常采用去除T细胞的造血细胞移植,但不利于免疫重建,更容易发生移植后感染。由于上述问题相互交织,至今也难于找到各方面均可兼顾的综合处理方案,因此严重影响了同种造血干细胞移植的治疗效果。
慢性移植物抗宿主病(cGVHD)是异基因造血干细胞移植(allo-HSCT)晚期最常见的并发症之一和主要死亡原因,约30~50%存活半年以上受者可出现cGVHD,相关死亡率高(约50%)。近年来研究认为,cGVHD是与异体免疫反应和自身免疫反应现象相关的重度免疫性疾病。经典的免疫抑制剂、单抗、反应停等疗法未有令人满意的疗效,且相关毒副作用大。因此,寻找一种安全、有效的疗法成为研究焦点之一。
骨髓间质干细胞(mesenehymal stem cells,MSCs)是骨髓中具有强大的体外增殖和分化能力的干细胞。近年更发现MSCs具有独特的免疫学特征。Klyushnenkova等发现人MSCs在体外同样可抑制同种异体混合淋巴细胞反应。而Bartholomew等率先报道了灵长类动物的MSCs可抑制同种异体混合淋巴细胞反应,体内注射供者来源的体外扩增MSCs可延长同种异体移植皮肤的寿命,显示MSCs具有独特的免疫学特性。
发明内容
本发明的目的在于:根据骨髓间质干细胞(MSCs)的免疫学特性与慢性移植物抗宿主病(cGVHD)发生机制,设计并提供一种用于预防与治疗慢性移植物抗宿主病的干细胞制剂,将该干细胞制剂应用于骨髓移植后的病人,可避免或者降低慢性移植物抗宿主病(cGVHD)的发生率,并且明显降低cGVHD严重程度,从而为综合解决造血干细胞移植后的问题提供了新的解决方案。
本发明所述的一种用于预防与治疗慢性移植物抗宿主病的干细胞制剂,为间质干细胞与造血干细胞的联合移植的干细胞制剂;
所述的间质干细胞的制备方法是:取同种异体健康骨髓,收获单个核细胞进行培养,借助间质干细胞的贴壁特性,反复换液、弃悬浮细胞,逐步获得纯的骨髓间质干细胞;通过形态学观察、流式细胞术分析、向成骨、成脂、神经样细胞分化的多向分化潜能鉴定,证实分离到的细胞为间质干细胞,并不断传代扩增,移植前细胞活度检测>95%;所述的造血干细胞的准备同常规骨髓移植方案;所述的干细胞制剂经静脉输入人体受体,间质干细胞的输入剂量为0.5-1.5×106细胞/Kg体重,造血干细胞的输入剂量同常规骨髓移植方案。
发明人是在参考造血干细胞移植剂量(CD34+细胞>3×106/kg体重,单个核细胞>3×108/kg体重)的基础上,确定上述间质干细胞的输入剂量,经实验证明,输入上述剂量的间质干细胞后,受体无不良反应,而且疗效确切。
应用本发明所述的干细胞制剂,即间质干细胞与造血干细胞的联合移植的干细胞制剂,可预防骨髓移植(BMT)后慢性移植物抗宿主病(cGVHD)的出现,不仅明显降低GVHD的发生率,而且一旦出现GVHD时可明显降低其严重程度,去除病人痛苦,减少免疫抑制药物使用,减少合并感染,减轻患者医疗费用。
具体实施方式
(一)骨髓间质干细胞(MSCs)供体的来源
一般选取20岁或20岁以下的健康移植供体或患者亲属,也可用胎儿骨髓源性MSCs。采集骨髓前供体需作必要体检及检测HIV、肝炎病毒及CMV等。
(二)骨髓间质干细胞(MSCs)的培养扩增
供体局部麻醉后,从髂前或髂后采集骨髓20-50ml,在GMP标准的细胞培养室处理,分离、培养MSCs,经3周左右传3-5代获得相应MSCs细胞量,约2-10×106/kg,并将部分标本送质控及病原学检测合格后应用于临床。
MSCs的具体分离培养方法:取同种异体健康骨髓,收获单个核细胞进行培养,借助MSCs的贴壁特性,反复换液、弃悬浮细胞,逐步获得纯的骨髓间质干细胞。通过形态学观察、流式细胞术分析、向成骨、成脂、神经样细胞分化的多向分化潜能鉴定,证实分离到的细胞为间质干细胞,并不断传代扩增,通常经3周左右传3-5代获得相应MSCs细胞量。
(三)造血干细胞的准备
造血干细胞的准备同常规骨髓移植方案。
(四)本发明所述的干细胞制剂的临床研究
对于移植后的病人(受体),在常规骨髓移植的基础上加入MSCs,同时经静脉输入受体。移植前处理造血干细胞的准备同常规骨髓移植方案;MSCs细胞活度检测>95%。将MSCs悬浮于生理盐水30ml,与造血干细胞同时经静脉快速(约20分钟)输注入病人体内。输注完毕,观察30分钟,如无不良反应,可回家。尽可能输注2-3次,间隔2-8周。
追踪观察:病人输注后1-2个月可起效,每4-8周随诊一次。
病人在输注前后定期作必要的血液生化、免疫学及组织病理检查,作为疗效观察指标。
治疗反应包括:(1)临床表现;(2)实验室检查;(3)其他指标。
(五)临床研究结果
1、临床病例提供:南方医科大学南方医院血液科、广东省人民医院血液科
2、临床病例观察:
应用本发明所述的干细胞制剂,即间质干细胞与造血干细胞的联合移植的干细胞制剂,对移植后的病人(受体)进行输注处理后,大多数病例不会出现慢性移植物抗宿主病(cGVHD),延缓了GVHD的出现,少数病例仍出现cGVHD。以下为发明人所观察的六例病人的情况:
上表中:“+”表示有症状,“-”表示无症状。
效果评价:
根据上述临床病例的观察,应用本发明所述的干细胞制剂,即间质干细胞与造血干细胞的联合移植的干细胞制剂,可预防骨髓移植(BMT)后慢性移植物抗宿主病(cGVHD)的出现,不仅明显降低GVHD的发生率,而且一旦出现GVHD时可明显降低其严重程度,去除病人痛苦,减少免疫抑制药物使用,减少合并感染,减轻患者医疗费用。
Claims (1)
1. 一种用于预防与治疗慢性移植物抗宿主病的干细胞制剂,其特征在于:为间质干细胞与造血干细胞的联合移植的干细胞制剂;
所述的间质干细胞的制备方法是:取同种异体健康骨髓,收获单个核细胞进行培养,借助间质干细胞的贴壁特性,反复换液、弃悬浮细胞,逐步获得纯的骨髓间质干细胞;通过形态学观察、流式细胞术分析、向成骨、成脂、神经样细胞分化的多向分化潜能鉴定,证实分离到的细胞为间质干细胞,并不断传代扩增,移植前细胞活度检测>95%;
所述的造血干细胞的准备同常规骨髓移植方案;
所述的干细胞制剂经静脉输入人体受体,间质干细胞的输入剂量为0.5~1.5×106细胞/Kg体重,造血干细胞的输入剂量同常规骨髓移植方案。
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