间充质干细胞在儿童异基因造血干细胞移植中的应用研究

发布时间：2018-11-19 21:33

【摘要】：一、人脐带间充质干细胞培养及超微结构观察 目的：分离培养足月新生儿脐带间充质干细胞，进行超微结构观察。 方法：用胶原酶消化法分离培养人脐带间充质干细胞,进行传代培养、扩增，显微镜下观察细胞形态。培养至3代的间充质干细胞进行免疫表型测定，进行成脂细胞、成骨细胞和成软骨细胞诱导分化实验，并在透射电镜、扫描电镜下进行超微结构的全面观察。 结果：人脐带间充质干细胞外观呈梭形和多角形，并可见细胞核；高表达CD44,CD73,CDl05，不表达CD34，CD45，CD31和HLA-DR；能够进行成脂、成骨、成软骨分化；扫描电镜下可见细胞表面有短而粗的微绒毛突起，透射电镜下可见到两种不同的细胞形态。一种是处于静止期，细胞核大，圆或卵圆形，仅有一个核仁，胞质内细胞器少；另一种是处于相对活跃期，同一个细胞内可以看到1个或2个细胞核，细胞器数量丰富、结构清晰，并可见扩张的线粒体。 结论：从脐带中成功分离培养的细胞，具有间充质干细胞的生物学特性，具有两种不同状态的超微结构。 二、脐带间充质干细胞治疗儿童异基因造血干细胞移植后急性移植物抗宿主病疗效观察 目的：观察人脐带间充质干细胞(mesenchymal stem cells,MSC)治疗儿童急性移植物抗宿主病疗效和安全性。 方法：用胶原酶消化法分离培养人脐带间充质干细胞,进行传代培养、扩增，培养至第3-5代用于临床治疗；5例急性白血病患儿经化疗达完全缓解。2例行亲缘HLA3/6相合的骨髓造血干细胞移植，，1例行同胞HLA全相合骨髓与外周血造血干细胞联合移植，1例行无血缘相关HLA4/6相合双份脐血造血干细胞移植，1例行无血缘相关HLA5/6相合单份脐血造血干细胞移植。患儿发生Ⅲ-Ⅳ度急性移植物抗宿主病（acute graft-versus-host disease,aGVHD），接受二线免疫抑制治疗无效后，行人脐带MSC（0.5×106/kg受者体重）治疗。 结果：5例急性白血病患儿均获造血重建，并发生皮肤、肝脏和胃肠道Ⅲ-Ⅳ度aGVHD，经二线免疫抑制治疗无效，输注人脐带间充质干细胞治疗后，皮疹消退，肝功能恢复正常，胃肠道症状好转。患儿均未发生输注相关不良反应。目前患儿均无原发病的复发，均处于无病生存状态。 结论：异基因造血干细胞移植是治疗儿童急性白血病的有效方法。脐带间充质干细胞治疗儿童急性移植物抗宿主病是安全有效的。
[Abstract]:1. Culture and ultrastructure observation of human umbilical cord mesenchymal stem cells objective: to isolate and culture umbilical cord mesenchymal stem cells from term neonates and observe the ultrastructure of human umbilical cord mesenchymal stem cells. Methods: human umbilical cord mesenchymal stem cells were isolated and cultured by collagenase digestion. The immunophenotype of mesenchymal stem cells (MSCs) cultured to passage 3 was determined. The differentiation of adipoblasts, osteoblasts and chondroblasts was studied. The ultrastructure of mesenchymal stem cells was observed under transmission electron microscope and scanning electron microscope. Results: the appearance of human umbilical cord mesenchymal stem cells was fusiform and polygonal, and the nucleus could be seen, and the high expression of CD44,CD73,CDl05, could not express CD34,CD45,CD31 and HLA-DR; in adipogenesis, osteogenesis and cartilage differentiation. Scanning electron microscopy showed that there were short and thick microvilli protuberances on the surface of the cells, and two kinds of cell morphology were observed under transmission electron microscope. One is at rest, the nucleus is large, round or oval, there is only one nucleolus, less organelles in the cytoplasm; On the other hand, one or two nuclei can be seen in the same cell in a relatively active phase, with abundant organelle, clear structure and dilated mitochondria. Conclusion: the cells isolated and cultured from umbilical cord have the biological characteristics of mesenchymal stem cells and have two different ultrastructures. 2. Therapeutic effect of umbilical cord mesenchymal stem cells on acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in children objective: to observe (mesenchymal stem cells, of human umbilical cord mesenchymal stem cells MSC) in the treatment of acute graft-versus-host disease in children. Methods: human umbilical cord mesenchymal stem cells were isolated and cultured by collagenase digestion. Five children with acute leukemia received complete remission after chemotherapy, 2 patients received bone marrow hematopoietic stem cell transplantation (BMSCs) matched with related HLA3/6, and 1 patient underwent sibling HLA homozygous bone marrow transplantation combined with peripheral blood hematopoietic stem cell transplantation (PBSCT). One patient was treated with unrelated HLA4/6 matched with double cord blood hematopoietic stem cell transplantation, and one patient with unrelated HLA5/6 matched with single umbilical cord blood hematopoietic stem cell transplantation. Acute graft-versus-host disease (acute graft-versus-host disease,aGVHD) occurred in children with grade 鈪

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