【摘要】：一、人臍帶間充質(zhì)干細(xì)胞培養(yǎng)及超微結(jié)構(gòu)觀察 目的：分離培養(yǎng)足月新生兒臍帶間充質(zhì)干細(xì)胞，進行超微結(jié)構(gòu)觀察。 方法：用膠原酶消化法分離培養(yǎng)人臍帶間充質(zhì)干細(xì)胞,進行傳代培養(yǎng)、擴增，顯微鏡下觀察細(xì)胞形態(tài)。培養(yǎng)至3代的間充質(zhì)干細(xì)胞進行免疫表型測定，進行成脂細(xì)胞、成骨細(xì)胞和成軟骨細(xì)胞誘導(dǎo)分化實驗，并在透射電鏡、掃描電鏡下進行超微結(jié)構(gòu)的全面觀察。 結(jié)果：人臍帶間充質(zhì)干細(xì)胞外觀呈梭形和多角形，并可見細(xì)胞核；高表達CD44,CD73,CDl05，不表達CD34，CD45，CD31和HLA-DR；能夠進行成脂、成骨、成軟骨分化；掃描電鏡下可見細(xì)胞表面有短而粗的微絨毛突起，透射電鏡下可見到兩種不同的細(xì)胞形態(tài)。一種是處于靜止期，細(xì)胞核大，圓或卵圓形，僅有一個核仁，胞質(zhì)內(nèi)細(xì)胞器少；另一種是處于相對活躍期，同一個細(xì)胞內(nèi)可以看到1個或2個細(xì)胞核，細(xì)胞器數(shù)量豐富、結(jié)構(gòu)清晰，并可見擴張的線粒體。 結(jié)論：從臍帶中成功分離培養(yǎng)的細(xì)胞，具有間充質(zhì)干細(xì)胞的生物學(xué)特性，具有兩種不同狀態(tài)的超微結(jié)構(gòu)。 二、臍帶間充質(zhì)干細(xì)胞治療兒童異基因造血干細(xì)胞移植后急性移植物抗宿主病療效觀察 目的：觀察人臍帶間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSC)治療兒童急性移植物抗宿主病療效和安全性。 方法：用膠原酶消化法分離培養(yǎng)人臍帶間充質(zhì)干細(xì)胞,進行傳代培養(yǎng)、擴增，培養(yǎng)至第3-5代用于臨床治療；5例急性白血病患兒經(jīng)化療達完全緩解。2例行親緣HLA3/6相合的骨髓造血干細(xì)胞移植，，1例行同胞HLA全相合骨髓與外周血造血干細(xì)胞聯(lián)合移植，1例行無血緣相關(guān)HLA4/6相合雙份臍血造血干細(xì)胞移植，1例行無血緣相關(guān)HLA5/6相合單份臍血造血干細(xì)胞移植�；純喊l(fā)生Ⅲ-Ⅳ度急性移植物抗宿主�。╝cute graft-versus-host disease,aGVHD），接受二線免疫抑制治療無效后，行人臍帶MSC（0.5×106/kg受者體重）治療。 結(jié)果：5例急性白血病患兒均獲造血重建，并發(fā)生皮膚、肝臟和胃腸道Ⅲ-Ⅳ度aGVHD，經(jīng)二線免疫抑制治療無效，輸注人臍帶間充質(zhì)干細(xì)胞治療后，皮疹消退，肝功能恢復(fù)正常，胃腸道癥狀好轉(zhuǎn)�；純壕窗l(fā)生輸注相關(guān)不良反應(yīng)。目前患兒均無原發(fā)病的復(fù)發(fā)，均處于無病生存狀態(tài)。 結(jié)論：異基因造血干細(xì)胞移植是治療兒童急性白血病的有效方法。臍帶間充質(zhì)干細(xì)胞治療兒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

本文編號：2343484