摘要
目的观察纤维连接蛋白-血小板生成素(FN-TPO)基因修饰的人骨髓间充质干细胞(mesenchymal stemcells,MSCs)支持脐血造血干细胞植入的能力。方法将20只严重联合免疫缺陷(SCID)小鼠经亚致死剂量射线辐射后随机分为实验组(n=5):FN-TPO基因修饰的骨髓MSCs联合脐血单个核细胞(CB-MNC)组共移植;对照组:包括单纯CB-MNC移植组(n=5)、CB-MNC联合未修饰骨髓MSCs共移植组(n=5)和仅输入不含血清的IMDM培养基的空白对照组。细胞移植后,持续观察各组小鼠4周,记录一般情况及生存率;通过检测不同时间点(辐射前,辐射后细胞移植前,细胞移植后2 d及1、2、3、4周)的小鼠外周血常规来反映小鼠造血系统恢复情况;4周后以流式细胞术和PCR等检测移植后小鼠体内的人源细胞的整合情况。结果辐射前、辐射后细胞移植前、移植后2 d及移植2周后各组动物外周血白细胞、红细胞、血红蛋白和血小板数均无明显差别;细胞移植1周,实验组小鼠外周血WBC、RBC、Hb和Plt分别为(0.83±0.15)×109/L、(9.84±0.36)×1012/L、147.50±4.80 g/L和(198.75±71.14)×109/L,均较对照组为高,差别均有统计学意义(P<0.05),且实验组小鼠外周血常规变化比较平稳。移植4周各组动物生存率,基因修饰和未修饰MSCs联合CB-MNC共移植组较单纯CB-MNC移植组明显为高(80%VS40%);存活下来的细胞移植小鼠骨髓和外周血中均检测到人源性CD45+细胞,实验组小鼠骨髓和外周血中人CD45+细胞比例(%)分别为:8.15±1.72和2.28±0.57,与单纯CB-MNC移植组的3.93±1.28和0.82±0.06相比差别均具有统计学意义(P<0.05);PCR检测移植后4周存活小鼠体内人beta-actin基因表达情况显示:小鼠外周血、骨髓及心、肝、脾、脑、肺等重要脏器基因组DNA中有人beta-actin基因存在。结论FN-TPO基因修饰的骨髓MSCs能够更有效的支持脐血造血干细胞植入。
Objective To observe the influence of Fibronectin-Thrombopoietin （FN-TPO） gene modified human bone marrow mesenchymal stem cells （MSCs） on the engraftment of cord blood hematopoietic stem cells. Methods FN-TPO gene modified human bone marrow MSCs combined with cord blood mononuclear cells （CB-MNC） were transplanted to sub- lethal dose treated severe combined immunodeficiency disease （SCID） mice. After transplantation, these mice were observed for 4 weeks. Peripheral blood cell counts were performed at different time point to assay the hematopoietic system status of the mice. Four weeks after the transplantation, human-sourced cell integration was assayed by flow cytometry （FCM） and polymerase chain reaction （PCR）. Results One week after the cell transplantation, every main index of the peripheral blood cell counts in the gene modified group was higher than that in the control groups （ P 〈0. 05）. Four weeks after the transplantation, survival rates of the co-transplantation groups （both gene modified and non-modified） were higher than the CB-MNC only transplanted group （80% vs 40% ）. Human CD45^＋ cells were found in bone marrow and peripheral blood of the survived transplanted mice. The percentage of human CD45^＋ cells in the bone marrow and peripheral blood of the gene modified co-transplantation group were 8.15 ± 1.72 and 2128±0.57, which were much higher than those in the CB-MNC only transplanted group （P 〈 0.05）. PCR assay showed that human beta-actin gene was expressed in the periph- eral blood, bone marrow, heart, liver, spleen and lung genome. Conclusion FN-TPO gene modified human bone marrow MSCs can effectively improve the engraftment of the human CB-MNC.
出处
《中国输血杂志》
CAS
CSCD
北大核心
2009年第2期94-98,共5页
Chinese Journal of Blood Transfusion
基金
国家自然科学基金资助项目(30100095)
