原代骨髓基质细胞联合细胞因子体外诱导ES-D_3细胞造血分化的研究  

Primary bone marrow stromal cells associated with cytokines induce ES-D_3 cells to differentiate to hematopoietic cells in vitro

作　　者：姜景岩[1] 孔北华[1] 李华[2] 沈柏均[3] 侯怀水[3] 时庆[3] 马秀峰[3] 

机构地区：[1]山东大学齐鲁医院妇产科,济南250012 [2]济南市中心医院妇产科 [3]山东大学齐鲁医院脐血库

出　　处：《临床血液学杂志》2006年第1期23-27,共5页Journal of Clinical Hematology

基　　金：国家自然科学基金资助项目(No:30271361)

摘　　要：目的：研究原代骨髓基质细胞联合细胞因子（VEGF，SCF和TPO）体外诱导ES-D3细胞造血分化的效率，探讨体外高效诱导胚胎干细胞生成造血干／祖细胞的方法。方法：取6～8周昆明鼠骨髓制备小鼠骨髓基质细胞（BMSC）饲养层。采用二步诱导法，先将ES-D3细胞诱导分化为拟胚体（EB），再将EB置于4种体系下诱导分化造血干／祖细胞；第1组：自发分化对照组；第2组：细胞因子组（VEGF加SCF加TPO）；第3组：BMSC饲养层组；第4组：BMSC饲养层加细胞因子组（BMSC加VEGF加SCF加TPO）。诱导分化1周的部分细胞行造血祖细胞集落培养。流式细胞仪检测诱导培养7d和14d的各组细胞中干细胞特异抗原（CD34）、祖细胞特异抗原（c-kit）、粒细胞表面抗原（CD11b）、红系细胞特异抗原（Ter119）阳性表达率。间接免疫荧光染色法显示CD34、c-kit、CD11b、Te〉119阳性细胞。RT-PCR检测造血转录因子GATA-2、FIk-1、SCL、p-H1和p-major珠蛋白的表达。结果：诱导7d生成的细胞均表达造血CD34和Pkit，诱导14d生成的细胞表达单核巨噬细胞、CD11b和Te〉119，且诱导细胞表达造血转录因子（GATA-2、SCL、β-H1、β-major）基因mRNA，培养在特定条件下可形成造血祖细胞集落。从生成造血细胞和造血集落的数量分析，骨髓基质细胞饲养层与细胞因子合用时诱导效率明显高于单用组和对照组（P〈0．05或0．01）。结论：骨髓基质细胞饲养层与细胞因子合用，体外可以促进ES-D3细胞的早期造血分化，产生的造血前体细胞可进一步形成造血集落并分化成熟，表达与造血转录和早期造血分化相关的基因。Objective：To study the efficiency of primary bone marrow stromal cells associated with cytokines, i.e. vascular endothelial growth factor （VEGF）, stem cell factor （SCF） and thrombopoietin （TPO） inducing ES-D3 cells to differentiate to hematopoietic cells in vitro, and to explore the method of inducing ES to differentiate to hematopoietic progenitors in vitro. Method..BM samples of 6～8 weeks old Kunming mice were used to prepare primary bone marrow stromal cells （BMSC） feeder layer. Embryoid body （EB） were derived from ES-D3 cells, and then induced under four different system： spontaneous differentiation control group, cytokines group （VEGF＋SCF--TPO）, BMSC group and BMSC--VEGF--SCF--TPO group. Some 7-day differentiated cells were used to carry through hematopoietic colonies assay. Flow cytometry were used to observe the positive rate and indirect immunofluorescence cell staining were used to detect the positive cells of CD34,c-kit,CD11b,Ter119 in 7/14-day differentiated cells. Hematopoietic transcription factors （Flk-1, GATA-2, SCL, β-H1 and β-major） were detected by RT-PCR. Result：7-day differentiated cells expressed hematopoietic progenitors antigen （CD34 and c-kit）, 14-day differentiated cells expressed monocyte and granulocyte antigen （CDllb）, red cell antigen （Ter119）. Differentiated cells also expressed transcription factors （Flk-1, GATA-2, SCL, β-H1 and β-major） and could generate hematopoietic colonies. The BMSC-- VEGF＋SCF｜TPO group got the highest inducing rate according to the numbers of hematopoietic progenitors and colonies （ P〈 0.05 or 0.01）. Conclusion： Bone marrow feeder layer associated with cytokines strongly promotes hematopoietic differentiation in vitro. Those induced hematopoietic progenitors could form hematopoietic colonies or differentiate mature cells. They also expressed genestromal cells associated with hematotopoietic transcription and differentiation.

关 键 词：胚胎 拟胚体 造血分化 

分 类 号：R321.4[医药卫生—人体解剖和组织胚胎学]