血液血管细胞生成素对胎儿骨髓造血和内皮干细胞作用的研究

The effect of human hemangiopoietin on human fetal bone marrow hematopoietic stem cells and endothelial stem cells

目的研究人血液血管细胞生成素(hemangiopoietin,HAPO)对胎儿骨髓细胞的作用,探讨其生物学特性。方法采用细胞液体培养、半固体培养、MTT方法、免疫荧光标记流式细胞仪测定、免疫组化、显微镜观察照相等方法。结果在液体培养3周的胎儿骨髓单个核细胞中,HAPO组中出现了大量小而圆的早期造血细胞,其中CD34+细胞含量比对照组高20%,对照组CD34+细胞为1.25×105个,而HAPO组CD34+细胞为3.93×106个。取胎儿骨髓悬浮造血细胞进行半固体培养,对照组不能形成CFU-GEMM,而HAPO组形成CFU-GEMM数达到(11.0±2.6)个;HAPO也协同SCF、IL-3、GM-SCF等生长因子促进集落形成,CFU总数是对照组2.6倍,CFU-GEMM数HAPO组是对照组2.1倍。MTT方法发现,HAPO对胎儿骨髓基质细胞也有促增殖作用,HAPO可使基质细胞增长21%;液体培养的胎儿骨髓基质细胞中,有内皮特异性标志的细胞均增高;在甲基纤维素半固体培养中HAPO使胎儿骨髓内皮细胞的集落数增高,并出现条索状排列的集落,有促进血管形成的趋势。进一步证明HAPO可直接促进CD34+KDR+细胞的增殖。结论HAPO对骨髓造血和血管内皮干细胞均有刺激增殖作用。

Objective To investigate the effect of human hemangiopoietin （HAPO） on human fetal bone marrow hematopoietic stem/progenitor cells （HSPC） and its biological function. Methods The liquid culture, semi-solid culture, MTT proliferation test, fluorescence activating cell sorter （FACS） detection,immunohistochemistry and microscope counting were performed. Results The FACS results showed that CD34^＋ cells in HAPO（100 ng/ml） group were 1.2 times higher than those in control group after culture for 3 weeks. Semi-solid culture of fetal bone marrow showed that HAPO （100 ng/ml） promoted the colony formation ：The number of CFU-GEMM was 11.0 ± 2.58 in HAPO group but 0 in control group;HAPO could also stimulate colony formation in coordination with other growth factors eg. SCF,IL-3,GM-SCF. The total number of CFU in HAPO group was 2.63 times higher than that in control group,CFU-GEMM was 2.1 times of that in control. The HAPO （100 ng/ml） could promote the proliferation of fetal bone marrow mesenchymal cells to 2.1 times. In the liquid fetal bone marrow mesenchymal cells culture system, the cells with endothelial markers appeared in higher percentage. In the methy cellulose semi-solid culture system, HAPO promoted the endothelial colony formation in fetal bone marrow cells. The presence of HAPO also promoted the existence of cord-like structure colony suggesting a trend toward vessel formation. It was further demonstrated that HAPO could directly stimulate the proliferation of CD34^＋KDR^＋ cells. Conclusion It is showed that HAPO has the effect on enhancing the proliferation of human fetal bone marrow hematopoietic stem cells and endothelial stem cells.