【作者】 吴昊；

【导师】 韩忠朝；

【作者基本信息】 中国协和医科大学 ， 内科学， 2010， 硕士

【摘要】 间充质干细胞(MSC)是一种具有自我更新能力和多向分化潜能的成体干细胞,它存在于多种组织中,如胎儿肝脏、胎血、胎儿骨髓、胎肺、脐血、胎盘、脐带、成人骨髓和脂肪等,已经成为组织工程和再生医学细胞治疗的主要候选干细胞之一。其中研究比较广泛的是骨髓间充质干细胞(BMMSC),但骨髓间充质干细胞需要进行骨穿才能得到,对供者有一定的损伤还有感染的可能。人脐带间充质干细胞(hUCMSC)与其他来源的间充质干细胞相比,更容易获得和在体外扩增,并且被病毒感染的概率明显降低,因此hUCMSC是众多来源间充质干细胞中更具有应用价值的种子细胞。急性早幼粒白血病(APL)不同与其他急性髓系白血病,具有独特的病理特征。在全反式维甲酸(ATRA)应用于治疗APL以前,病人极容易因大量出血而死亡。由于APL细胞具有因染色体易位产生的PML/RARA融合基因,因此早幼粒细胞不能向成熟的单核细胞或粒细胞分化。为了深入研究APL和其他的白血病,研究者从病人体内分离或对正常细胞进行分子生物学操作得到了多种白血病细胞系,如：HL-60、NB-4、KG-1a和K562等。HL-60细胞是从M-2型病人体内分离得到的早幼粒细胞,在不同的刺激下可向成熟的粒系细胞或单核／巨噬细胞分化,是个很好的研究急性白血病和粒系细胞分化的细胞模型。本研究探讨了人脐带间充质干细胞(hUCMSC)对全反式维甲酸(ATRA)诱导HL-60细胞分化以及对HL-60增殖的影响。HL-60细胞分为4组：未经ATRA处理的对照组、与hUCMSC细胞共培养的hUCMSC组,经ATRA处理的ATRA组以及经ATRA处理并与hUCMSC细胞共培养的ATRA+hUCMSC组。在规定时间采用Cell Counting Kit-8 (CCK8)检测对照组和hUCMSC组HL-60细胞的增殖情况；在光镜下观察各组细胞形态、硝基四氮唑蓝(NBT)阳性细胞；应用real-timePCR方法检测c-myc基因表达水平以及流式细胞术检测CDllb表面标志来比较各组中HL-60细胞的分化情况。结果表明：共培养体系中hUCMSC细胞能抑制HL-60的增殖(hUCMSC:HL-60为1：1时,48h P<0.05,72h P<0.01; hUCMSC:HL-60为1：5时,72h P＜0.05)。在2μmol/L ATRA的刺激下,ATRA+hUCMSC组与ATRA组相比,ATRA+hUCMSC组出现更多具有中性粒细胞形态的HL-60细胞并具更高的NBT阳性率(P<0.05); ATRA+hUCMSC组中HL-60细胞c-myc基因表达水平更低(P<0.05); ATRA+hUCMSC组中HL-60细胞表面CD11b的表达水平更高(48hP＜0.05,72h P＜0.01)。此外我们还研究了hUCMSC对1,25(OH)2D3诱导HL-60细胞分化的影响。在1,25(OH)2D3的刺激下,用流式细胞技术检测HL-60细胞表面CD11b和CD14的表达水平。与hUCMSC共培养后,HL-60的CD11b表达水平明显上升了,但其CD14的表达水平没有明显变化。结论：脐带间充质干细胞能抑制HL-60的增殖,增强ATRA对HL-60细胞的诱导分化作用并且hUCMSC细胞自身也能诱导HL-60细胞分化成熟。更多还原

【Abstract】 This study was aimed to investigate the enhancement of all-trans retinoic acid-induced HL-60 leukemia cell differentiation by umbilical cord mesenchymal stem cell. The HL-60 cells were divided into four groups:control group without ATRA treatment, hUCMSC group co-cultured with hUCMSCs, ATRA group with ATRA treatment as well as ATRA+hUCMSC group with ATRA treatment and co-cultivation with hUCMSCs. The proliferation of control group and hUCMSC group was compared by Cell Counting Kit-8 (CCK8). The morphology of HL-60 cells and NBT positive rate in four groups were observed and compared by microscopy, the c-myc expression of HL-60 cells in different groups was evaluated by real-time PCR, and the CDllb expression on HL-60 cells in different groups were detected by flow cytometry. The results showed that, in the co-culturing system, hUCMSCs can inhibit the proliferation of HL-60 (hUCMSC:HL-60 is 1:1,48h P<0.05,72h P<0.01; hUCMSC:HL-60 is 1:5, 72h P<0.05). With ATRA treatment, compared ATRA+hUCMSC group with ATRA group, there are more neutrophil like HL-60 cells and higher NBT positive rate (P<0.05) in ATRA+hUCMSC group. The c-myc expression of HL-60 cells in ATRA+hUCMSC group was lower than ATRA group (P<0.05). Furthermore, HL-60 cells in ATRA+hUCMSC group had stronger CD11b expression than ATRA group (48h P<0.05, 72h P<0.01). It is concluded that hUCMSC not only can inhibition the proliferation of HL-60 cells, but also can enhance the differentiation potential of ATRA on HL-60 cells.In addition, we also investigate the effects of hUCMSCs on the HL-60 differetiation induced by 1,25(OH)2D3. When treated by 1,25(OH)2D3, we use flow cytometry to test the expression of CD11b and CD 14 on the HL-60 cells. After co-culturing on the hUCMSC, there is an enhancement of CDllb on the HL-60 cells, but there is no significant change of the expression of CD 14. In some degree, hUCMSCs also can enhance the differentiation effects of 1,25(OH)2D3, while 1,25(OH)2D3 can induce HL-60 cells to neutrophil like cells rather than macrophage cells on hUCMSCs.更多还原