人脐带间充质干细胞对白血病细胞增殖分化的作用

Effect of Human Umbilical Cord-derived Mesenchymal Stem Cells on Proliferation and Differentiation of Leukemia Cells

目的:探讨人脐带间充质干细胞对急性髓系白血病M3型细胞NB4细胞系增殖分化的影响及其相关机制。方法:分离及培养HUC-MSC细胞和NB4细胞,建立以HUC-MSC细胞为基质细胞、NB4细胞为目的细胞的直接共培养体系。应用CCK-8法和细胞计数法检测细胞增殖情况;应用瑞氏吉姆萨染色和NBT还原实验检测细胞分化状态;应用ELISA法检测各组上清中IL-6表达水平;应用实时定量PCR技术检测IL-6相关基因CDKN1A、CCND1、Survivin、STAT3的转录水平。结果:两种细胞共培养后在倒置显微镜下可见到共生良好。与正常NB4细胞相比,同HUC-MSC共培养的NB4细胞的增殖能力减弱;HUC-MSC能促进ATRA诱导的NB4细胞分化。在共培养组上清中IL-6水平显著升高。与单纯培养的NB4细胞相比,共培养组和ATRA诱导组的NB4细胞中细胞周期素D1基因(CCN D1)转录水平均不同程度下降(NB4:NM:NA:NMA=1:0.35:0.70:0.20),CDKN1A表达水平不同程度增高(NB4:NM:NA:NMA=1:3.21:1.41:5.32)。结论:间充质干细胞可能通过IL-6信号通路抑制NB4细胞的增殖,促进ATRA诱导的NB4细胞的分化。

Objective：To investigate the effect of human umbilical cord-derived mesenchymal stem cells（HUCMSC） on the proliferation and differentiation of NB4 treated with all-trans retinoid acid（ATRA） and its underlining mechanisms.Methods：Human umbilical cord mesenchymal stem cells were isolated from umbilical cord of newborns.Co-culture system was established by HUC-MSC and NB4 in vitro.The experiment was divided into 4 groups ＇.NB4group（NB4 cells alone）,NM group（NB4 cells co-cultured with HUC-MSC）,NA group（NB4 cells treated with ATRA）,NMA group（NB4 cells co-cultured with HUC-MSC and treated with ATRA）.NB4 cells were counted by a microscopy,NB4 proliferation was monitored by CCK-8 assay,NB4 differentiation was assessed by Wright ＇s staining and nitroblue tetrazolium reduction test.IL-6 levels in the culture supernatant of different groups were tested by ELISA kit.Quantitative PCR was used to detect the transcription level of CDKN1 A,CCND1 and Survivin.Results：NB4 and HUC-MSC in the co-culturing systems were in good condition with a slight repression of NB4 proliferation by HUCMSC.HUC-MSC could collaborate with ATRA to induce significant NB4 differentiation.Consistent with this finding,IL-6 expression levels of co-cultured groups were remarkably higher than that in any other groups or the group of HUCMSC alone.The quantitative PCR analysis showed that the levels of CDKN1 A and CCND1 mRNA expression were increased or decreased respectively in the co-cultured groups.Conclusion：HUC-MSC co-culture can reduce proliferation but promote the differentiation of NB4 cells,suggesting that this effect may be closely related with the secretion of IL-6which can affect the expression of some factors in vitro.