骨形成蛋白4对小鼠骨髓造血干/祖细胞体外扩增的作用研究

Effect of BMP4 on expansion of mouse hematopoietic stem/progenitor cells in vitro

目的探究骨形成蛋白4(BMP4)对小鼠造血干/祖细胞(HSPC)体外扩增的作用。方法用磁珠分选法富集小鼠骨髓细胞中的c⁃Kit+造血细胞,分成对照组(添加基础培养基)及实验组(每天补充40 ng/ml BMP4)进行体外培养。培养4 d后,进行细胞计数及存活率分析,并结合流式细胞术分析细胞中HSPC的比例及数量,采用造血集落形成实验分析细胞的造血集落形成能力。在此基础上,结合BMP4信号通路抑制剂LDN193189(500 nmol/L)处理,通过流式细胞术检测阻断该信号通路对细胞中HSPC比例的影响,并结合实时荧光定量PCR(qRT⁃PCR)分析细胞中毒性应激和凋亡相关基因表达水平。结果c⁃Kit+造血细胞体外培养4 d后,对照组及实验组细胞数量分别是起始接种数量的(3.20±0.33)倍及(5.00±0.61)倍。结合流式细胞术对细胞中HSPC比例分析的结果,培养4 d后对照组及实验组Lineage^(-)Sca⁃1^(+)c⁃Kit^(+)(LSK)细胞数量分别较起始接种细胞中LSK细胞数量扩增了(2.62±0.09)倍及(5.67±0.11)倍。造血集落形成实验结果表明,对照组形成了(49.25±6.99)个集落,而实验组可形成(69.95±3.40)个集落。培养基中添加LDN193189(500 nmol/L)培养4 d后,LSK细胞数量为(3.16±0.43)×10^(4)个/孔,较未添加LDN193189组下降了(38.27±7.44)%。qRT⁃PCR结果表明,与对照组相比,BMP4处理组降低了蛋白质毒性应激相关基因和促凋亡相关基因的表达。结论BMP4处理能够促进小鼠骨髓造血细胞的体外扩增,并有效提高细胞中HSPC的增殖及造血细胞集落形成能力。

Objective To explore the effect of bone morphogenetic protein 4(BMP4)on the expansion of mouse hema⁃topoietic stem/progenitor cells(HSPCs)in vitro.Methods c⁃Kit+hematopoietic cells in mouse bone marrow were enriched with immune microbeads before being divided into the control group(without BMP4 addition)and the experimental group(supplemented with 40 ng/ml BMP4 every day).After 4 days of culture,the viability of the cells was analyzed.The proportions and numbers of HSPCs in the cultured cells were calculated by flow cytometry.The ability of these cells to form hematopoietic colonies was investigated via hematopoietic colony⁃forming unit assay.Along with treatment with BMP4 signaling pathway inhibitor LDN193189(500 nmol/L),flow cytometry was used to detect the effect of blocking this signaling pathway on the proportions of HSPCs,while quantitative reverse transcription PCR(qRT⁃PCR)was used to analyze the expression levels of proteotoxic stress⁃related genes and apoptosis⁃related genes.Results After 4⁃day culture of c⁃Kit+hematopoietic cells,the total cell numbers in the control group and the experimental group increased(3.20±0.33)fold and(5.00±0.61)fold,respectively.The results of flow cytometry analysis of the proportions of HSPCs in each group suggested that Lineage^(-)Sca⁃1^(+)c⁃Kit^(+)(LSK)cells in the control group and the experimental group increased(2.62±0.09)fold and(5.67±0.11)fold,respectively.Hematopoietic colony⁃forming unit assay showed that(49.25±6.99)colonies were formed in the control group,compared with(69.95±3.40)colonies in the experimental group.The BMP4 signal pathway blocking experiment showed that the number of LSK cells was(3.16±0.43)×10^(4) per well after 4⁃day culture supplemented with LDN193189(500 nmol/L),which was a(38.27±7.44)%decrease compared with the group without LDN193189.qRT⁃PCR detection results showed that compared with the control group,the expression levels of proteotoxic stress and apoptosis⁃related genes were decreased in the BMP4 treatment group.Conclusion BMP4 treatment can promote the expansion of mouse bone marrow hematopoietic cells in vitro,and improve the proliferation and colony formation ability of murine HSPC.