目的研究衰老骨髓基质细胞对骨髓造血细胞增殖分化能力的影响,为阐述机体造血微环境衰老对造血干/祖细胞增殖的影响提供实验依据。方法全骨髓贴壁法体外培养大鼠骨髓基质细胞,分为对照组和衰老组。衰老组:常规培养基内加入30 mg/m L D-...目的研究衰老骨髓基质细胞对骨髓造血细胞增殖分化能力的影响,为阐述机体造血微环境衰老对造血干/祖细胞增殖的影响提供实验依据。方法全骨髓贴壁法体外培养大鼠骨髓基质细胞,分为对照组和衰老组。衰老组:常规培养基内加入30 mg/m L D-半乳糖作用48 h。CCK-8法测定BMSCs增殖;流式细胞术分析细胞周期;β-半乳糖苷酶(SA-β-Gal)染色观察衰老BMSCs百分率;Western blot检测P16、P21和P53蛋白表达。骨髓造血细胞与BMSCs共培养,集落计数检测髓系多向性造血祖细胞(CFU-Mix)增殖分化。ELISA检测BMSCs培养上清液中IL-1β、GM-CSF和SCF含量;DCFH-DA流式荧光检测BMSC活性氧簇(ROS)水平;酶学法检测BMSCs内过氧化物丙二醛(MDA)含量和总超氧化物歧化酶(SOD)活性。结果与对照组相比,D-半乳糖诱导BMSCs衰老,细胞阻滞于G0/G1期(P<0.01),增殖能力显著下降,SA-β-Gal染色阳性率升高(P<0.01);衰老相关蛋白P16、P21和P53表达明显上调(P<0.01)。与衰老BMSCs共培养的骨髓造血细胞增殖分化能力减弱。衰老BMSCs内ROS、MDA氧化损伤指标上升,SOD抗氧化指标下降(P<0.01);BMSCs培养上清液IL-1β、GM-CSF和SCF含量明显下降(P<0.01)。结论衰老骨髓基质细胞抑制造血细胞增殖、分化能力,其机制可能与骨髓基质细胞氧化损伤,分泌活性因子改变有关。展开更多
The tyrosine kinase receptor III, c-Kit/stem cell factor receptor and its ligand, human stem cell factor (huSCF) are the predominant regulator of mitogenesis in the hematopoietic stem and progenitor cells. However, ga...The tyrosine kinase receptor III, c-Kit/stem cell factor receptor and its ligand, human stem cell factor (huSCF) are the predominant regulator of mitogenesis in the hematopoietic stem and progenitor cells. However, gain-of-function mutations alter c-Kit auto-regulatory mechanisms to aberrant c-Kit signaling, leading to the onset or progression of cancerous transformations. The most common mutation of c-Kit is the substitution of aspartic acid residue in position 816 to valine (D816V), which is majorly responsible for its ligand-independent constitutive activation, and is implicated in hematopoietic malignancies. Currently, molecular targeted therapy is increasingly becoming a hot spot due to its specificity and low toxicity. As the molecular mechanisms responsible for D816V-c-Kit mediated tumorogenicity are largely unknown, in this study, we aimed to investigate the D816V-c-Kit signaling mediated downstream molecular targets. Specifically, we created c-Kit active mutant form D816V and performed inducible gene expression of mutant D816V-c-Kit in monomyelocytic cell line U937. Mutant D816V-c-Kit expressing cells revealed significantly enhanced cellular mitogenic activity compared to wild-type c-Kit expressing cells independent of huSCF. To examine the molecular targets regulating tumorogenic proliferation, we evaluated the consequences of mutant D816V-c-Kit expression on downstream gene expression profile by high throughput microarray technology. The levels of some of the relevant genes (PIK3CB, eIF4B, PRKCDBP, MOAP1) were validated by quantitative polymerase chain reaction. SLA, STAT5B, MAP3K2 and MAPK14 emerged as important downstream molecular targets of mutant D816V-c-Kit. Further, by dissecting the signaling pathways, we also demonstrated that the D816V-c-Kit mediated hematopoietic cell proliferation is dependent on molecular target p38 MAP kinase.展开更多
目的观察辐射后骨髓基质细胞（MSC）高迁移率族蛋白B1（high mobility group box 1，HMGB1）的释放，研究HMGB1对人脐血造血干细胞（HSC）增殖分化的影响。方法体外培养人骨髓MSC，利用ELISA方法检测经12Gyγ射线照射后培养上清中HMGB1...目的观察辐射后骨髓基质细胞（MSC）高迁移率族蛋白B1（high mobility group box 1，HMGB1）的释放，研究HMGB1对人脐血造血干细胞（HSC）增殖分化的影响。方法体外培养人骨髓MSC，利用ELISA方法检测经12Gyγ射线照射后培养上清中HMGB1含量的变化。HMGB1与CD34^＋的人脐血HSC体外液体共培养6d，通过流式细胞术检测CD34^＋细胞分化指标（CD13、CD14、CD11c、CD41、CD71）的变化。集落形成实验观察HMGB1对HSC增殖分化的影响。结果辐射后，骨髓MSC培养上清中HMGB1含量为（4．3±0．9）ng／ml，较对照组HMGB1含量[（0．4±0．2）ng／m1]明显升高（P〈0．01）。脐血CD34^＋细胞表面表达HMGBl受体RAGE、TLR2和TLR4。HSC与HMGBI共培养6d后，与对照组比较，红系（CD71）和粒单系（CD13、CD14、CD11c）标记的表达明显增强，分别为CD13（18．4±3．8）％和（32．6±5．9）％、CD14（12．6±2．7）％和（25．4±4．4）％、CD11c（9．8±2．1）％和（20．3±3．9）％、CD71（26．6±4．6）％和（47．1±7．4）％，而巨核系标记CD41的表达[（1．1±0．4）％和（1．3±0．5）％]无明显变化。集落形成实验示共培养14d后，红系集落、粒一巨噬细胞集落和总集落的生成较对照组明显增多（P〈0．05），联用抗-TLR2和抗-TLR4抗体可部分抑制这一作用。结论辐射促进骨髓MSC释放HMGB1，胞外HMGB1可以促进HSC的增殖分化。辐射后，骨髓MSC释放的HMGB1与造血恢复或造血重建的关系值得进一步研究。展开更多
文摘The tyrosine kinase receptor III, c-Kit/stem cell factor receptor and its ligand, human stem cell factor (huSCF) are the predominant regulator of mitogenesis in the hematopoietic stem and progenitor cells. However, gain-of-function mutations alter c-Kit auto-regulatory mechanisms to aberrant c-Kit signaling, leading to the onset or progression of cancerous transformations. The most common mutation of c-Kit is the substitution of aspartic acid residue in position 816 to valine (D816V), which is majorly responsible for its ligand-independent constitutive activation, and is implicated in hematopoietic malignancies. Currently, molecular targeted therapy is increasingly becoming a hot spot due to its specificity and low toxicity. As the molecular mechanisms responsible for D816V-c-Kit mediated tumorogenicity are largely unknown, in this study, we aimed to investigate the D816V-c-Kit signaling mediated downstream molecular targets. Specifically, we created c-Kit active mutant form D816V and performed inducible gene expression of mutant D816V-c-Kit in monomyelocytic cell line U937. Mutant D816V-c-Kit expressing cells revealed significantly enhanced cellular mitogenic activity compared to wild-type c-Kit expressing cells independent of huSCF. To examine the molecular targets regulating tumorogenic proliferation, we evaluated the consequences of mutant D816V-c-Kit expression on downstream gene expression profile by high throughput microarray technology. The levels of some of the relevant genes (PIK3CB, eIF4B, PRKCDBP, MOAP1) were validated by quantitative polymerase chain reaction. SLA, STAT5B, MAP3K2 and MAPK14 emerged as important downstream molecular targets of mutant D816V-c-Kit. Further, by dissecting the signaling pathways, we also demonstrated that the D816V-c-Kit mediated hematopoietic cell proliferation is dependent on molecular target p38 MAP kinase.
文摘目的观察辐射后骨髓基质细胞（MSC）高迁移率族蛋白B1（high mobility group box 1，HMGB1）的释放，研究HMGB1对人脐血造血干细胞（HSC）增殖分化的影响。方法体外培养人骨髓MSC，利用ELISA方法检测经12Gyγ射线照射后培养上清中HMGB1含量的变化。HMGB1与CD34^＋的人脐血HSC体外液体共培养6d，通过流式细胞术检测CD34^＋细胞分化指标（CD13、CD14、CD11c、CD41、CD71）的变化。集落形成实验观察HMGB1对HSC增殖分化的影响。结果辐射后，骨髓MSC培养上清中HMGB1含量为（4．3±0．9）ng／ml，较对照组HMGB1含量[（0．4±0．2）ng／m1]明显升高（P〈0．01）。脐血CD34^＋细胞表面表达HMGBl受体RAGE、TLR2和TLR4。HSC与HMGBI共培养6d后，与对照组比较，红系（CD71）和粒单系（CD13、CD14、CD11c）标记的表达明显增强，分别为CD13（18．4±3．8）％和（32．6±5．9）％、CD14（12．6±2．7）％和（25．4±4．4）％、CD11c（9．8±2．1）％和（20．3±3．9）％、CD71（26．6±4．6）％和（47．1±7．4）％，而巨核系标记CD41的表达[（1．1±0．4）％和（1．3±0．5）％]无明显变化。集落形成实验示共培养14d后，红系集落、粒一巨噬细胞集落和总集落的生成较对照组明显增多（P〈0．05），联用抗-TLR2和抗-TLR4抗体可部分抑制这一作用。结论辐射促进骨髓MSC释放HMGB1，胞外HMGB1可以促进HSC的增殖分化。辐射后，骨髓MSC释放的HMGB1与造血恢复或造血重建的关系值得进一步研究。
