B-ALL细胞影响骨髓间充质干细胞分化的实验研究

发布时间：2018-05-05 08:11

  本文选题：儿童急性白血病 + Jagged1　； 参考：《重庆医科大学》2015年博士论文

【摘要】：第一部分Notch信号通路相关配体Jaggedl在儿童急性白血病中的表达特点及意义目的：探讨Notch信号通路相关配体Jaggedl在急性白血病中的表达特点及临床意义。方法：收集88例初诊儿童急性白血病骨髓液(包括AML25例,B-ALL52例,T-ALL11例),18例ITP患儿骨髓液为对照组。应用Real-Time PCR法检测各组骨髓单个核细胞中Jagged1 mRNA表达水平。结果：1)Jaggedl在AML组、B-ALL组、T-ALL组及对照组均有表达；2)AML组Jaggedl表达水平高于对照组,差异无统计学意义(P0.05,n=25)；3)B-ALL组Jaggedl表达水平高于对照组,差异有统计学意义(P0.05,n=52)；4)T-ALL组Jaggedl表达水平低于对照组,差异有统计学意义(P0.05,n=11)；5)在B-ALL组中,高危组Jaggedl表达水平高于在标危组与中危组,差异有统计学意义(P0.05)；结论：Notch相关配体Jaggedl在不同类型儿童急性白血病中的表达水平不一致,与对照组相比,B-ALL中呈高表达,T-ALL呈低表达,AML中呈正常表达,提示在ALL中存在Jaggedl的表达异常。第二部分B-ALL细胞影响骨髓间充质干细胞向成骨细胞分化及其机制研究目的：研究B-ALL细胞对骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)成骨分化的影响,并探讨Notch信号通路在此过程中的作用。方法：采用B-ALL细胞与BMSCs体外共培养模型模拟体内微环境,以BMSCs单独培养及正常骨髓单个核细胞与BMSCs共培养作为对照。成骨诱导条件下共培养3天后,Real time PCR及Western-blot方法检测各组成骨分化标志物OPN、OCN、Runx2 mRNA和蛋白表达差异；成骨诱导条件下共培养14天后,茜素红S染色检测BMSCs的矿化能力,碱性磷酸酶(Alkaline Phosphatase, ALP)试剂盒检测ALP活性。最后通过重组蛋白Jagged1与中和抗体anti-Jagged1-Ab激活与抑制BMSCs中Notch信号通路,探讨B-ALL细胞是否通过Jaggedl激活BMSCs中Notch信号通路影响BMSCs向成骨细胞分化。结果：1)B-ALL细胞与BMSCs共培养组中,成骨分化标志OPN、 OCN表达水平较对照组降低,同时ALP活性及矿化能力较对照组减低；2)B-ALL细胞与BMSCs共培养后,BMSCs中Notch信号通路下游基因Hes1表达水平较对照组上调,提示BMSCs中的Notch细胞通路被激活；3)B-ALL细胞与BMSCs共培养体系中加入anti-Jagged1-Ab中和抗体后,Notch信号通路被抑制,同时成骨分化标志OPN、OCN表达上调；4)BMSCs中加入重组蛋白Jaggedl能够激活Notch信号通路,同时成骨分化标志OPN、OCN表达下降；5)BMSCs中Notch信号通路被抑制后,其下游基因Hesl表达下调,而成骨分化特异转录因子Runx2蛋白表达上调；结论：B-ALL细胞可抑制BMSCs向成骨细胞分化,其可能的机制是B-ALL细胞通过Jagged1激活BMSCs中的Notch信号通路,其下游基因Hes1活化降低成骨分化转录因子Runx2蛋白表达水平,从而抑制BMSCs向成骨细胞分化。第三部分B-ALL细胞影响骨髓间充质干细胞向内皮细胞分化及其机制研究目的：研究B-ALL对骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)内皮分化的影响,并探讨在此过程中可能存在的机制。方法：采用B-ALL细胞与BMSCs体外共培养模型模拟体内微环境,以BMSCs单独培养及正常骨髓单个核细胞与BMSCs共培养作为对照。共培养三天与七天后,Real time PCR检测各组内皮分化标志物Flk-1、CD31、vWFmRNA表达差异；Western-blot方法检测各组内皮分化特异标志CD31、Flk-1蛋白表达差异；再通过共培养中加入中和抗体anti-Jagged1-Ab抑制BMSCs中Notch信号通路,验证B-ALL细胞是否通过Jaggedl激活BMSCs中Notch信号通路影响BMSCs向内皮细胞分化。最后采用ELISA检测各组培养上清中VEGF分泌蛋白表达水平,探讨B-ALL细胞影响BMSCs向内皮分化的机制。结果：1)B-ALL细胞与BMSCs共培养三天后,BMSCs内皮分化标志Flk-1、CD31、vWF mRNA表达水平及Flk-1、CD31蛋白表达水平较对照组升高,但无显著差异(P0.05)；2) B-ALL细胞与BMSCs共培养七天后,BMSCs内皮分化标志Flk-1、CD31、vWF mRNA表达水平及Flk-1、CD31蛋白表达水平较对照组升高,有显著差异(P0.05); 3) B-ALL细胞与BMSCs共培养后,Notch信号通路被激活；4)共培养组中加入anti-Jagged1-Ab中和抗体后,Notch信号通路被抑制,但Flk-1、CD31、vWF mRNA表达水平及Flk-1、CD31蛋白表达水平无明显改变；5)采用ELISA检测共培养组中VEGF分泌蛋白表达水平,发现共培养组中VEGF表达水平上调,同时B-ALL细胞与BMSCs中VEGF表达水平相应的升高,推测BMSCs内皮分化机制可能与共培养环境中VEGF分泌蛋白水平升高相关。结论：B-ALL细胞可促进BMSCs向内皮细胞分化,其可能的机制是与B-ALL细胞与BMSCs共培养后促进共培养环境中VEGF分泌蛋白水平升高有关,而Notch信号通路在此过程并不起主要作用。
[Abstract]:The first part of the expression of Notch signaling pathway related ligand Jaggedl in acute leukemia in children and its purpose: To explore the expression and clinical significance of Notch signaling pathway related ligand Jaggedl in acute leukemia. Methods: to collect 88 cases of acute leukemia in early diagnosis of children (including AML25, B-ALL52, T-ALL11 cases), 18 The expression of Jagged1 mRNA expression in bone marrow mononuclear cells of each group was detected by Real-Time PCR method. Results: 1) Jaggedl was expressed in AML group, B-ALL, T-ALL group and control group; 2) Jaggedl expression level in AML group was higher than that of control group, and there was no statistical significance (P0.05, 3); 3) The difference was statistically significant (P0.05, n=52), and 4) the expression level of Jaggedl in group T-ALL was lower than that in the control group (P0.05, n=11), and 5) in the B-ALL group, the level of Jaggedl expression in high-risk group was higher than that in the risk group and the middle risk group, and the difference has the significance of overall planning (P0.05); conclusion: Notch related ligand Jaggedl is in different classes. The expression level of acute leukemia in children was not consistent. Compared with the control group, the expression of B-ALL was high, T-ALL showed low expression and normal expression in AML, suggesting the existence of Jaggedl expression in ALL. The second part of B-ALL cells influenced the differentiation and mechanism of bone marrow mesenchymal stem cells to osteoblasts and the study of B-ALL cells. The effect of bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells (BMSCs)) on osteogenesis and the role of Notch signaling pathway in this process. Methods: a co culture model of B-ALL cells and BMSCs in vitro was used to simulate the microenvironment in vivo, and BMSCs alone and normal bone marrow mononuclear cells and BMSCs co culture were used as control. 3 days after induction of osteogenesis, Real time PCR and Western-blot methods were used to detect the differences in the expression of OPN, OCN, Runx2 mRNA and protein, and the mineralization ability of BMSCs was detected by alizarin red S staining, and alkaline phosphatase (Alkaline Phosphatase) kit was used to detect the activity of the bone differentiation markers. After the activation and inhibition of the Notch signaling pathway in BMSCs by recombinant protein Jagged1 and neutralizing antibody anti-Jagged1-Ab, the effect of B-ALL cells on the differentiation of BMSCs into osteoblasts by Jaggedl activation of the Notch signaling pathway in BMSCs. Results: 1) B-ALL cells and BMSCs co culture group, the osteogenic differentiation marker OPN, the expression level is lower than the control group. At the same time, the activity and mineralization of ALP decreased compared with the control group; 2) after co culture of B-ALL cells with BMSCs, the Hes1 expression level of the downstream gene of Notch signaling pathway in BMSCs was up up, suggesting that the Notch cell pathway in BMSCs was activated; 3) Notch signals were connected with anti-Jagged1-Ab neutralization antibody in the co culture system of B-ALL cells and BMSCs. The pathway was inhibited, and the expression of osteogenic differentiation OPN, OCN expression was up-regulated; 4) the addition of recombinant protein Jaggedl in BMSCs could activate the Notch signaling pathway, while the osteogenic differentiation marker OPN, OCN expression decreased; and the Notch signal pathway in BMSCs was inhibited, and the downstream gene Hesl expression was down regulated, while the expression of Runx2 protein expression of the specific transcription factor of osteogenic differentiation was up to up. Conclusion: B-ALL cells can inhibit the differentiation of BMSCs into osteoblasts, and the possible mechanism is that B-ALL cells activate the Notch signaling pathway in BMSCs through Jagged1, and the downstream gene Hes1 activation reduces the level of Runx2 protein expression in the osteogenic differentiation transcription factor, thus inhibiting the differentiation of BMSCs to osteoblast. The third part of B-ALL cells affects bone marrow mesenchymal stem cells. Study on the effect of B-ALL on endothelial differentiation of bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) and explore the possible mechanism in this process. Methods: using B-ALL cells and BMSCs external co culture model to simulate the microenvironment in vivo, with BMSCs single. Single culture and normal bone marrow mononuclear cells were co cultured with BMSCs as control. Three days and seven days after co culture, Real time PCR was used to detect the differences in the expression of Flk-1, CD31, vWFmRNA in each group of endothelial differentiation. Western-blot method was used to detect the differential expression of CD31 and Flk-1 protein in each group of endothelial differentiation, and then the neutralization resistance was added in co culture. The body anti-Jagged1-Ab inhibits the Notch signaling pathway in BMSCs, and verifies whether B-ALL cells can affect the differentiation of BMSCs into the endothelial cells by activating the Notch signaling pathway in BMSCs through Jaggedl. Finally, the expression of VEGF secreted protein in the supernatant of each group is detected by ELISA, and the mechanism of B-ALL cells to affect the differentiation of BMSCs to the endothelial cells. Results: 1) Three days after co culture with BMSCs, the expression level of Flk-1, CD31, vWF mRNA, Flk-1, CD31 protein expression level and CD31 protein expression level were higher than that of the control group, but there was no significant difference (P0.05); 2) B-ALL cells and BMSCs were cultured for seven days, and the expression level and the expression level of BMSCs endothelial cells were compared with the control group. There were significant differences (P0.05); 3) after co culture of B-ALL cells with BMSCs, Notch signaling pathway was activated; 4) after adding anti-Jagged1-Ab neutralization antibody in co culture group, Notch signaling pathway was suppressed, but Flk-1, CD31, vWF mRNA expression level and Flk-1, CD31 protein expression level was not obviously changed; 5) 5) The expression level of secreted protein showed that the expression level of VEGF was up-regulated in the co culture group, and the level of VEGF expression in B-ALL cells and BMSCs increased correspondingly. It was suggested that the mechanism of BMSCs endothelial differentiation may be related to the increase of VEGF secreting protein level in co culture environment. Conclusion: B-ALL cells can promote the differentiation of BMSCs into endothelial cells, and the possible mechanism is that the mechanism is that B-ALL cells can promote the differentiation of BMSCs into endothelial cells. The co culture of B-ALL cells and BMSCs promoted the increase of VEGF secreted protein level in co culture environment, and Notch signaling pathway did not play a major role in this process.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R733.7

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