本文選題：兒童急性白血病 + Jagged1　； 參考：《重慶醫(yī)科大學(xué)》2015年博士論文

【摘要】：第一部分Notch信號(hào)通路相關(guān)配體Jaggedl在兒童急性白血病中的表達(dá)特點(diǎn)及意義目的：探討Notch信號(hào)通路相關(guān)配體Jaggedl在急性白血病中的表達(dá)特點(diǎn)及臨床意義。方法：收集88例初診兒童急性白血病骨髓液(包括AML25例,B-ALL52例,T-ALL11例),18例ITP患兒骨髓液為對(duì)照組。應(yīng)用Real-Time PCR法檢測(cè)各組骨髓單個(gè)核細(xì)胞中Jagged1 mRNA表達(dá)水平。結(jié)果：1)Jaggedl在AML組、B-ALL組、T-ALL組及對(duì)照組均有表達(dá)；2)AML組Jaggedl表達(dá)水平高于對(duì)照組,差異無統(tǒng)計(jì)學(xué)意義(P0.05,n=25)；3)B-ALL組Jaggedl表達(dá)水平高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05,n=52)；4)T-ALL組Jaggedl表達(dá)水平低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05,n=11)；5)在B-ALL組中,高危組Jaggedl表達(dá)水平高于在標(biāo)危組與中危組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；結(jié)論：Notch相關(guān)配體Jaggedl在不同類型兒童急性白血病中的表達(dá)水平不一致,與對(duì)照組相比,B-ALL中呈高表達(dá),T-ALL呈低表達(dá),AML中呈正常表達(dá),提示在ALL中存在Jaggedl的表達(dá)異常。第二部分B-ALL細(xì)胞影響骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞分化及其機(jī)制研究目的：研究B-ALL細(xì)胞對(duì)骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)成骨分化的影響,并探討Notch信號(hào)通路在此過程中的作用。方法：采用B-ALL細(xì)胞與BMSCs體外共培養(yǎng)模型模擬體內(nèi)微環(huán)境,以BMSCs單獨(dú)培養(yǎng)及正常骨髓單個(gè)核細(xì)胞與BMSCs共培養(yǎng)作為對(duì)照。成骨誘導(dǎo)條件下共培養(yǎng)3天后,Real time PCR及Western-blot方法檢測(cè)各組成骨分化標(biāo)志物OPN、OCN、Runx2 mRNA和蛋白表達(dá)差異；成骨誘導(dǎo)條件下共培養(yǎng)14天后,茜素紅S染色檢測(cè)BMSCs的礦化能力,堿性磷酸酶(Alkaline Phosphatase, ALP)試劑盒檢測(cè)ALP活性。最后通過重組蛋白Jagged1與中和抗體anti-Jagged1-Ab激活與抑制BMSCs中Notch信號(hào)通路,探討B(tài)-ALL細(xì)胞是否通過Jaggedl激活BMSCs中Notch信號(hào)通路影響B(tài)MSCs向成骨細(xì)胞分化。結(jié)果：1)B-ALL細(xì)胞與BMSCs共培養(yǎng)組中,成骨分化標(biāo)志OPN、 OCN表達(dá)水平較對(duì)照組降低,同時(shí)ALP活性及礦化能力較對(duì)照組減低；2)B-ALL細(xì)胞與BMSCs共培養(yǎng)后,BMSCs中Notch信號(hào)通路下游基因Hes1表達(dá)水平較對(duì)照組上調(diào),提示BMSCs中的Notch細(xì)胞通路被激活；3)B-ALL細(xì)胞與BMSCs共培養(yǎng)體系中加入anti-Jagged1-Ab中和抗體后,Notch信號(hào)通路被抑制,同時(shí)成骨分化標(biāo)志OPN、OCN表達(dá)上調(diào)；4)BMSCs中加入重組蛋白Jaggedl能夠激活Notch信號(hào)通路,同時(shí)成骨分化標(biāo)志OPN、OCN表達(dá)下降；5)BMSCs中Notch信號(hào)通路被抑制后,其下游基因Hesl表達(dá)下調(diào),而成骨分化特異轉(zhuǎn)錄因子Runx2蛋白表達(dá)上調(diào)；結(jié)論：B-ALL細(xì)胞可抑制BMSCs向成骨細(xì)胞分化,其可能的機(jī)制是B-ALL細(xì)胞通過Jagged1激活BMSCs中的Notch信號(hào)通路,其下游基因Hes1活化降低成骨分化轉(zhuǎn)錄因子Runx2蛋白表達(dá)水平,從而抑制BMSCs向成骨細(xì)胞分化。第三部分B-ALL細(xì)胞影響骨髓間充質(zhì)干細(xì)胞向內(nèi)皮細(xì)胞分化及其機(jī)制研究目的：研究B-ALL對(duì)骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)內(nèi)皮分化的影響,并探討在此過程中可能存在的機(jī)制。方法：采用B-ALL細(xì)胞與BMSCs體外共培養(yǎng)模型模擬體內(nèi)微環(huán)境,以BMSCs單獨(dú)培養(yǎng)及正常骨髓單個(gè)核細(xì)胞與BMSCs共培養(yǎng)作為對(duì)照。共培養(yǎng)三天與七天后,Real time PCR檢測(cè)各組內(nèi)皮分化標(biāo)志物Flk-1、CD31、vWFmRNA表達(dá)差異；Western-blot方法檢測(cè)各組內(nèi)皮分化特異標(biāo)志CD31、Flk-1蛋白表達(dá)差異；再通過共培養(yǎng)中加入中和抗體anti-Jagged1-Ab抑制BMSCs中Notch信號(hào)通路,驗(yàn)證B-ALL細(xì)胞是否通過Jaggedl激活BMSCs中Notch信號(hào)通路影響B(tài)MSCs向內(nèi)皮細(xì)胞分化。最后采用ELISA檢測(cè)各組培養(yǎng)上清中VEGF分泌蛋白表達(dá)水平,探討B(tài)-ALL細(xì)胞影響B(tài)MSCs向內(nèi)皮分化的機(jī)制。結(jié)果：1)B-ALL細(xì)胞與BMSCs共培養(yǎng)三天后,BMSCs內(nèi)皮分化標(biāo)志Flk-1、CD31、vWF mRNA表達(dá)水平及Flk-1、CD31蛋白表達(dá)水平較對(duì)照組升高,但無顯著差異(P0.05)；2) B-ALL細(xì)胞與BMSCs共培養(yǎng)七天后,BMSCs內(nèi)皮分化標(biāo)志Flk-1、CD31、vWF mRNA表達(dá)水平及Flk-1、CD31蛋白表達(dá)水平較對(duì)照組升高,有顯著差異(P0.05); 3) B-ALL細(xì)胞與BMSCs共培養(yǎng)后,Notch信號(hào)通路被激活；4)共培養(yǎng)組中加入anti-Jagged1-Ab中和抗體后,Notch信號(hào)通路被抑制,但Flk-1、CD31、vWF mRNA表達(dá)水平及Flk-1、CD31蛋白表達(dá)水平無明顯改變；5)采用ELISA檢測(cè)共培養(yǎng)組中VEGF分泌蛋白表達(dá)水平,發(fā)現(xiàn)共培養(yǎng)組中VEGF表達(dá)水平上調(diào),同時(shí)B-ALL細(xì)胞與BMSCs中VEGF表達(dá)水平相應(yīng)的升高,推測(cè)BMSCs內(nèi)皮分化機(jī)制可能與共培養(yǎng)環(huán)境中VEGF分泌蛋白水平升高相關(guān)。結(jié)論：B-ALL細(xì)胞可促進(jìn)BMSCs向內(nèi)皮細(xì)胞分化,其可能的機(jī)制是與B-ALL細(xì)胞與BMSCs共培養(yǎng)后促進(jìn)共培養(yǎng)環(huán)境中VEGF分泌蛋白水平升高有關(guān),而Notch信號(hào)通路在此過程并不起主要作用。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R733.7

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