SALL4在儿童白血病中的作用及芹菜素对SALL4表达的影响

发布时间：2018-01-26 07:20

本文关键词： SALL4 急性白血病儿童危险度微小残留病芹菜素 U937 细胞增殖凋亡 SALL4　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：第一部分SALL4在儿童白血病中的表达及临床意义目的探讨人婆罗双树样基因4（SALL4）在儿童白血病中的表达及临床意义。方法采用real-time PCR和免疫细胞化学技术检测50例新诊儿童白血病，包括急性前B淋巴细胞白血病（Pre-B-ALL）24例，急性T淋巴细胞白血病(T-ALL)4例，急性髓细胞白血病（AML）22例及15例免疫性血小板减少性紫癜（immune thrombocytopenic purpura, ITP）患儿骨髓单个核细胞（BMMNC）SALL4mRNA及蛋白的表达量；动态观察5例白血病患儿新诊和完全缓解后SALL4mRNA变化；分析SALL4mRNA表达水平与临床特征的关系。结果 1. SALL4mRNA在新诊Pre-B-ALL、AML的表达量分别为13.89（1.00-63.15）、11.12（2.31-56.59），显著高于对照组1.00（0.29-1.71）（P0.01），T-ALL的表达量1.48（0.87-4.81）与对照组无显著差异。 2. SALL4蛋白在新诊前B-ALL、 AML、T-ALL的表达阳性率分别为83.33%（20/24）、86.36%（19/22）、0（0/4），在15例对照儿童中全部呈阴性表达。在Pre-B-ALL、 AML中的表达与对照组相比，差异有统计学意义（P0.01）。在T-ALL和对照组之间无显著差异（P0.05），与real-time PCR结果一致。 3.动态观察的5例白血病患儿完全缓解后SALL4的表达量0.98（0.22-1.09）较新诊时28.64（11.20-87.46）显著降低（P0.01）。 4. SALL4mRNA的高表达与外周血高白细胞计数、高危分型、诱导化疗末期MRD正相关（r=0.424、r=0.403、 r=0.393, P均0.05）；与发病年龄，性别，肝、脾、淋巴结肿大等因素无相关性（P均0.05）。结论 1. SALL4在新诊Pre-B-ALL、AML中存在高表达，完全缓解后表达量显著降低，可能促进了儿童Pre-B-ALL、 AML的发生发展。 2. SALL4在Pre-B-ALL、 AML中存在高表达，，在T-ALL中阴性表达，提示T-ALL和Pre-B-ALL、 AML有不同的发病机制。 3. SALL4高表达与外周血高白细胞计数、高危分型、诱导化疗末期MRD正相关，与发病年龄，性别，肝、脾、淋巴结肿大等因素不相关，有望成为监测治疗和判断预后的新指标。目的探讨芹菜素对U937细胞增殖、凋亡及SALL4表达的影响。方法以不同浓度芹菜素（0、20、40、60μmol/L）处理对数生长期的U937细胞，倒置显微镜观察细胞形态；CCK-8法检测细胞的增殖活力；流式细胞术检测细胞周期和凋亡； Real-time PCR检测SALL4、C-MYC、CCND1mRNA表达水平；Western blot检测SALL4、BCL-2、Caspase-3蛋白表达水平。结果与对照组相比，芹菜素处理组细胞碎片增多；细胞增殖受抑制，呈时间和剂量依赖性（P0.05）； G2/M期细胞比例增加（P0.05）；凋亡率增加（P0.05）。芹菜素处理U937细胞36h后，SALL4、C-MYC、CCND1mRNA表达下调，SALL4、BCL-2蛋白表达降低，Caspase-3蛋白表达增加，与对照组相比，差异有统计学意义（P0.05）。结论芹菜素对U937细胞有抑制增殖、诱导凋亡的作用，并能使U937细胞阻滞在G2/M期，其机制可能与下调转录因子SALL4，调控凋亡相关蛋白BCL-2、Caspase-3，抑制癌基因C-MYC、CCND1的表达有关。
[Abstract]:The expression of SALL4 in childhood leukemia and its clinical significance Objective to investigate the expression and clinical significance of double tree like gene 4 (SALL 4) in childhood leukemia. Methods real-time PCR and immunocytochemistry were used to detect 50 cases of newly diagnosed childhood leukemia, including 24 cases of Pre-B-ALL leukemia. There were 4 cases of T-ALL with acute T lymphocyte leukemia. Immune thrombocytopenic purpura was found in 22 cases of acute myeloid leukemia and 15 cases of immune thrombocytopenic purpura. The expression of BMMNCpSALL4 mRNA and protein in bone marrow mononuclear cells of children with ITP; The changes of SALL4mRNA in 5 children with leukemia after new diagnosis and complete remission were observed dynamically. To analyze the relationship between SALL4mRNA expression level and clinical features. Results 1. The expression of SALL4mRNA in newly diagnosed Pre-B-ALL SALL4mRNA was 13.89 ~ 1.00-63.15). 11.12A2.31-56.59, which was significantly higher than that of the control group (1.00 ~ 0.29-1.71, P 0.01). There was no significant difference in T-ALL expression between the control group and the control group. 2. The positive rates of SALL4 protein expression in B-ALL and AML-T-ALL were 83.33 / 20 / 24 / 86.36 / 22, respectively. 0 / 4%, all of the 15 control children were negative, and the expression of AML in Pre-BALL was higher than that in the control group. There was no significant difference between T-ALL and control group (P 0.05), which was consistent with the result of real-time PCR. 3. The expression of SALL4 in 5 children with leukemia after complete remission was 0.98 ~ 0.22-1.09), which was higher than that in newly diagnosed children (28.64 / 11.20-87.46). P0.01C was significantly decreased. 4. The high expression of SALL4mRNA was positively correlated with the high leukocyte count in peripheral blood, high risk typing, and the positive correlation between MRD and MRD 0.403 at the end of induced chemotherapy. R = 0.393, P = 0.05; There was no correlation with age, sex, liver, spleen and lymph node enlargement (P < 0.05). Conclusion 1. High expression of SALL4 was found in newly diagnosed Pre-B-ALLN AML, and the expression level decreased significantly after complete remission, which may promote the development of Pre-B-ALL in children. The occurrence and development of AML. 2. High expression of SALL4 in Pre-B-ALL and AML, negative expression in T-ALL, suggesting T-ALL and Pre-B-ALL. AML has different pathogenesis. 3. The high expression of SALL4 was positively correlated with peripheral blood leukocyte count, high risk typing, MRD at the end of induced chemotherapy, but not with age, sex, liver, spleen, lymphadenopathy and so on. It is expected to be a new indicator for monitoring treatment and judging prognosis. Objective to investigate the effects of apigenin on the proliferation, apoptosis and SALL4 expression of U937 cells. Methods U937 cells at logarithmic growth stage were treated with different concentrations of apigenin (40 渭 mol / L), and the morphology of U937 cells was observed by inverted microscope. The proliferative activity of the cells was detected by CCK-8 assay. Cell cycle and apoptosis were detected by flow cytometry. Real-time PCR was used to detect the expression of CCND1 mRNA in SALL4 and C-MYC1mRNA. Western blot was used to detect the expression of Caspase-3 protein in SALL4 and BCL-2. Results compared with the control group, the number of cell fragments increased in apigenin treated group. Cell proliferation was inhibited in a time-and dose-dependent manner (P 0.05). The proportion of G _ 2 / M phase cells increased (P 0.05). The apoptotic rate was increased in U937 cells treated with apigenin for 36 hours, and the expression of SALL4 C-MYCfCCND1 mRNA was down-regulated in U937 cells. The expression of BCL-2 protein decreased and the expression of Caspase-3 increased, compared with the control group, the difference was statistically significant (P 0.05). Conclusion apigenin can inhibit the proliferation and induce apoptosis of U937 cells and block U937 cells in G _ 2 / M phase. The mechanism may be related to the down-regulation of transcription factor SALL4. The regulation of apoptosis-related protein BCL-2 and Caspase-3 inhibits the expression of oncogene C-MYCnD1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R733.7

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