本文關(guān)鍵詞： SALL4 急性白血病 兒童 危險(xiǎn)度 微小殘留病 芹菜素 U937 細(xì)胞 增殖 凋亡 SALL4　出處：《重慶醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分SALL4在兒童白血病中的表達(dá)及臨床意義 目的探討人婆羅雙樹樣基因4（SALL4）在兒童白血病中的表達(dá)及臨床意義。 方法采用real-time PCR和免疫細(xì)胞化學(xué)技術(shù)檢測(cè)50例新診兒童白血病，包括急性前B淋巴細(xì)胞白血病（Pre-B-ALL）24例，急性T淋巴細(xì)胞白血病(T-ALL)4例，急性髓細(xì)胞白血病（AML）22例及15例免疫性血小板減少性紫癜（immune thrombocytopenic purpura, ITP）患兒骨髓單個(gè)核細(xì)胞（BMMNC）SALL4mRNA及蛋白的表達(dá)量；動(dòng)態(tài)觀察5例白血病患兒新診和完全緩解后SALL4mRNA變化；分析SALL4mRNA表達(dá)水平與臨床特征的關(guān)系。 結(jié)果 1. SALL4mRNA在新診Pre-B-ALL、AML的表達(dá)量分別為13.89（1.00-63.15）、11.12（2.31-56.59），顯著高于對(duì)照組1.00（0.29-1.71）（P0.01），T-ALL的表達(dá)量1.48（0.87-4.81）與對(duì)照組無(wú)顯著差異。 2. SALL4蛋白在新診前B-ALL、 AML、T-ALL的表達(dá)陽(yáng)性率分別為83.33%（20/24）、86.36%（19/22）、0（0/4），在15例對(duì)照兒童中全部呈陰性表達(dá)。在Pre-B-ALL、 AML中的表達(dá)與對(duì)照組相比，差異有統(tǒng)計(jì)學(xué)意義（P0.01）。在T-ALL和對(duì)照組之間無(wú)顯著差異（P0.05），與real-time PCR結(jié)果一致。 3.動(dòng)態(tài)觀察的5例白血病患兒完全緩解后SALL4的表達(dá)量0.98（0.22-1.09）較新診時(shí)28.64（11.20-87.46）顯著降低（P0.01）。 4. SALL4mRNA的高表達(dá)與外周血高白細(xì)胞計(jì)數(shù)、高危分型、誘導(dǎo)化療末期MRD正相關(guān)（r=0.424、r=0.403、 r=0.393, P均0.05）；與發(fā)病年齡，性別，肝、脾、淋巴結(jié)腫大等因素?zé)o相關(guān)性（P均0.05）。 結(jié)論 1. SALL4在新診Pre-B-ALL、AML中存在高表達(dá)，完全緩解后表達(dá)量顯著降低，可能促進(jìn)了兒童Pre-B-ALL、 AML的發(fā)生發(fā)展。 2. SALL4在Pre-B-ALL、 AML中存在高表達(dá)，，在T-ALL中陰性表達(dá)，提示T-ALL和Pre-B-ALL、 AML有不同的發(fā)病機(jī)制。 3. SALL4高表達(dá)與外周血高白細(xì)胞計(jì)數(shù)、高危分型、誘導(dǎo)化療末期MRD正相關(guān)，與發(fā)病年齡，性別，肝、脾、淋巴結(jié)腫大等因素不相關(guān)，有望成為監(jiān)測(cè)治療和判斷預(yù)后的新指標(biāo)。 目的探討芹菜素對(duì)U937細(xì)胞增殖、凋亡及SALL4表達(dá)的影響。 方法以不同濃度芹菜素（0、20、40、60μmol/L）處理對(duì)數(shù)生長(zhǎng)期的U937細(xì)胞，倒置顯微鏡觀察細(xì)胞形態(tài)；CCK-8法檢測(cè)細(xì)胞的增殖活力；流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期和凋亡； Real-time PCR檢測(cè)SALL4、C-MYC、CCND1mRNA表達(dá)水平；Western blot檢測(cè)SALL4、BCL-2、Caspase-3蛋白表達(dá)水平。 結(jié)果與對(duì)照組相比，芹菜素處理組細(xì)胞碎片增多；細(xì)胞增殖受抑制，呈時(shí)間和劑量依賴性（P0.05）； G2/M期細(xì)胞比例增加（P0.05）；凋亡率增加（P0.05）。芹菜素處理U937細(xì)胞36h后，SALL4、C-MYC、CCND1mRNA表達(dá)下調(diào)，SALL4、BCL-2蛋白表達(dá)降低，Caspase-3蛋白表達(dá)增加，與對(duì)照組相比，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論芹菜素對(duì)U937細(xì)胞有抑制增殖、誘導(dǎo)凋亡的作用，并能使U937細(xì)胞阻滯在G2/M期，其機(jī)制可能與下調(diào)轉(zhuǎn)錄因子SALL4，調(diào)控凋亡相關(guān)蛋白BCL-2、Caspase-3，抑制癌基因C-MYC、CCND1的表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R733.7

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