【作者】 张小玲；

【导师】 胡群； 周木想；

【作者基本信息】 华中科技大学 ， 儿科学， 2010， 博士

【摘要】 目的：癌基因双微体2(Murine Double Minute 2, MDM2)在肿瘤发生、发展和化疗耐药性产生过程中发挥重要作用。本实验室前期的研究表明,组成性高表达MDM2的急性淋巴细胞白血病和神经母细胞瘤患者的预后往往较差。我们初期的研究显示,小檗碱能特异性下调肿瘤细胞MDM2表达。MDM2具有E3连接酶活性,当MDM2与DAXX和HAUSP处于结合状态时,作为E3连接酶的MDM2泛素化并降解肿瘤抑制子p53。而当MDM2与DAXX和HAUSP处于分离状态时,作为E3连接酶的MDM2泛素化并降解MDM2自身,从而激活p53。因此,本研究的目的：1.小檗碱对儿童肿瘤细胞增殖、凋亡的影响；2.小檗碱对儿童肿瘤细胞MDM2、p53表达影响及其分子机制；3.小檗碱是否具不同于传统化疗药物阿霉素抗肿瘤作用机制,即是否通过抑制DAXX下调癌基因MDM2表达。方法：1.选取20种儿童急性淋巴细胞白血病细胞系为研究对象,用WST方法检测不同浓度的小檗碱对20种ALL细胞系生长抑制作用,并比较分析小檗碱IC50与MDM2、p53表达状态之间的关系。2.选取对传统化疗药物阿霉素耐药的ALL细胞系EU-1、REH和神经母细胞瘤细胞NB-1691和NB-1643为模型。用Western-blot方法检测不同浓度和不同时间条件下小檗碱作用细胞后,MDM2、p53、Bcl-2等相关蛋白表达的变化。3.选取对传统化疗药物阿霉素耐药的ALL细胞系EU-1为模型。用RT-PCR方法检测小檗碱以相同浓度处理不同时间条件下细胞MDM2、p53、p21、PUMA、GAPDH等相关基因mRNA表达变化。4.选取对传统化疗药物阿霉素耐药的ALL细胞系EU-1为模型。用Western-blot方法检测小檗碱以不同浓度处理24h条件下EU-1细胞p53及其下游靶基因p21、PUMA、GAPDH等蛋白表达变化。5.CHX(内源性蛋白合成抑制剂)作用于细胞后,用Western-blot检测小檗碱作用于EU-1、UOC-B1细胞前后MDM2、p53、Bcl-2蛋白半衰期变化。6.选取对传统化疗药物阿霉素耐药的ALL细胞系EU-1为模型。用Western-blot方法检测小檗碱以不同浓度处理24h条件下EU-1细胞凋亡关键蛋白酶caspase 3及其底物PARP是否被剪切。7.Western-blot方法检测8种(其中包括4种ALL细胞和4种NB细胞)细胞系DAXX、MDM2、HAUSP蛋白组成性表达,小檗碱处理前后DAXX、MDM2、HAUSP蛋白变化；比较分析以上蛋白表达与小檗碱细胞增殖抑制率之间的相互关系。8.以NB-1691为模型,用小分子干扰RNA (DAXX-siRNA)转染EU-4细胞下调DAXX表达,Western-blot方法鉴定转染DAXX-siRNA效果,用Western-blot检测转染DAXX-siRNA前后MDM2、HAUSP表达变化。9.用免疫共沉淀方法检测不同浓度小檗碱处理EU-1细胞24h后MDM2泛素化水平。10.构建无E3泛素化酶活性的MDM2质粒C464A,wt-MDM2质粒,相同条件下将C464A,wt-MDM2和空载体转染EU-4细胞后,Western-blot检测小檗碱对转染后EU-4细胞MDM2表达的影响。11.Annexin V凋亡试剂盒检测小檗碱处理不同浓度和不同时间条件下细胞凋亡情况。结果：1.小檗碱抑制肿瘤细胞MDM2表达,该抑制作用与p53表达状态无关。2.小檗碱诱导wt-p53／MDM2肿瘤细胞p53激活。3.小檗碱诱导p53下游基因p21和PUMA激活。4.小檗碱并非在转录水下调MDM2基因表达。5.小檗碱促进MDM2蛋白的降解从而稳定p53功能。6. DAXX表达在小檗碱诱导的MDM2表达下调和细胞死亡过程中是必需的。7.小檗碱诱导的细胞死亡与ALL细胞MDM2表达正相关而与p53表达无关。8.小檗碱诱导的细胞死亡与MDM2表达相关。9.小檗碱显著诱导wt-p53／高表达MDM2急性淋巴细胞白血病ALL细胞凋亡。结论：小檗碱下调MDM2表达并显著诱导wt-p53/MDM2高表达的儿童肿瘤细胞凋亡。目的：探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)的四种受体TR1-R4在儿童急性白血病(AL)中的表达,及其与临床分型和预后的关系。方法：应用半定量逆转录聚合酶链反应(RT-PCR)测定22例急性白血病患儿和10例骨髓正常的非恶性血液病患儿TR1-R4 mRNA的表达情况。结果：AL组死亡受体DR4(TR1)和DR5(TR2) mRNA的表达明显高于正常对照组(P<0.01)；而AL组蒙骗受体DcR1(TR3)和DcR2 (TR4) mRNA的表达明显低于正常对照组(P<0.05)。AL组与正常对照组均表达DR5,但AL组内DR5表达相对量明显高于DR4,差异具有统计学意义(P<0.05)。AL患儿不同亚型之间TRAIL受体表达差异无统计学意义。结论：TRAIL受体TR1-R4在儿童急性白血病中表达有明显差异性,TRAIL及其受体在介导白血病细胞凋亡中起重要作用。目的：探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)联合小檗碱诱导Molt-4细胞凋亡作用及其对NF-κB/P65表达的影响。方法：采用MTT比色法测定TRAIL单独和联合应用小檗碱时对Molt-4细胞的生长抑制率；用流式细胞术和光镜细胞形态观察来检测凋亡；应用免疫印迹法检测单独和联合用药组细胞凋亡相关蛋白酶caspase3,caspase8及NF-κB/P65表达。结果：(1)MTT检测结果发现小檗碱可增加TRAIL对Molt-4细胞的生长抑制率,且呈时间和剂量依赖性(P<0.05)。(2)流式细胞术可以检测到凋亡,光镜细胞形态观察可见凋亡特异性形态改变。(3) Western blot结果显示：单独用TRAIL组及TRAIL联合小檗碱用药组caspase3, caspase8活化程度随TRAIL浓度依次增加,联合用药组活化作用更强；单独用TRAIL组NF-κB/P65表达随剂量增加而增加。联合小檗碱用药组NF-KB/P65表达与单独用TRAIL组相比则明显受抑制。结论：(1)小檗碱可协同TRAIL诱导Molt-4细胞凋亡。(2)小檗碱协同TRAIL诱导Molt-4细胞凋亡的分子机制涉及抑制NF-κB/P65表达和caspase3, caspase8剪切活化。更多还原

【Abstract】 Objective:The oncoprotein MDM2 plays critical roles in cancer initiation, progression and the development of resistance to therapy. We studied pediatric cancer patients, including those with acute lymphoblastic leukemia (ALL) and neuroblastoma (NB), finding that patients with a poor prognosis commonly had cancer cells that expressed constitutively highlevels of MDM2. Our preliminary data supports a previously unrecognized mechanism of action for Berberine:It specifically downregulates MDM2 in cancer cells. An important function of MDM2 is the ubiquitination (as an E3 ligase) and degradationof the tumor suppressor p53, due to interactions with DAXX and HAUSP. Interestingly, disruption of these interactions can result in self-ubiquitination of MDM2 and p53 activation. So, we want to elucidate the following questions:1. Effect of berberine on the proliferation and apoptosis of pediatric tumor cells.2. Effect of berberine on the MDM2、p53 expression of pediatric tumor cells and its underlying mechanism.3. To test the hypothesis that regulation of MDM2 by DAXX inhibition is the major mechanism by which Berberine differs from Dox in exerting its anti-cancer effect. Methods:1.20 ALL cell lines were selected as study model. Proliferation inhibition of berberine on those cell lines were determined by WST assay and calculate IC50 of each cell line. Analyze the relationship between IC50 and the expression of MDM2、p53 on each cell line.2. The neuroblastoma cell lines(NB-1691 and NB-1643) and the conventional chemotherapeutic drug doxorubicin-resistant acute lymphoblastic leukemia (ALL) cell lines (EU-1、REH) were selected as study model. Western-blot was used to detect the protein expression changes of MDM2、p53、Bcl-2 of each cell line in different dose or time treatment of berberine.3. The conventional chemotherapeutic drug doxorubicin-resistant ALL cell line EU-1 was selected as study model. RT-PCR was used to detect the mRNA expression changes of MDM2、p53、p21、PUMA、GAPDH of of EU-1 cells treated by constant concentration of berberine for various time.4. The conventional chemotherapeutic drug doxorubicin-resistant ALL cell line EU-1 was selected as study model. Western-blot was used to detect the protein expression changes of p53 downstream targets:p21 and PUMA of EU-1 cells treated by various concentration of berberine for 24h.5. The protein synthesis inhibitor cycloheximide (CHX) were utilized to treat cells. Western-blot was used to detect the protein half-life changes of MDM2、p53、Bcl-2 of EU-1、UOC-B1 cells treated by berberine.6. The conventional chemotherapeutic drug doxorubicin-resistant ALL cell line EU-1 was selected as study model. Western-blot was used to check wheatear the apoptosis associated protein caspase 3 and its substrate PARP of EU-1 cells were cleaved after berberine treatment.7. Western-blot was used to detect the constitutional expression of DAXX、MDM2 and HAUSP in 8 cell lines including 4 ALL cell lines and 4 NB cell lines. Western-blot was used to detect the protein changes of DAXX and MDM2 in EU-1 cells treated by various concentration of berberine for 24h. Analyze the relationships between DAXX、MDM2 and HAUSP expression and the proliferation inhibition of berberine on each cell line.8. The NB-1691 cell line was selected as study model. DAXX-siRNA was transfected into NB-1691 cells to knock down DAXX expression. Western-blot was used to detect the changes of DAXX、MDM2 and HAUSP expression after DAXX-siRNA transfection.9. Co-immunoprecipitation was used to detect the ubiquitination changes of MDM2 of EU-1 cells treated by various concentration of berberine for 24h.10. Construct MDM2 without ubiquitin E3 ligase activity plasmid C464A and transfect the C464A plasmid, wt-MDM2 plasmid and the empty vector in to EU-4 cells.western-blot was used to detect the MDM2 expression of those transfected cells followed by berberine treatment.11. Annexin V apoptosis detection kit was used to detect the apoptosis of tumor cells in different dose or time treatment of berberine.Results:1. BBR inhibits MDM2 expression, regardless of p53 status in different ALL cells2. Induction of p53 by BBR in wt-p53/MDM2-expressing cells3. BBR activates p53 function by inducing its transcriptional targets4. MDM2 transcription is not repressed by BBR5. BBR promotes degradation of the MDM2 protein and p53 stabilization6. The expression of DAXX is absolutely required for BBR to down-regulate MDM2 and induce cell death7. Cytotoxicity of BBR is positively associated with MDM2 expression level in ALL cell lines, regardless of their p53 status.8. BBR-induced cytotoxicity associated with MDM2 expression9. BBR strongly induces apoptosis in ALL cells with wt-p53 and MDM2 overexpression Conclusoins:Berberine downregulates MDM2, inducing potent apoptosis of human cancer cells with wt-p53/MDM2 overexpression1. Expression of TRAIL receptors and its significance in children with acute leukemiaObjective:To explore the expression of TRAIL receptors (TR1-R4) and its significance in clinic classification and prognosis of children with acute leukemia.Method:Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TRAIL receptors on leukemic cells of 22 children with acute leukemia.10 cases of children with no-malignant blood diseases were served as control group.Results:The mRNA expression of DR4 and DR5 in AL group was dramatically higher than that in control group (p<0.05). However, the mRNA expression of DcR1 and DcR2 in AL group was significantly lower than that in control group (p<0.05). DR5 was expressed in both AL group and control group, but its mRNA relative amount was markedly higher than that of DR4 in AL group. No significant differences of TR1-R4 expressions were observed in different clinical sub-types.Conclusion:There are significant differences between TRAIL receptors TR1-R4 in children with acute leukemia. TRAIL and its receptors play an important role in TRAIL and its receptors mediated apoptosis of leukemic cells. 2. Berberine Augments TRAIL-induced Apoptosis of Leukemic CellsObjective:To investigate the apoptosis-inducing effect of TRAIL(TNF Related Apoptosis-Inducing Ligand) and berberine on the leukemic cell line Molt-4 and the relationship between the NF-κB/P65 expression and the synergistic effect.Method:MTT assays were used to measure the cytotoxic effects of TRAIL used alone or combined with the Chinese herb berberine. The apoptosis was detected by flow cytometry and microscopy. Western blotting was used to detect the expressions of caspase3,caspase8 and NF-κB/P65 in different chemotherapeutic groups.Results:(1) The MTT assay suggested berberine augments TRAIL-induced apoptosis in Molt-4 cells in a dose and time dependent way(P<0.05).(2)The Apoptosis can be detected by flow cytomery; Morphological observation also showed the typical apoptotic changes.(3) Western blot showed that the activity of caspase3 and 8 were up regulated when treated with either TRAIL alone or TRAIL combined with berberine. However, in the combinational use of berberine the caspase3, caspase8 activity was up regulated much more than in the absence of berberine. When treated with TRAIL alone, the expression of NF-κB/P65 was increased in a dose and time-dependent way. When berberine was added (50mg/L), the NF-κB/P65 activation was suppressed.Conclusion:(1)TRAIL combined with berberine can induce apoptosis of Molt-4 cells in a synergistic effect.(2)The suppression of NF-κB/P65 expression, caspase3 and caspase8 activation were involved in the mechanism of the synergistic effect.更多还原