【作者】 郭冬梅；

【导师】 纪春岩；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 第一部分siRNA介导的Notchl下调对T-ALL细胞增殖凋亡影响研究背景:T细胞急性淋巴细胞白血病（T-ALL）是一类严重威胁人类健康的血液系统恶性疾患,在儿童及成人发病率较高。目前主要采取联合化疗来治疗白血病,但患者长期生存率仍较低,尤其是在成人T-ALL亟需完善治疗措施以改善预后。白血病的发生是由于癌基因的激活,抑癌基因的失活,导致细胞异常增殖、分化障碍和凋亡受阻。涉及T-ALL发病的分子机制非常复杂。Notch信号与肿瘤的关系最早发现于急性T淋巴细胞白血病,t（7;9）（q34;q34.3）染色体易位使9号染色体上Notch 1基因的胞内段编码区与7号染色体上T细胞受体（T cell receptor,TCR）β基因的增强子和启动子区融合,导致Notch信号组成型激活,引起了肿瘤的发生。最近有学者报道,约50%T-ALL病人存在Notchl激活的突变,说明Notchl的异常激活是T-ALL的重要发病机制。γ分泌酶抑制剂（gamma secretase inhibitor,GSI）能够有效抑制Notchl的激活,但近期多数研究报道GSI只对人或鼠的一小部分细胞株起作用。此外,GSI作用不专一,可以阻断所有Notch受体并影响其他涉及增殖的蛋白。因此,我们利用RNA干扰技术特异性下调Notch表达深入探讨Notchl在T-ALL的作用及机制。研究目的:研究Notchl信号对T-ALL细胞增殖、凋亡和周期等生物学行为的影响,以揭示T-ALL发病机制并为白血病的靶向治疗开辟新的途径。研究方法:1.体外培养T-ALL细胞系SupT1,Jurkat。Yrizo1方法提取组织总RNA,在一定的反应体系中逆转录成cDNA,PCR方法检测Notchl,HES-1在mRNA水平的表达情况。2.免疫细胞化学染色:体外培养T-ALL细胞系SupT1和Jurkat,细胞固定后用Notch1抗体对标本进行免疫染色,以检测Notch1在T-ALL细胞中的表达情况。3.siRNA片段的转染:取指数生长期细胞消化计数后按8.16×10⁵/ml接种于24孔细胞培养板中,后应用Lipofectamine2000将针对Notch1的siRNA片段转染T-ALL细胞SupT1和Jurkat。72小时后real-time PCR方法检测siRNA片段对Notch1 mRNA表达的阻断效率。4.MTT方法检测siRNA片段对T-ALL细胞增殖的影响:取指数生长期细胞消化计数后按5×10³个/孔接种于96孔细胞培养板中,后转染siRNA片段。转染后24、48、72小时MTT方法检测小干扰片段对细胞增殖的影响。5.流式细胞术检测siRNA片段对细胞周期的影响:取指数生长期细胞接种于细胞培养板中,后转染siRNA片段,转染后72小时收集细胞,PI染色后利用流式细胞仪检测细胞周期的变化。6.流式细胞术检测siRNA片段对细胞凋亡率的影响:取指数生长期细胞接种于细胞培养板中,转染siRNA片段,转染后72小时收集细胞,AnnexinV/PI双染后利用流式细胞仪检测细胞凋亡率的变化。7.MTT方法检测siRNA片段对T-ALL细胞化疗药物敏感性的影响:取指数生长期细胞接种于细胞培养板中,24小时后转染siRNA片段。转染后24小时加入不同浓度的化疗药物阿霉素孵育48小时,MTT检测细胞增殖情况。8.Western blot检测通路相关蛋白表达情况:取指数生长期细胞接种于10cm培养皿,24小时后转染siRNA片段,转染后72小时收集细胞,提取细胞总蛋白,Western blot检测通路相关蛋白的表达情况。研究结果:1.PCR结果显示Notch1和HES-1均在T-ALL细胞系SupT1和Jurkat中表达。2.免疫细胞化学染色方法结果显示两种细胞均存在Notch1蛋白的表达且存在不同定位。3.针对Notch1的siRNA片段可以有效下调Notch1的表达:siRNA转染后72小时,SupT1和Jurkat细胞干扰组Notch1 mRNA的表达较对照组分别下降91.8%±4.56%和90.2%±7.84%（P＜0.05）。SupT1和Jurkat细胞干扰组Notch1蛋白亦出现明显的下调。4.MTT检测发现在48、72及96 h,siRNA介导的Notch1的下调明显的抑制SupT1细胞的增殖（P＜0.05）,而Jurkat细胞的增殖没有受到明显的影响（P＞0.05）。5.SupT1转染siRNA后72小时,流式细胞仪检测细胞周期分布,干扰组为:G₀/G₁:62.85%,而对照组细胞的周期分布为:G₀/G₁:46.17%,与对照组比较G₀/G₁细胞增多,下调Notchl的表达能够导致SupT1的细胞周期阻滞于G₀/G₁期,而对Jurkat细胞的周期无明显作用。6.SupT1细胞转染siRNA后72小时,AnnexinV/PI双染后流式细胞仪检测细胞凋亡率,干扰组早期、晚期凋亡率分别为9.01%、12.41%,对照组凋亡率为4.13%、4.84%;Jurkat转染siRNA后72小时,干扰组凋亡率与对照组无明显改变。下调Notch1的表达能够显著增加SupT1细胞的凋亡率。7.SupT1和Jurkat细胞转染后24小时,加入阿霉素作用48小时。结果显示SupT1细胞干扰组与对照组阿霉素的IC50值分别为0.05μg/ml和0.21μg/ml（P＜0.05）。SupT1细胞干扰组与对照组相比阿霉素诱导的凋亡率增加了2.4倍。Jurkat细胞干扰组与对照组相比无明显差异（P＞0.05）。8.SupT1细胞干扰组cyclin D1及CDK2蛋白出现明显的下调,p21蛋白的表达则明显增高。Jurkat细胞干扰组cyclin D1及CDK2蛋白未出现明显的改变,p21蛋白的表达略增高。SupT1细胞干扰组p-Akt蛋白明显降低,而Jurkat细胞干扰组与对照组相比无明显改变。结论:1.siRNA可以有效阻断Notch1的表达。2.siRNA介导的Notch1表达下调能够引起细胞周期阻滞和细胞凋亡率增加,从而抑制T-ALL细胞的增殖。3.siRNA介导的Notch1表达下调能够增加T-ALL细胞对阿霉素的药物敏感性。4.siRNA介导的Notch1表达下调增加细胞凋亡与药敏的机制可能与抑制Akt生存通路相关。5.Notch1与Akt通路存在相互联系,Notch信号调节Akt信号。第二部分Notch1信号在基质细胞介导耐药中的作用研究背景:化疗耐药问题在治疗ALL和其他血液系统恶性疾患过程中日趋严重。近来,越来越多的证据表明肿瘤细胞与其微环境的相互作用导致了白血病和淋巴瘤的化疗耐药。然而,骨髓基质细胞（Bone marrow stromal cells,BMSCs）介导的肿瘤化疗耐药机制仍然不清楚。为了阐述这个问题,我们聚焦于研究Notch受体家族,其在BMSCs及造血细胞广泛表达,并起重要作用。Notch信号传导通路是在进化过程中高度保守的信号传导系统,由Notch受体和配体组成。Notch基因编码4种跨膜蛋白分子,分别为Notch1、Notch2、Notch3和Notch 4,作为受体与相应配体（Delta-like 1、Delta-like 3、Delta-like 4、Jagged 1和Jagged 2）结合。位于细胞膜表面的Notch受体与相应配体结合后,引发分别由TACE（TNF-α-converting enzyme）和γ-secretase/presenilin complex介导的水解过程,释放其活性片段Notch-IC。Notch-IC转至细胞核内,与DNA结合蛋白CBF1/Su（H）/Lag1（CSF）结合后,激活其下游靶基因HES、Hey等的转录。有研究结果显示Notch1在成熟细胞抑制T细胞受体诱导的凋亡,在胸腺细胞抑制地塞米松诱导的凋亡,并且在T细胞起到抗凋亡的作用。愈来愈多的证据表明存在Notch1激活的肿瘤对化疗耐药。研究目的:研究骨髓基质细胞对T-ALL细胞药物诱导凋亡的影响;探讨Notch1信号在基质细胞介导的耐药中的作用,以揭示T-ALL耐药机制并为白血病的耐药逆转开辟新的途径。研究方法:通过Ficoll分离法分离骨髓单个核细胞,体外培养扩增出BMSCs;应用脂质体介导的方法将siRNA转入T-ALL细胞株Jurkat细胞,后与间充质细胞共培养;免疫磁珠分选共培养后的白血病细胞;RQ-PCR法检测基因的表达;流式细胞术检测细胞凋亡;Western blot检测其蛋白的表达情况。研究结果:1.骨髓基质细胞保护Jurkat细胞药物诱导的凋亡。Jurkat细胞与基质细胞共培养,然后加入细胞毒药物,检测凋亡。共培养的Jurkat细胞与悬浮细胞培养组加入1μM地塞米松凋亡率分别为17.30%,67.91%;加入阿霉素凋亡率分别为9.46%,22.19%。2.基质细胞表达Notch1配体,Jurkat细胞中的Notch1信号共培养后进一步激活。在共培养后检测Notch1与下游靶基因HES-1蛋白的表达。Jurkat细胞悬浮培养或与基质细胞共培养48h,共培养的Jurkat细胞与悬浮培养组比较Notch1激活形式蛋白Notch1-ICD及HES-1蛋白表达增多。3.Western blot检测发现共培养的Jurkat细胞与悬浮培养组比较磷酸化Akt蛋白表达上升。4.Notch1 siRNA有效下调了共培养Jurkat细胞Notch1的表达。Notch1 siRNA的效率由实时定量PCR及Western blot判定。Notchl的蛋白表达几乎被NotchlsiRNA完全敲除。HES-1蛋白的表达亦明显降低。实时定量PCR亦在mRNA水平证实Notch1 siRNA有效下调Jurkat细胞Notch1和HES-1基因的表达。5.siRNA转染Jurkat细胞,后与基质细胞共培养,24h后加入地塞米松或阿霉素进行凋亡检测。凋亡结果显示Notch1-IC siRNA转染共培养的Jurkat细胞与siRNA对照片段转染组比较增加化疗药物地塞米松或阿霉素诱导的细胞凋亡。6.Western blot检测结果显示干扰组磷酸化Akt蛋白表达下调。结论:1.骨髓基质细胞保护Jurkat细胞药物诱导的凋亡。2.白血病细胞与基质细胞的相互作用激活Notch1-HES和Akt通路。3.下调Notch1恢复共培养Jurkat细胞的药物敏感性,其机制涉及Akt信号通路的抑制。更多还原

【Abstract】 Section I The effects of down-regulation of Notch1 mediated by siRNA on the proliferation and apoptosis of T-ALL cellsBackground:T-cell acute lymphoblastic leukemia （T-ALL） ranks a serious type of hematological malignancies in patients, constituting a substantial fraction of ALL, both in children and in adults. Current treatment is primarily based on combination chemotherapy with low long-term survival rate particularly in adult patients, emphasizing the need for improved therapy. A large body of evidence has been accumulated showing that oncogene activation and antioncogene inactivation can lead uncontrolled proliferation, defective differentiation and abnormal apoptosis, and then leukemias develop. The molecular mechanisms underpinning T-ALL are likely to be complex.The involvement of Notch1 was first observed in a rare t（7;9）（q34;q34.3） translocation, which brings an activated form of the Notch-1 receptor gene under the control element of the T-cell receptor gene. More recently, it was shown that more than 50% of all T-ALL patients carried Notch1 gain-of-function mutations that generate an activated form of Notch. Mutant Notch1 could represent an important new target for therapy of T-ALL patients. Since the generation of activated Notch1 can be inhibited by gamma-secretase inhibitors （GSIs）, GSIs are valuable tools for delineating the cell biological function of the Notch cascade, but the efficacy of this strategy has been questioned, as GSIs seem to be active in only a small fraction of human and mouse T-ALL cell lines with constitutive Notch1 activity. GSI are not specific for Notch1 and turn off all four Notch receptors. Furthermore, gamma-secretase might affect other proteins involved in proliferation. Therefore, it is important to explore the role of Notch1 in T-ALL using RNA interference technology.Objective:To investigate Notch1 on the effects of biological behavior of T-ALL cell proliferation, apoptosis and cell cycle, so as to reveal the pathogenesis of T-ALL and provide thebasis for molecular targeted therapy.Materials and Methods:1. Cell culture. Human T-ALL cell lines SupT1, Jurkat were cultured in RPMI medium 1640. Total RNA was extracted by Trizol according to the manufacturer’s instructions. Synthesis of first-strand cDNA was carried out with Revert Aid? First Strand cDNA Synthesis Kit. RT-PCR was used to detect the expression of Notchl,HES-1 mRNA.2. Immunocytochemical stain. SupT1 and Jurkat cells on cover slides were fixed in 95% ethanol, and then the expression of Notch1 protein was detected using Notch1 antibody according to the manufacturer’s instructions.3. SiRNA transfection. 8-16×10⁵/ml of SupT1 and Jurkat cells were seeded in 24-well plates. And then cells were treated with 100nM Notch1 siRNA/control siRNA. Seventy-two hours after siRNA transfection, cells were used for MTT, real-time PCR, western blot, and other experiments.4. Cell growth inhibition by MTT assay. SupT1 and Jurkat cells were incubated at a density of 5,000 cells/well in 96-well plates, and subsequently transfected with Notch1 siRNA or control siRNA. 24, 48, 72, 96 hours after tansfection, 20μl of MTT was added each well. 4 additional hours later, color development was measured on a microplate reader at 570 nm.5. Flow cytometry and cell cycle analysis. Notch1 siRNA, or control siRNA transfected SupT1, Jurkat cells were collected and stained with PI. DNA content was detected on a FACS Calibur.6. Apoptosis Assay. 72 hours after transfection, Notch1 siRNA/control siRNA transfected SupT1 and Jurkat cells were collected and were labeled with annexin V-biotin followed by PI. Annexin V/PI were measured by FACS Calibur and analyzed with the Modfit Software.7. For the chemotherapeutic drug assays, 24 hours after transfection, cells were exposed to different concentration of adriamycin respectively for 48 hours, and then cells were collected and analyzed as above.8. Western Blot Analysis. Total protein was extracted from transfected cells with Notch1 siRNA/control siRNA cells. Total proteins were fractionated using SDS-PAGE and transferred onto nitrocellulose membrane. After incubation with blocking buffer, the membrane was incubated with primary and secondary antibodies. The protein bands were detected using the enhanced chemiluminesence detection system.Results:1. Notch1 and HES-1 mRNAs were all expressed in human T-ALL cell lines SupT1 and Jurkat.2. Immunocytochemical stain results show that Notch1 protein is expressed and located at different places in human T-ALL cell lines SupT1 and Jurkat.3. Notch1 siRNA effectively down-regulated the expression level of Notch1 in human T-ALL cells. Notch1 and HES1 mRNAs were decreased by more than 90% in both SupT1 and Jurkat cells. Protein levels were also greatly reduced in Notch1 siRNA transfected cells compared with control siRNA transfected cells.4. Down-regulation of Notch1 expression by siRNA inhibited the proliferation of human T-ALL cells. Cell viability was determined by the MTT assay after transfection with Notch1 siRNA or control siRNA. Down-regulation of Notch1 expression by siRNA markedly inhibited cell proliferation in SupT1 cells at 48, 72 and 96 h （P<0.05）, whereas no apparent changes in Jurkat cells occurred （P> 0.05）.5. Down-regulation of Notch1 expression by siRNA induced G₀/G₁ phase cell cycle arrest in SupT1 cells. The Notch1 siRNA transfected SupT1 cells demonstrated a phase arrest pattern （62.85% vs. 46.17%） at 72 hours after transfection as compared with control cells. No alteration in cell cycle distribution was observed in Jurkat cells.6. Down-regulation of Notch1 expression by siRNA led to apoptosis in human T-ALL cells. 72 hours after transfection, cells were stained with Annexin V/PI, and analyzed by flow cytometry. The early and late apoptosis rates were 9.01% and 12.41% in Notch1 siRNA-transfected SupT1 cells, compared with 4.13% and 4.84% in control siRNA-transfected group respectively. There was no apparent change in early and late apoptosis of Jurkat cells.7. Down-regulation of Notch1 expression by siRNA increased chemosensitivity. We have used siRNA to specifically knock down the expression of Notch1 in SupT1 and Jurkat cells, and then examined cell viability and drug-induced apoptosis. Results showed that the IC50 value of adriamycin in SupT1 cells transfected with control siRNA was 0.21 （μg/ml） and SupT1 cells transfected with Notch1 siRNA was more sensitive, with an IC₅₀ value of 0.05 （μg/ml）. The combination of Notch1 siRNA and adriamycin group increased the apoptosis rate 2.4 fold in comparison with the combination of control siRNA and adriamycin group in SupT1 cells. However, there was no significant difference in Jurkat cells between the combination of Notch1 siRNA and adriamycin group and the combination of control siRNA and adriamycin group.8. Down-regulation of Notch1 expression affected the levels of cyclin proteins and p-Akt protein. Results showed that p-Akt, cyclin D1 and CDK2 protein expressions were decreased while p21 protein expression was increased in Notch1 siRNA-transfected SupT1 cells. However, there was no significant change of the expressions of p-Akt, cyclin D1 and CDK2 proteins, and p21 protein expression was slightly increased in Notch1 siRNA-transfected Jurkat cells.Conclusion:1. Notch1 siRNA effectively down-regulated the expression level of Notch1.2. Down-regulation of Notch1 mediated by siRNA can inhibit the proliferation of T-ALL cells by increasing apoptosis and inducing cell cycle arrest. 3. Down-regulation of Notch1 mediated by siRNA can increase T-ALL cells chemosensitivity to adriamycin.4. These effects may be through the repressing of Akt pathway.5. Notch pathway interacts with Akt pathway and Notch signaling can regulate Akt signaling.Section II The role of Notch1 in BMSCs mediated drug resistance of T-ALL cellsBackground:Resistance to chemotherapy is one of the most serious problems in treating patients with ALL and other hematologic malignancies. Recently, increasing evidence suggests that the interaction between tumor cells and elements of their microenvironment results in resistance to chemotherapy in leukemia and myeloma. However, the molecular mechanisms of BMSCs-mediated drug resistance of tumor cells are poorly understood.To address this question, we have focused on the Notch family of receptors and transcriptional regulators that are critically important for interaction between BMSCs and hematopoietic cells. Notch signaling is a pathway highly conserved through evolution which regulates various physiological processes, including stem cell maintenance, differentiation, proliferation and apoptosis. In mammals, key components of the Notch pathway include four transmembrane receptors （Notch 1-4） and five ligands （Delta-like 1, Delta-like 3, Delta-like 4, Jagged 1, Jagged 2）. Direct binding of a ligand from a signaling cell to a Notch receptor on the membrane of the receiving cell initiates two successive proteolytic cleavages by TACE （TNF-α-converting enzyme） and theγ-secretase/presenilin complex, which ultimately results in the release of the intracellular domain （Notch-IC）. Notch-IC then translocates into the nucleus and directly interacts with the DNA binding protein CBFl/Su（H）/Lagl（CSF） that activates the transcription of target genes including the HES （hairy/enhancer-of-split） and hey.Notch1 has been shown to inhibit T-cell receptor-induced apoptosis in mature cells and dexamethasone-mediated apoptosis in thymocytes and to be antiapoptotic in T cells. More and more evidence suggests cancers with activated Notch1 signal are chemo-resistant. We suggest that Notch1 signaling may play an important role in the drug resistance mediated by stromal cells.Objective:To investigate BMSCs on the effect of drug-induced apoptosis of T-ALL cells and explore the role of Notch1 signaling in the drug resistance mediated by stromal cells, so as to reveal the pathogenesis of T-ALL and provide the basis for molecular targeted therapy for drug resistance.Materials and Methods:BMSCs were isolated and culture-expanded from human bone marrow; Jurkat cells were transfected by siRNA using the Lipofectamine 2000 reagent, and then co-cultured with BMSCs; co-cultured Jurkat cells were selected using CD3+ magnetic micro beads for real-time reverse transcription-PCR, apoptosis analysis and Western blot; real time quantitative polymerase chain reaction （RQ-PCR） was used to detect the mRNA; Flow cytometry was used to detect apoptosis; Western blot were applied to detect the protein expression.Results:1. BMSCs protect drug-induced apoptosis in Jurkat cells. Jurkat cells were co-cultured with BMSCs followed by cytotoxic drugs. Jurkat cells cocultured with BMSCs were resistant to dexamethasone and adriamycin treatment. Treatment of Jurkat cells with 1μM dexamethasone induced 67.91% apoptosis of cells maintained in suspension, compared with a value of 17.30% of cells co-cultured with BMSCs. Similarly, Jurkat cells contact with BMSCs followed by adriamycin also underwent less apoptosis than those in suspension （9.46% vs. 22.19%）.2. Notch1 ligands are detected in BMSCs and Notch-1 signaling is further activated in Jurkat cells following contact with BMSCs. The protein expression of Notch-1 and its target gene HES-1 were examined. Jurkat cells were incubated for 48 hours in suspension or on the monolayer of BMSCs. Notch1-ICD （the activated form of Notch1） and HES-1 protein were increased in Jurkat cells with BMSCs compared with those in suspension.3. P-Akt is up-regulated in Jurkat cells following contact with BMSCs. Western blot analysis with antibodies specific for phosphorylated Akt was performed. Level of phosphorylated Akt was elevated in co-cultured Jurkat cells compared with that in suspension.4. Notch1 siRNA effectively down-regulates the expression level of Notch1 in Jurkat. The efficiency of Notch1 siRNA for knockdown of Notch1 mRNA and protein was confirmed by real time RT-PCR and Western blot. Notch1 protein level was significantly reduced and almost completely knocked down by Notch1 siRNA compared with control siRNA-transfected cells in Jurkat cells. And Knock-down of Notch1 in Jurkat cells resulted in decreased protein levels of Notch target gene HES-1. Knock-down of Notch1 and HES-1 in Jurkat cells with siRNA paralleled the reduced amount of specific mRNA, as demonstrated by real time RT-PCR amplification5. Down-regulation of Notch1 enhances the effect of chemotherapy on co-cultured Jurkat cells. Notch1 was down-regulated by siRNA in Jurkat cells, and then drug-induced apoptosis of co-cultured Jurkat cells was examined. Control siRNA-or Notch1 siRNA-transfected Jurkat cells following contact with BMSCs were exposed to dexamethasone or adriamycin as described, drug-induced apoptotic rates was detected. Notch1 siRNA-transfected Jurkat cells following contact with BMSCs were significantly susceptible to apoptosis induced by dexamethasone or adriamycin compared with control siRNA-transfected Jurkat cells following contact with BMSCs6. Down-regulation of Notch1 affects the level of p-Akt protein. p-Akt protein was examined by Western blot analysis. When Notch1 was down-regulated in Jurkat cells following contact with BMSCs, the expression of p-Akt was decreased. Conclusion:1. BMSCs protect drug-induced apoptosis in Jurkat cells2. Interactions of leukemic and BMSCs result in activation of Notch1-HES and Akt signaling in leukemic cells.3. Down-regulation of Notch1 restores of sensitivity to chemotherapy of co-cultured Jurkat cells. The possible mechanisms of restoration of sensitivity to chemotherapy of Jurkat cells following contact with BMSCs by down-regulation of Notch1 could be associated with repressed the activity of Akt signaling.更多还原