【作者】 徐波；

【导师】 王保捷；

【作者基本信息】 中国医科大学 ， 法医学， 2006， 博士

【摘要】 前言 急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)是儿童肿瘤中的常见病,随着联合化疗药物的应用,白血病的治愈率在不断提高,但仍有20%病人因临床耐药而复发。糖皮质激素(glucocorticoid,GC)如地塞米松等能有效诱导凋亡,尤其对淋巴细胞影响巨大,已成为治疗白血病的重要抗肿瘤药。目前GC诱导细胞凋亡的机理及耐药机制仍不完全清楚,多数研究认为GC与其受体结合后激活细胞核内的相关基因,通过转录激活或转录抑制基因的表达来调节细胞凋亡。GC耐药机制异常复杂,GC作用通路的任何环节异常均可导致耐药发生,包括GC代谢酶异常、GC受体基因表达异常、GC受体相关蛋白异常、GC作用的靶基因异常,以及能干扰GC反应的其他信号途径的参与等。在GC诱导凋亡及其耐药机制的研究中,Bcl-2家族因具有调节凋亡作用而备受关注。Bcl-2家族分为抗凋亡及促凋亡成员,抗凋亡成员包括Bcl-2、Bcl-xL等,促凋亡成员包括Bax、Bak、Bid、Bad、Bim、Puma、Noxa等,其中Bim、Puma等仅含BH3区的BH3-only促凋亡成员成为Bcl-2家族的研究热点。微阵列分析发现,淋巴细胞中GC能诱导促凋亡蛋白Bim的表达。有报道GC作用于Bim或Puma基因敲除的小鼠后,不能诱导小鼠体内淋巴细胞凋亡,说明Bim及Puma是体内GC诱导淋巴细胞凋亡的重要启动因子。研究发现Bcl-2家族BH3区模拟物HA14-1能诱导细胞凋亡,并能增强肿瘤细胞对化疗药的敏感性。Bcl-2家族成员Bim及Puma在细胞凋亡过程中发挥重要作用,但在GC诱导的白血病细胞凋亡中所起的作用尚不完全清楚,且目前的研究仅限于动物的体内实验及有限的细胞系,与临床的关联不紧密。对Bcl-2家族模拟物HA14-1在GC耐药ALL细胞中的作用及诱导ALL细胞凋亡的途径,目前的研究仍然有限。更多还原

【Abstract】 introductionAcute lymphoblastic leukemia (ALL) is the common disease in childhood malignancy. With the use of combination chemotherapy, the cure rate of leukemia is increasing. However, the remaining 20% patients are still suffering from relapse because of drug resistance. Glucocorticoids ( GCs) such as dexametha-sone (Dex) can effectively induce apoptosis and have become important anti-cancer drugs in the treatment of leukemia. To date, mechanisms of glucocorti-coid-induced apoptosis and its drug resistance remain poorly understood. Most studies report glucocorticoid binds with its receptor (GR) , activates associated genes in nucleus and regulates apoptosis by transactivation or transrepression of target genes. Mechanisms of glucocorticoid resistance are very complicated. Any defects along GC signaling pathway can result in drug resistance, such as insufficient GC receptor ligand, abnormality of GR gene expression, abnormality of GR-associated proteins or target genes, and involvement of other signaling pathway that can interfere GC action. In the studies of GC action and mechanisms of GC resistance, Bcl-2 family members were extremely paid attention to due to the function in regulating apoptosis. Bcl-2 family can be classified into anti-apoptot-ic and pro-apoptotic members. Anti-apoptotic members include Bcl-2, Bcl-xL. Whilst pro-apoptotic members include Bax, Bak, Bid, Bad, Bim, Puma and Noxa, of which the BH3-only pro-apoptotic members stand out. Recent studies have found the pro-apoptotic BH3-only members Bim and Puma play an essential role in apoptosis. In GC-induced apoptosis of haematopoietic cells, it was foundthat GC could induce Bim expression with the help of microarray analysis. Studies using gene knockout mice have revealed that Bim or Puma deficiency can effectively protect most lymphocytes from GC-induced killing, which further demonstrated that Bim or Puma are key initiators of GC-induced apoptosis in lymph-oid cells. Researchers have found that BH3 mimetics HA14-1 can induce apoptosis and increase the sensitivity of tumor cells to chemotherapy drugs. Studies have revealed that Bcl-2 family members Bim and Puma play a critical role in apoptosis, however, it is still not very clear its role in GC-induced apoptosis in ALL cells. Furthermore, most studies have focused on the in-vitro experiments of animals or several limited cell lines and are not closely relevant with clinical outcome. Besides, the role of BH3 mimetics HA14-1 in GC-resistant ALL cells and its pathway to induce apoptosis remains poorly understood.In this study, we tried to engraft human ALL cells into the immunodeficient mice and establish xenograft leukemia cell lines that were closely related with clinical patients. The protein and mRNA expression of BH3-only members Bim and Puma were detected following Dex treatment. The roles of Bim and Puma and their effects on drug resistance were investigated in the treatment of ALL by GC. We further determined the cytotoxicity of HA14-1 on ALL cells, its effect on Dex resistant ALL cells and the pathway to induce apoptosis in CEM and Nalm6 cells. Hence, this report provides possibility to find the causes of GC resistance and also theoretical basis for a successful therapeutic strategy.Methods1. To establish leukemia experimental models and culture xenograft ALL cells.2. Employing MTT colorimetric assay to detect cell viability of xenografts with various concentration of Dex treatment for 72h. And define the in vitro sensitivity of xenograft ALL cells to Dex and its relevance with clinical patients.3. Using flow cytometry to detect the effect of various concentrations or various time course of Dex treatment on the cell viability of xenografts.4. Using Western blot analysis and real-time quantitative RT-PCR to inves-tigate the expression of Bim and Puma at both protein and mRNA levels after different time course of Dex treatment.5. Using MTT colorimetric assay to investigate cell viability of xenograft ALL cells, CEM as well as Nalm6 cells following different concentrations of HA14-1 treatment. And further detect cell viability of xenograft ALL cells with the combination treatment of HA14-1 and Dex.6. Using flow cytometer to detect the mechamisms of apoptosis in CEM and Nalm6 cell lines after HA14-1 treatment, including the assessment of caspase activity and loss of mitochondrial membrane potential.7. Statistical analysis. Each experiment was repeated in triplicate. Data were analyzed by SPSSl 1.0 statistical software and represented means standard error. Anova analysis was used to determine difference between groups. P < 0. 05 was set as statistical difference.Results1. Cell viability detected by MTT colorimetric assay.Xenograft cells ALL-3 and ALL-16, which derived from good clinical outcome patient samples, were sensitive to Dex. Cell viability was lower than 20% when exposed to more than lnM Dex with IC^values lower than lOnM, 3.01nM and 1.25nM respectively. While xenograft ALL-2 and ALL-19, which derived from poor clinical patient samples, were resistant to Dex. Cell viability was approximately 70% when exposed to more than lnM Dex treatment, with ICy, values higher than 10|xM that were 1000-fold higher than that of the sensitive xeno-grafts.2. Cell viability detected by flow cytometry.Cell viability of ALL-3 and ALL-16 decreased greatly as Dex concentrations increased, with IC^values lower than lOOnM, 36. 8lnM and 2. 23nM respectively. However, cell viability of ALL-2 and ALL-19 did not decrease obviously as Dex concentrations increased, with IC^ values higher than 10|xM. Following ljiM Dex treatment for various time points, cell viability of ALL-3 and ALL-16 decreased dramatically, especially at 72h, but no obvious decrease was detectedin ALL-2 and ALL-19.3. Effect of Dex on Bim protein and mRNA expression in xenograft cells Following IjxM Dex treatment, Bim protein increased obviously within 8hand reached the peak within 20h in the sensitive ALL-3 and ALL-16. However, no induction of Bim protein was detected in the resistant ALL-2 and ALL-19 at various time points. In the sensitive xenograft, Bim mRNA increased markedly within 2h and reached the peak within 16h following Dex treatment. While there was no obvious increase of Bim mRNA in ALL-2 and only a slight increase significantly lower than the sensitive in ALL-19 (P <0.01). There was significant difference of Bim protein and mRNA expression between the sensitive and resistant xenografts ( P < 0.01).4. Effect of Dex on Puma protein and mRNA expression in xenograft cells Following lfxM Dex treatment, no induction of Puma protein and mRNAwas detected in both the sensitive and resistant xenografts.5. Effect of HA14-1 on xenograft cells detected by MTT assayHA14-1 can have cytotoxicity after 48h treatment. Cell viability of xenograft cells, CEM and Nalm6 cells decreased as the concentration of HA14-1 increased. IC^ values of xenograft were between 16.41jxM and 43.10|xM. While ICsq values of CEM and Nalm6 were 6. 13jjlM and 12.69|xM respectively. After the combination treatment of 10|xM, 25yM HA14-1 with IjxM Dex for ALL-2, or 25jxM HA 14-1 with Dex for ALL-19, cell viability decreased significantly compared with that of treatment with HA14-1 alone (P <0.05).6. The HA 14-1-induced apoptosis pathway detected by flow cytometer Following 20jiM of HA14-1 treatment on CEM, 20|xM and 30u.M of HA14-1 on Nalm6, both the caspase activity and loss of mitochondrial membrane potential increased dramatically within 12h and remained high until 48h.Conclusions1. Xenografts ALL-3 and ALL-16 were sensitive to Dex while xenografts ALL-2 and ALL-19 were resistant to Dex. The in vitro sensitivity of xenografts to Dex was consistarit with clinical outcome and were affected by Dex concentrationand treatment time in the Dex-sensitive xenografts.2. Dex can induce Bim expression in the Dex-sensitive xenografts ALL-3 and ALL-16, however, it cannot induce Bim expression in the resistant xenografts ALL-2 and ALL-19 in vitro. Bim plays an essential role in the regulation of GC-induced apoptosis in xenografts ALL-2, 3, 16, 19.3. Dex cannot induce Puma upregulation in xenografts ALL-2, 3, 16, 19 in vitro. Bcl-2 family members except Puma may regulate GC-induced apoptosis.4. HA14-1 can have the cytotoxicity to xenografts ALL-2, 3, 16, 19 , as well as CEM and Nalm6 cells. CEM and Nalm6 were more sensitive to HA14-1 than xenograft ALL cells. HA14-1 can increase the sensitivity of GC-resistant xenografts ALL-2 and ALL-19 to dexamethasone.5. HA14-1 induces apoptosis in CEM and Nalm6 cells by the activation of caspase and loss of mitochondrial membrane potential.更多还原