Sam68对T-ALL细胞增殖和凋亡的作用研究

发布时间：2018-05-27 13:01

本文选题：Sam68 + 急性T淋巴细胞白血病　；参考：《北京协和医学院》2016年博士论文

【摘要】：背景急性淋巴细胞白血病(ALL)是一种遗传异质性疾病,以骨髓、外周血和其他器官中未成熟淋巴细胞增殖为特征,ALL通过淋巴细胞表面抗原来进行免疫学分型,大致可分为前B细胞ALL,成熟B细胞ALL和T细胞ALL。T细胞ALL (T-ALL)是起源于T细胞淋巴母细胞的遗传异质性疾病,发生于骨髓,外周血或胸腺,淋巴结和结外组织。表达不成熟T细胞免疫表型,最常见免疫抗原为CD3,包括胞浆CD3和细胞表面CD3。T-ALL患者肿瘤细胞停滞在不同分化阶段,根据不同免疫表型分为四类：早期前体T-ALL,前体T-ALL,皮质T-ALL和髓质T-ALL。与B-ALL患者比较,T-ALL患者预后较差,近年随着化疗的进展,儿童T-ALL患者治愈率接近75%,成年患者为50%。这种提高主要归功于对该病的分子遗传学和病理学的深入理解,根据危险分层调整的治疗和新型靶向药物的出现。但耐药或复发的T-ALL患者预后仍然很差。深入研究急性淋巴白血病发生发展过程中的分子机制有助于血液肿瘤的临床治疗。在T-ALL形成中,不同基因水平的变化协同作用更改了胸腺内T细胞异常生长、增殖、凋亡、分化的过程。多种癌基因和功能性失活突变共同参与其发生发展。T-ALL病理发展过程的遗传多变性进一步体现在一些普遍存在的细胞遗传学和分子遗传学的改变,从而引起特定生物进程的变化,如细胞周期信号传导通路,细胞生长和增殖,染色体重组,T细胞分化和自我更新等。Sam68 (Src-associated in mitosis 68 kDa)属于STAR家族,具有KH、SH3等多种结构域,既是一种RNA结合蛋白,又是衔接蛋白,通过与多种蛋白和RNA相互结合,参与细胞信号转导、RNA转录后修饰等分子进程,在细胞增殖、凋亡及自噬等多种细胞行为中发挥重要作用。研究表明Sam68基因的异常表达与肿瘤发生发展有密切关系,Sam68不仅在多种肿瘤中表达增高,与肿瘤生长、侵袭、转移、凋亡等关系密切,其在乳腺癌、肾癌等多种实体瘤细胞中的表达异常身高与临床不良预后有关。此外,Sam68参与T细胞活化,参与TCR信号转导通路,甚至可能促进混合型白血病的恶性转化。但是目前Sam68基因在急性T淋巴细胞白血病中的作用尚未见报道。Sam68的表达是否与T-ALL的发生、发展和维持有关,以及其在T-ALL中作用的分子机制需要进一步探索。目的以急性T-ALL患者骨髓细胞和T-ALL细胞系Jurkat、CCRF-CEM为研究对象,探讨Sam68异常表达对T-ALL的影响,在此基础上研究相关调控机制,为理解T-ALL的发病机制,寻找新的治疗靶点提供更多研究基础。方法对2014年6月～2015年4月中国医学科学院血液学研究所血液病医院收治32例T-ALL患者骨髓标本进行研究,并收集9例正常人骨髓标本为对照。分离单个核细胞,应用荧光实时定量PCR方法检测Sam68基因的表达水平。1.应用荧光实时定量PCR方法和Western Blot检测T-ALL细胞系Jurkat和CCRF-CEM中Sam68表达水平。2. 利用RNA干扰技术,构建靶向Sam68的特异性干扰载体,稳定转染Jurkat和CCRF-CEM细胞,下调细胞内Sam68表达水平。利用pLKO-Tet-On质粒对目的质粒敲降作用需强力霉素诱导的特性,去除强力霉素后检测Sam68恢复效率。利用荧光实时定量PCR方法和Western Blot检测Sam68表达调控效率。3.采用MTT实验检测Sam68表达变化对Jurkat和CCRF-CEM细胞生长增殖活性的影响,利用细胞集落形成实验检测Sam68表达变化对Jurkat和CCRF-CEM细胞的单细胞克隆增殖能力。利用Hoechst染色、Annexin V/FITC-PI双染方法检测Sam68表达变化对Jurkat和CCRF-CEM凋亡情况的影响。利用Western Blot检测Sam68表达变化对细胞周期和凋亡相关蛋白表达影响以及AKT/mTOR通路信号分子表达变化情况。结果1. 32例T-ALL患者中Sam68mRNA表达水平明显高于正常对照组。2. T-ALL相关Jurkat和CCRF-CEM细胞中Sam68mRNA和蛋白表达水平明显高于正常对照。3. 成功构建了利用shRNA干扰技术导致Sam68敲降的稳定Jurkat和CCRF-CEM细胞系,且Sam68表达可随强力霉素去除而恢复。4. MTT实验结果提示Sam68敲降后Jurkat和CCRF-CEM细胞生长增殖活性受到抑制,Sam68恢复后Jurkat和CCRF-CEM受抑制增殖情况明显改善。细胞集落实验结果显示Sam68敲降后Jurkat和CCRF-CEM细胞的克隆形成能力下降,Sam68恢复后下降的克隆形成情况明显改善。细胞凋亡实验结果显示Sam68敲降后Jurkat和CCRF-CEM细胞凋亡明显增加,Sam68恢复后凋亡水平下降。伴随Sam68敲降所致上述功能变化,细胞周期相关蛋白p21表达上调,CDK2表达下降,凋亡相关蛋白Bad表达上调,Bcl-xl下调,caspase-3, caspase-9, PARP的剪切活化程度明显增加,AKT/mTOR信号通路中,p-AKT, p-FOXO1, mTOR和p-p70S6k水平明显下调,而总的AKT, FOXO1和p70S6k水平无明显变化。去除强力霉素和恢复Sam68表达后,上述蛋白均明显恢复,与阴性对照差异不显著,无统计学意义。结论T-ALI患者、Jurkat和CCRF-CEM细胞株中均存在Sam68表达异常增高,提示Sam68可能参与T-ALL病理过程。Sam68表达变化引起了T-ALL细胞株Jurkat和CCRF-CEM曾殖和凋亡变化,且这种变化至少是通过对p21, CDK2, Bcl-xl,Bad和Caspase家族所致线粒体内源性凋亡通路的影响实现的。AKT/mTOR通路可能在Sam68介导的Jurkat和CCRF-CEM细胞增殖和凋亡相关变化中发挥了重要作用。本研究进一步阐明了T-ALL发生发展过程中相关分子机制。
[Abstract]:Background acute lymphoblastic leukemia (ALL) is a genetic heterozygous disease characterized by the proliferation of immature lymphocytes in bone marrow, peripheral blood and other organs. ALL is immunologically typed by lymphocyte surface antigen. It can be roughly divided into B cell ALL, mature B cell ALL and T cell ALL.T cells ALL (T-ALL) originating from T cells The genetic heterogeneity of lymphoblastic cells, occurring in bone marrow, peripheral blood or thymus, lymph nodes and extranodal tissues. Expression of immature T cell immunophenotype, the most common immuno antigen is CD3, including cytoplasmic CD3 and cell surface CD3.T-ALL tumor cells stagnated at different differentiation stages and divided into four categories according to different immunophenotypes: early precursors T-ALL, precursor T-ALL, cortical T-ALL and medullary T-ALL. compared with B-ALL patients, T-ALL patients have poor prognosis. In recent years, with the progress of chemotherapy, the cure rate of children with T-ALL is close to 75%. The improvement of adult patients for 50%. is mainly due to the deep understanding of the molecular genetics and pathology of the disease, according to the treatment and new target of dangerous stratified adjustment. The appearance of the drug appears. But the prognosis of T-ALL patients with resistance or recurrence is still poor. The molecular mechanism in the development of acute lymphoblastic leukemia is helpful to the clinical treatment of blood tumors. In the formation of T-ALL, the synergistic effect of different gene levels changes the abnormal growth, proliferation, apoptosis and differentiation of T cells in the thymus. The genetic polymorphism of multiple oncogenes and functional inactivation mutations involved in the pathogenesis of.T-ALL is further manifested in some common changes in cytogenetics and molecular genetics, resulting in changes in specific biological processes, such as cell cycle signaling pathways, cell growth and proliferation, and chromosomes. .Sam68 (Src-associated in mitosis 68 kDa), such as recombination, T cell differentiation and self renewal, belongs to the STAR family, with a variety of domains such as KH and SH3. It is both a RNA binding protein and a cohesive protein. By combining with a variety of proteins and RNA, it participates in cell signal transduction, RNA after transcriptional modification and other molecular processes, in cell proliferation, apoptosis and The study shows that the abnormal expression of Sam68 gene is closely related to the development of tumor. The expression of Sam68 is not only increased in many kinds of tumor, but also closely related to tumor growth, invasion, metastasis and apoptosis. The expression of abnormal height and clinical expression in many solid tumor cells such as breast cancer and renal cancer. In addition, Sam68 participates in the activation of T cells, participates in the TCR signal transduction pathway and may even promote the malignant transformation of mixed leukemia. However, the role of the Sam68 gene in acute T lymphocytic leukemia has not yet been reported whether the expression of.Sam68 is associated with the development and maintenance of T-ALL, and the role of.Sam68 in T-ALL. The molecular mechanism needs to be further explored. Objective to study the effect of abnormal expression of Sam68 on T-ALL in acute T-ALL patients' bone marrow cells and T-ALL cell line Jurkat and CCRF-CEM. On the basis of this, the related regulatory mechanism is studied to provide more research basis for understanding the pathogenesis of T-ALL and finding new therapeutic targets. Method to 2014 From June to April 2015, 32 cases of T-ALL patients were treated in the hematological Hospital of the hematology Institute of the Institute of Hematology of the Chinese Academy of Medical Sciences. 9 normal human bone marrow specimens were collected as control. Mononuclear cells were isolated and the expression level of Sam68 gene was detected by real time fluorescence quantitative PCR method. The real-time quantitative PCR method and Western Bl were used for the detection of the expression level of the gene. Ot detected the expression level of Sam68 in the T-ALL cell lines Jurkat and CCRF-CEM using RNA interference to construct a specific interference carrier of the target Sam68, stable transfection of Jurkat and CCRF-CEM cells and down regulation of the Sam68 expression level in the cells. Detection of Sam68 recovery efficiency. Using fluorescence real-time quantitative PCR method and Western Blot detection of Sam68 expression regulation efficiency.3., MTT test was used to detect the effect of Sam68 expression change on the growth and proliferation activity of Jurkat and CCRF-CEM cells. Cell colony formation test was used to detect the proliferation of Sam68 expression on the proliferation of single cell clone. Ability. The effects of Sam68 expression changes on the apoptosis of Jurkat and CCRF-CEM were detected by Hoechst staining and Annexin V/FITC-PI double staining. The effects of Sam68 expression on the cell cycle and apoptosis related protein expression were detected by Western Blot and the expression of AKT/mTOR pathway signal molecules expression was detected by Western Blot. The expression level of Sam68mRNA and protein in Jurkat and CCRF-CEM cells was significantly higher than that of normal control group. The expression level of Sam68mRNA and protein in Jurkat and CCRF-CEM cells was significantly higher than that of normal control.3.. The stable Jurkat and CCRF-CEM cell lines were successfully constructed by shRNA interference technique, and Sam68 expression could be recovered with the removal of the strong force mycin. The proliferation activity of Jurkat and CCRF-CEM cells was inhibited after Sam68 knockout, and the proliferation of Jurkat and CCRF-CEM was significantly improved after Sam68 recovery. Cell colony assay showed that the clone formation ability of Jurkat and CCRF-CEM cells decreased after Sam68 knock down, and the formation of descended clones after Sam68 recovery was obviously improved. Cell apoptosis was obviously improved. The experimental results showed that the apoptosis of Jurkat and CCRF-CEM cells increased significantly after Sam68 knockdown, and the apoptosis level decreased after the Sam68 recovery. The expression of cell cycle related protein p21 was up, CDK2 expression decreased, and the expression of Bad was up regulated, Bcl-xl down-regulation, Caspase-3, caspase-9, PARP. The level of p-AKT, p-FOXO1, mTOR and p-p70S6k decreased significantly in the AKT/mTOR signaling pathway, while the total AKT, FOXO1 and p70S6k levels were not significantly changed. After the removal of doxycycline and the recovery of Sam68 expression, the above proteins were obviously restored, and there was no significant difference from the negative control. Conclusion T-ALI patients, Jurkat and CCRF-CEM thin were no significant. There is an abnormal increase in the expression of Sam68 in the cell lines, suggesting that Sam68 may participate in the change of.Sam68 expression in the pathological process of T-ALL, which causes the changes in the colonization and apoptosis of T-ALL cell line Jurkat and CCRF-CEM, and this change is at least through the influence of p21, CDK2, Bcl-xl, Bad and Caspase. The pathway may play an important role in Sam68 mediated proliferation and apoptosis related changes in Jurkat and CCRF-CEM cells. This study further elucidates the molecular mechanisms of T-ALL development and development.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.71

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