本文選題：Sam68 + 急性T淋巴細胞白血病��； 參考：《北京協(xié)和醫(yī)學院》2016年博士論文

【摘要】：背景急性淋巴細胞白血病(ALL)是一種遺傳異質(zhì)性疾病,以骨髓、外周血和其他器官中未成熟淋巴細胞增殖為特征,ALL通過淋巴細胞表面抗原來進行免疫學分型,大致可分為前B細胞ALL,成熟B細胞ALL和T細胞ALL。T細胞ALL (T-ALL)是起源于T細胞淋巴母細胞的遺傳異質(zhì)性疾病,發(fā)生于骨髓,外周血或胸腺,淋巴結(jié)和結(jié)外組織。表達不成熟T細胞免疫表型,最常見免疫抗原為CD3,包括胞漿CD3和細胞表面CD3。T-ALL患者腫瘤細胞停滯在不同分化階段,根據(jù)不同免疫表型分為四類：早期前體T-ALL,前體T-ALL,皮質(zhì)T-ALL和髓質(zhì)T-ALL。與B-ALL患者比較,T-ALL患者預后較差,近年隨著化療的進展,兒童T-ALL患者治愈率接近75%,成年患者為50%。這種提高主要歸功于對該病的分子遺傳學和病理學的深入理解,根據(jù)危險分層調(diào)整的治療和新型靶向藥物的出現(xiàn)。但耐藥或復發(fā)的T-ALL患者預后仍然很差。深入研究急性淋巴白血病發(fā)生發(fā)展過程中的分子機制有助于血液腫瘤的臨床治療。在T-ALL形成中,不同基因水平的變化協(xié)同作用更改了胸腺內(nèi)T細胞異常生長、增殖、凋亡、分化的過程。多種癌基因和功能性失活突變共同參與其發(fā)生發(fā)展。T-ALL病理發(fā)展過程的遺傳多變性進一步體現(xiàn)在一些普遍存在的細胞遺傳學和分子遺傳學的改變,從而引起特定生物進程的變化,如細胞周期信號傳導通路,細胞生長和增殖,染色體重組,T細胞分化和自我更新等。Sam68 (Src-associated in mitosis 68 kDa)屬于STAR家族,具有KH、SH3等多種結(jié)構(gòu)域,既是一種RNA結(jié)合蛋白,又是銜接蛋白,通過與多種蛋白和RNA相互結(jié)合,參與細胞信號轉(zhuǎn)導、RNA轉(zhuǎn)錄后修飾等分子進程,在細胞增殖、凋亡及自噬等多種細胞行為中發(fā)揮重要作用。研究表明Sam68基因的異常表達與腫瘤發(fā)生發(fā)展有密切關(guān)系,Sam68不僅在多種腫瘤中表達增高,與腫瘤生長、侵襲、轉(zhuǎn)移、凋亡等關(guān)系密切,其在乳腺癌、腎癌等多種實體瘤細胞中的表達異常身高與臨床不良預后有關(guān)。此外,Sam68參與T細胞活化,參與TCR信號轉(zhuǎn)導通路,甚至可能促進混合型白血病的惡性轉(zhuǎn)化。但是目前Sam68基因在急性T淋巴細胞白血病中的作用尚未見報道。Sam68的表達是否與T-ALL的發(fā)生、發(fā)展和維持有關(guān),以及其在T-ALL中作用的分子機制需要進一步探索。目的以急性T-ALL患者骨髓細胞和T-ALL細胞系Jurkat、CCRF-CEM為研究對象,探討Sam68異常表達對T-ALL的影響,在此基礎上研究相關(guān)調(diào)控機制,為理解T-ALL的發(fā)病機制,尋找新的治療靶點提供更多研究基礎。方法對2014年6月～2015年4月中國醫(yī)學科學院血液學研究所血液病醫(yī)院收治32例T-ALL患者骨髓標本進行研究,并收集9例正常人骨髓標本為對照。分離單個核細胞,應用熒光實時定量PCR方法檢測Sam68基因的表達水平。1.應用熒光實時定量PCR方法和Western Blot檢測T-ALL細胞系Jurkat和CCRF-CEM中Sam68表達水平。2. 利用RNA干擾技術(shù),構(gòu)建靶向Sam68的特異性干擾載體,穩(wěn)定轉(zhuǎn)染Jurkat和CCRF-CEM細胞,下調(diào)細胞內(nèi)Sam68表達水平。利用pLKO-Tet-On質(zhì)粒對目的質(zhì)粒敲降作用需強力霉素誘導的特性,去除強力霉素后檢測Sam68恢復效率。利用熒光實時定量PCR方法和Western Blot檢測Sam68表達調(diào)控效率。3.采用MTT實驗檢測Sam68表達變化對Jurkat和CCRF-CEM細胞生長增殖活性的影響,利用細胞集落形成實驗檢測Sam68表達變化對Jurkat和CCRF-CEM細胞的單細胞克隆增殖能力。利用Hoechst染色、Annexin V/FITC-PI雙染方法檢測Sam68表達變化對Jurkat和CCRF-CEM凋亡情況的影響。利用Western Blot檢測Sam68表達變化對細胞周期和凋亡相關(guān)蛋白表達影響以及AKT/mTOR通路信號分子表達變化情況。結(jié)果1. 32例T-ALL患者中Sam68mRNA表達水平明顯高于正常對照組。2. T-ALL相關(guān)Jurkat和CCRF-CEM細胞中Sam68mRNA和蛋白表達水平明顯高于正常對照。3. 成功構(gòu)建了利用shRNA干擾技術(shù)導致Sam68敲降的穩(wěn)定Jurkat和CCRF-CEM細胞系,且Sam68表達可隨強力霉素去除而恢復。4. MTT實驗結(jié)果提示Sam68敲降后Jurkat和CCRF-CEM細胞生長增殖活性受到抑制,Sam68恢復后Jurkat和CCRF-CEM受抑制增殖情況明顯改善。細胞集落實驗結(jié)果顯示Sam68敲降后Jurkat和CCRF-CEM細胞的克隆形成能力下降,Sam68恢復后下降的克隆形成情況明顯改善。細胞凋亡實驗結(jié)果顯示Sam68敲降后Jurkat和CCRF-CEM細胞凋亡明顯增加,Sam68恢復后凋亡水平下降。伴隨Sam68敲降所致上述功能變化,細胞周期相關(guān)蛋白p21表達上調(diào),CDK2表達下降,凋亡相關(guān)蛋白Bad表達上調(diào),Bcl-xl下調(diào),caspase-3, caspase-9, PARP的剪切活化程度明顯增加,AKT/mTOR信號通路中,p-AKT, p-FOXO1, mTOR和p-p70S6k水平明顯下調(diào),而總的AKT, FOXO1和p70S6k水平無明顯變化。去除強力霉素和恢復Sam68表達后,上述蛋白均明顯恢復,與陰性對照差異不顯著,無統(tǒng)計學意義。結(jié)論T-ALI患者、Jurkat和CCRF-CEM細胞株中均存在Sam68表達異常增高,提示Sam68可能參與T-ALL病理過程。Sam68表達變化引起了T-ALL細胞株Jurkat和CCRF-CEM曾殖和凋亡變化,且這種變化至少是通過對p21, CDK2, Bcl-xl,Bad和Caspase家族所致線粒體內(nèi)源性凋亡通路的影響實現(xiàn)的。AKT/mTOR通路可能在Sam68介導的Jurkat和CCRF-CEM細胞增殖和凋亡相關(guān)變化中發(fā)揮了重要作用。本研究進一步闡明了T-ALL發(fā)生發(fā)展過程中相關(guān)分子機制。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R733.71

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