自噬基因Atg3和Atg5在儿童急性白血病骨髓单个核细胞中的表达及临床意义

发布时间：2018-07-11 11:50

本文选题：急性白血病 + 自噬相关基因　；参考：《郑州大学》2013年硕士论文

【摘要】：白血病是造血组织的恶性疾病,在儿童肿瘤中较为常见,其发病率呈逐年增高的趋势,随着治疗方案的进一步完善,大部分患儿尚能获得长期无病生存,但仍有较高的复发率或死亡率；目前的化疗只能有限的延缓白血病患儿的生命,很难从根本上扼制白血病细胞的复发与播散,因此有必要寻找更好的方法靶向治疗白血病,提高患儿总体的生存质量和生存率。自噬(autophagy)与肿瘤的关系是近几年来研究的热点,大量的研究发现,自噬对肿瘤有抑制及促进的双重作用,一方面,在肿瘤发生早期,自噬可通过增强自噬性细胞死亡,清除或修复受损的蛋白质或DNA,来抑制肿瘤的发生；另一方面,自噬在缺血缺氧的肿瘤细胞中,通过降解受损的细胞器及蛋白质为其存活提供能量,促进肿瘤细胞逃逸,利于肿瘤细胞的长期存活。在不同的肿瘤以及肿瘤发生的不同阶段,自噬的作用不同；因此,自噬与肿瘤关系密切,研究二者之间的联系为肿瘤的治疗提供了新的可行方向。目前,国内外对自噬与白血病关系的研究已逐渐深入,可通过药物诱导或抑制自噬来治疗白血病,有研究发现,在儿童ALL中自噬活性增高,通过抑制自噬来达到治疗白血病的目的,但自噬是如何诱发或抑制白血病细胞,其分子机制目前仍未明确。自噬发生过程严格受自噬相关基因(Atg)调控。随着研究的深入,已经发现大量新的自噬基因,如beclin1、ambra1、Atg3、Atg5等,检测这些基因在肿瘤中的作用,可能成为治疗肿瘤和解决耐药的新策略。自噬体的形成是自噬的关键,依赖于两个泛素样结合系统,Atg12-Atg5共轭系统和Atg3、Atg4、Atg7、Atg8脂化系统。其中Atg3、Atg5在自噬体的形成中起关键作用。Atg5在自噬过程中起调控作用,与保守蛋白Atg12结合,形成Atg12-Atg5蛋白结合系统,利于自噬体膜的延伸。Atg3在整个自噬过程起E2酶催化作用,促进Atg8与磷脂酰肌醇结合,参与自噬体形成的各阶段。通过检测Atg3及Atg5在儿童AL细胞中的表达,来推断自噬在儿童AL白血病中的作用,为其治疗提供新的方向及策略。目的检测初治组、缓解组、复发组急性白血病(AL)患儿及对照组患儿骨髓单个核细胞(bone marrow mononuclear cell, BMMNC)的自噬率,同时检测自噬相关基因Atg3、Atg5的表达情况,探讨自噬与儿童AL发生发展、复发的关系,揭示自噬相关基因在儿童急性白血病细胞肿瘤逃逸机制中的作用,为儿童AL提供新的治疗靶点和治疗策略。方法收取2011年3月至2012年5月期间在郑州大学第一附属医院儿科血液病区确诊AL的患儿骨髓标本74例,初治组患儿37例(男21例,女16例),中位年龄6岁(2-12岁),经化疗完全缓解组28例(男16例,女12例),中位年龄5岁(2-10岁)；难治复发组9例(男7例,女2例),中位年龄6岁(2-10岁)。对照组选取非肿瘤性疾病(如：免疫性血小板减少症、过敏性紫癜)患儿骨髓标本28例(男17例,女11例),中位年龄6岁(3-13岁)。各组患儿的年龄、性别、细胞免疫分型差异无统计学意义(P0.05),资料具有可比性。用淋巴细胞分离液分离出骨髓单个核细胞,经单丹磺酰尸胺(MDC)染色后制备玻片,于Olympus EX-71荧光显微镜下观察细胞自噬现象；采用流式细胞仪检测细胞自噬率；采用RT-PCR技术检测自噬基因Atg3mRNA和Atg5mRNA的表达。结果 1.荧光显微镜下观察到初治组、难治复发组的细胞自噬现象较多见,而对照组、缓解组自噬现象较少见。流式细胞术检测结果发现,初治组、难治复发组的自噬率分别为(17.07±2.31)%、(15.37±1.59)%,明显高于对照组的(2.71±1.57)%,差异有统计学意义(P0.05),缓解自噬率为(3.48±1.94)%,与对照组相比差异无统计学意义(P0.05)；难治复发组的自噬率显著高于缓解组,差异有统计学意义(P0.05)。 2. Atg3mRNA和Atg5mRNA在初治组和难治复发组的表达均高于对照组,差异均有统计学意义(P0.008)；缓解组和对照组相比差异无显著性(P0.008)；二者在难治复发组的表达值明显高于缓解组,差异具有统计学意义(P0.008)。结论 1.初治组和难治复发组BMMNC的自噬活性增强,自噬相关基因Atg3mRNA、Atg5mRNA的表达均上调,揭示Atg3、Atg5基因激活诱导的自噬活性增强可能与儿童白血病的发生、发展和耐药机制有关。 2.自噬活性增强可能促进白血病细胞逃逸,维持白血病细胞的长久存活。
[Abstract]:Leukemia is a malignant disease of hematopoietic tissues , which is common in children ' s tumors . The incidence of leukemia is increasing year by year . With the further improvement of the treatment plan , most of the children still have long - term disease - free survival , but still have higher recurrence rate or mortality ;
At present the chemotherapy can only delay the life of the leukemia children with limited delay , it is difficult to fundamentally choke the relapse and spread of the leukemia cells , so it is necessary to find a better method to target the leukemia and improve the overall survival quality and survival rate of the children .

The relationship between autophagy and tumor is a hot spot in recent years . A large number of studies have found that autophagy plays a role in inhibiting and promoting the tumor . On the one hand , autophagy can inhibit the occurrence of tumor by increasing autophagy cell death , clearing or repairing damaged protein or DNA in the early stage of tumor .
on the other hand , autophagy can provide energy for survival of tumor cells by degrading damaged organelle and protein , promote the escape of tumor cells and facilitate the long - term survival of tumor cells .
Therefore , the relationship between autophagy and tumor is closely related . The study of the relationship between autophagy and leukemia provides a new feasible direction . At present , the research on the relationship between autophagy and leukemia has been gradually deepened , and it has been found that the autophagy activity in childhood ALL is increased , and the purpose of treating leukemia can be achieved by inhibiting autophagy , but autophagy is the way to induce or inhibit leukemic cells , and its molecular mechanism is still not clear .

Atg5 plays a key role in the formation of autophagy . Atg5 plays a key role in the formation of autophagy . Atg5 plays a key role in the formation of autophagy . Atg5 plays a key role in the formation of autophagy . Atg5 plays a key role in the formation of autophagy . Atg5 plays a key role in the formation of autophagy . Atg5 plays a key role in the formation of autophagy . Atg5 plays a key role in the formation of autophagy . Atg5 plays a key role in the formation of autophagy . Atg5 plays a key role in the formation of autophagy . Atg5 plays a key role in the formation of autophagy .

Purpose

To detect the autophagy rate of bone marrow mononuclear cells ( BMMNC ) in children with acute leukemia ( AL ) and control group , and to examine the relationship between autophagy and the development and recurrence of childhood AL , and to reveal the role of autophagy - related genes in childhood acute leukemia cell tumor escape mechanism and to provide new treatment targets and treatment strategies for children AL .

method

74 cases of bone marrow specimens were collected from children with AL in the pediatric hematology area of the First Affiliated Hospital of Zhengzhou University from March 2011 to May 2012 . Among them , 37 cases ( 21 males and 16 females ) , median age 6 years ( 2 - 12 years ) were received , 28 were complete remission group ( 16 males and 12 females ) , median age was 5 years ( 2 - 10 years ) ;
In the control group , there were 28 cases ( 17 males , 11 females ) and middle age 6 years ( 3 - 13 years old ) .
Flow cytometry was used to detect the autophagy of cells .
The expression of Atg3mRNA and Atg5 mRNA was detected by RT - PCR .

Results

1 . In the control group , the autophagy of refractory recurrent group was significantly higher than that in the control group ( 17.07 卤 2.31 ) % , ( 15.37 卤 1 . 59 ) % , which was significantly higher than that in the control group ( 2 . 71 卤 1 . 57 ) % , which was significantly higher than that of the control group ( 2 . 71 卤 1 . 57 ) % , which was significantly higher than that of the control group ( P < 0 . 05 ) .
The autophagy rate of refractory recurrent group was significantly higher than that in the remission group ( P0.05 ) .

2 . The expression of Atg3mRNA and Atg5 mRNA in primary treatment group and refractory recurrent group was higher than that of control group ( P0.05 ) .
There was no significant difference between the remission group and the control group ( P 0 . 008 ) .
The expression values of both in refractory recurrent group were significantly higher than those in the remission group ( P 0 . 008 ) .

Conclusion

1 . The autophagy activity of BMMNC , Atg3 mRNA and Atg5 mRNA were up - regulated in primary treatment group and refractory recurrent group . At3 and Atg5 gene activation induced autophagy activity was probably related to the occurrence , development and drug resistance mechanism of childhood leukemia .

2 . Autophagocytosis may promote the escape of leukemic cells and maintain the long - term survival of leukemic cells .
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R733.71

【参考文献】