本文選題：急性白血病 + 自噬相關(guān)基因��； 參考：《鄭州大學(xué)》2013年碩士論文

【摘要】：白血病是造血組織的惡性疾病,在兒童腫瘤中較為常見,其發(fā)病率呈逐年增高的趨勢(shì),隨著治療方案的進(jìn)一步完善,大部分患兒尚能獲得長期無病生存,但仍有較高的復(fù)發(fā)率或死亡率；目前的化療只能有限的延緩白血病患兒的生命,很難從根本上扼制白血病細(xì)胞的復(fù)發(fā)與播散,因此有必要尋找更好的方法靶向治療白血病,提高患兒總體的生存質(zhì)量和生存率。 自噬(autophagy)與腫瘤的關(guān)系是近幾年來研究的熱點(diǎn),大量的研究發(fā)現(xiàn),自噬對(duì)腫瘤有抑制及促進(jìn)的雙重作用,一方面,在腫瘤發(fā)生早期,自噬可通過增強(qiáng)自噬性細(xì)胞死亡,清除或修復(fù)受損的蛋白質(zhì)或DNA,來抑制腫瘤的發(fā)生；另一方面,自噬在缺血缺氧的腫瘤細(xì)胞中,通過降解受損的細(xì)胞器及蛋白質(zhì)為其存活提供能量,促進(jìn)腫瘤細(xì)胞逃逸,利于腫瘤細(xì)胞的長期存活。在不同的腫瘤以及腫瘤發(fā)生的不同階段,自噬的作用不同；因此,自噬與腫瘤關(guān)系密切,研究二者之間的聯(lián)系為腫瘤的治療提供了新的可行方向。目前,國內(nèi)外對(duì)自噬與白血病關(guān)系的研究已逐漸深入,可通過藥物誘導(dǎo)或抑制自噬來治療白血病,有研究發(fā)現(xiàn),在兒童ALL中自噬活性增高,通過抑制自噬來達(dá)到治療白血病的目的,但自噬是如何誘發(fā)或抑制白血病細(xì)胞,其分子機(jī)制目前仍未明確。 自噬發(fā)生過程嚴(yán)格受自噬相關(guān)基因(Atg)調(diào)控。隨著研究的深入,已經(jīng)發(fā)現(xiàn)大量新的自噬基因,如beclin1、ambra1、Atg3、Atg5等,檢測(cè)這些基因在腫瘤中的作用,可能成為治療腫瘤和解決耐藥的新策略。自噬體的形成是自噬的關(guān)鍵,依賴于兩個(gè)泛素樣結(jié)合系統(tǒng),Atg12-Atg5共軛系統(tǒng)和Atg3、Atg4、Atg7、Atg8脂化系統(tǒng)。其中Atg3、Atg5在自噬體的形成中起關(guān)鍵作用。Atg5在自噬過程中起調(diào)控作用,與保守蛋白Atg12結(jié)合,形成Atg12-Atg5蛋白結(jié)合系統(tǒng),利于自噬體膜的延伸。Atg3在整個(gè)自噬過程起E2酶催化作用,促進(jìn)Atg8與磷脂酰肌醇結(jié)合,參與自噬體形成的各階段。通過檢測(cè)Atg3及Atg5在兒童AL細(xì)胞中的表達(dá),來推斷自噬在兒童AL白血病中的作用,為其治療提供新的方向及策略。 目的 檢測(cè)初治組、緩解組、復(fù)發(fā)組急性白血病(AL)患兒及對(duì)照組患兒骨髓單個(gè)核細(xì)胞(bone marrow mononuclear cell, BMMNC)的自噬率,同時(shí)檢測(cè)自噬相關(guān)基因Atg3、Atg5的表達(dá)情況,探討自噬與兒童AL發(fā)生發(fā)展、復(fù)發(fā)的關(guān)系,揭示自噬相關(guān)基因在兒童急性白血病細(xì)胞腫瘤逃逸機(jī)制中的作用,為兒童AL提供新的治療靶點(diǎn)和治療策略。 方法 收取2011年3月至2012年5月期間在鄭州大學(xué)第一附屬醫(yī)院兒科血液病區(qū)確診AL的患兒骨髓標(biāo)本74例,初治組患兒37例(男21例,女16例),中位年齡6歲(2-12歲),經(jīng)化療完全緩解組28例(男16例,女12例),中位年齡5歲(2-10歲)；難治復(fù)發(fā)組9例(男7例,女2例),中位年齡6歲(2-10歲)。對(duì)照組選取非腫瘤性疾病(如：免疫性血小板減少癥、過敏性紫癜)患兒骨髓標(biāo)本28例(男17例,女11例),中位年齡6歲(3-13歲)。各組患兒的年齡、性別、細(xì)胞免疫分型差異無統(tǒng)計(jì)學(xué)意義(P0.05),資料具有可比性。用淋巴細(xì)胞分離液分離出骨髓單個(gè)核細(xì)胞,經(jīng)單丹磺酰尸胺(MDC)染色后制備玻片,于Olympus EX-71熒光顯微鏡下觀察細(xì)胞自噬現(xiàn)象；采用流式細(xì)胞儀檢測(cè)細(xì)胞自噬率；采用RT-PCR技術(shù)檢測(cè)自噬基因Atg3mRNA和Atg5mRNA的表達(dá)。 結(jié)果 1.熒光顯微鏡下觀察到初治組、難治復(fù)發(fā)組的細(xì)胞自噬現(xiàn)象較多見,而對(duì)照組、緩解組自噬現(xiàn)象較少見。流式細(xì)胞術(shù)檢測(cè)結(jié)果發(fā)現(xiàn),初治組、難治復(fù)發(fā)組的自噬率分別為(17.07±2.31)%、(15.37±1.59)%,明顯高于對(duì)照組的(2.71±1.57)%,差異有統(tǒng)計(jì)學(xué)意義(P0.05),緩解自噬率為(3.48±1.94)%,與對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義(P0.05)；難治復(fù)發(fā)組的自噬率顯著高于緩解組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 2. Atg3mRNA和Atg5mRNA在初治組和難治復(fù)發(fā)組的表達(dá)均高于對(duì)照組,差異均有統(tǒng)計(jì)學(xué)意義(P0.008)；緩解組和對(duì)照組相比差異無顯著性(P0.008)；二者在難治復(fù)發(fā)組的表達(dá)值明顯高于緩解組,差異具有統(tǒng)計(jì)學(xué)意義(P0.008)。 結(jié)論 1.初治組和難治復(fù)發(fā)組BMMNC的自噬活性增強(qiáng),自噬相關(guān)基因Atg3mRNA、Atg5mRNA的表達(dá)均上調(diào),揭示Atg3、Atg5基因激活誘導(dǎo)的自噬活性增強(qiáng)可能與兒童白血病的發(fā)生、發(fā)展和耐藥機(jī)制有關(guān)。 2.自噬活性增強(qiáng)可能促進(jìn)白血病細(xì)胞逃逸,維持白血病細(xì)胞的長久存活。
[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 鹵 2.31 ) % , ( 15.37 鹵 1 . 59 ) % , which was significantly higher than that in the control group ( 2 . 71 鹵 1 . 57 ) % , which was significantly higher than that of the control group ( 2 . 71 鹵 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 .
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R733.71

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