本文選題：急性白血病 + 自噬相關基因　； 參考：《鄭州大學》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,隨著治療方案的進一步完善,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胞的復發(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)與腫瘤的關系是近幾年來研究的熱點,大量的研究發(fā)現(xiàn),自噬對腫瘤有抑制及促進的雙重作用,一方面,在腫瘤發(fā)生早期,自噬可通過增強自噬性細胞死亡,清除或修復受損的蛋白質(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胞中,通過降解受損的細胞器及蛋白質(zhì)為其存活提供能量,促進腫瘤細胞逃逸,利于腫瘤細胞的長期存活。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)外對自噬與白血病關系的研究已逐漸深入,可通過藥物誘導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)或抑制白血病細胞,其分子機制目前仍未明確。 自噬發(fā)生過程嚴格受自噬相關基因(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鍵,依賴于兩個泛素樣結合系統(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鍵作用。Atg5在自噬過程中起調(diào)控作用,與保守蛋白Atg12結合,形成Atg12-Atg5蛋白結合系統(tǒng),利于自噬體膜的延伸。Atg3在整個自噬過程起E2酶催化作用,促進Atg8與磷脂酰肌醇結合,參與自噬體形成的各階段。通過檢測Atg3及Atg5在兒童AL細胞中的表達,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 檢測初治組、緩解組、復發(fā)組急性白血病(AL)患兒及對照組患兒骨髓單個核細胞(bone marrow mononuclear cell, BMMNC)的自噬率,同時檢測自噬相關基因Atg3、Atg5的表達情況,探討自噬與兒童AL發(fā)生發(fā)展、復發(fā)的關系,揭示自噬相關基因在兒童急性白血病細胞腫瘤逃逸機制中的作用,為兒童AL提供新的治療靶點和治療策略。 方法 收取2011年3月至2012年5月期間在鄭州大學第一附屬醫(yī)院兒科血液病區(qū)確診AL的患兒骨髓標本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)；難治復發(fā)組9例(男7例,女2例),中位年齡6歲(2-10歲)。對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、細胞免疫分型差異無統(tǒng)計學意義(P0.05),資料具有可比性。用淋巴細胞分離液分離出骨髓單個核細胞,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胞自噬現(xiàn)象；采用流式細胞儀檢測細胞自噬率；采用RT-PCR技術檢測自噬基因Atg3mRNA和Atg5mRNA的表達。 結果 1.熒光顯微鏡下觀察到初治組、難治復發(fā)組的細胞自噬現(xiàn)象較多見,而對照組、緩解組自噬現(xiàn)象較少見。流式細胞術檢測結果發(fā)現(xiàn),初治組、難治復發(fā)組的自噬率分別為(17.07±2.31)%、(15.37±1.59)%,明顯高于對照組的(2.71±1.57)%,差異有統(tǒng)計學意義(P0.05),緩解自噬率為(3.48±1.94)%,與對照組相比差異無統(tǒng)計學意義(P0.05)；難治復發(fā)組的自噬率顯著高于緩解組,差異有統(tǒng)計學意義(P0.05)。 2. Atg3mRNA和Atg5mRNA在初治組和難治復發(fā)組的表達均高于對照組,差異均有統(tǒng)計學意義(P0.008)；緩解組和對照組相比差異無顯著性(P0.008)；二者在難治復發(fā)組的表達值明顯高于緩解組,差異具有統(tǒng)計學意義(P0.008)。 結論 1.初治組和難治復發(fā)組BMMNC的自噬活性增強,自噬相關基因Atg3mRNA、Atg5mRNA的表達均上調(diào),揭示Atg3、Atg5基因激活誘導的自噬活性增強可能與兒童白血病的發(fā)生、發(fā)展和耐藥機制有關。 2.自噬活性增強可能促進白血病細胞逃逸,維持白血病細胞的長久存活。
[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 .
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R733.71

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