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  本文關(guān)鍵詞： L-asp 增殖抑制 細胞周期 白血病 融合基因　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的探討門冬酰胺酶(L-asp)對Jurkat、K562、K562/ADM三種白血病細胞的體外增殖抑制作用及細胞周期的影響。方法體外培養(yǎng)Jurkat、K562、K562/ADM三種細胞,取對數(shù)期且生長良好的細胞用L-asp進行干預(yù)。Jurkat細胞用不同濃度L-asp(0.02U/ml、0.1 U/ml、0.5 U/ml、2.5 U/ml、12.5 U/ml),K562和K562/ADM細胞用不同濃度L-asp(0.1 U/ml、1 U/ml、10 U/ml、100 U/ml、1000 U/ml)分別處理24h、48h、72h,細胞增殖抑制率采用CCK-8法檢測,細胞周期采用流式細胞儀檢測。結(jié)果1.(1)相同時間下不同濃度L-asp作用于Jurkat、K562細胞和K562/ADM細胞,隨著濃度的增加細胞增殖抑制率增加,差異有統(tǒng)計學(xué)意義(P0.01);相同濃度L-asp作用于Jurkat細胞、K562細胞和K562/ADM細胞不同時間,隨著時間的延長細胞增殖抑制率增加,差異有統(tǒng)計學(xué)意義(P0.01)。(2)相同濃度L-asp作用相同時間時,K562細胞與K562/ADM細胞抑制率相比,除0.1U/ml L-asp作用24h、48h抑制率無明顯差別(P0.05),在其他濃度時間作用下,L-asp對K562/ADM細胞的抑制率大于K562細胞,差異有統(tǒng)計學(xué)意義(P0.05)。(3)相同時間下L-asp對不同白血病細胞增值抑制率的作用,JurkatK562/ADMK562。2.24h時Jurkat、K562、K562/ADM細胞的IC50分別為0.62ug/ml,766.34ug/ml,16.10ug/ml;48h時Jurkat、K562、K562/ADM細胞的IC50分別為0.11ug/ml,399.55ug/ml,6.17ug/ml;72h時Jurkat、K562、K562/ADM細胞的IC50分別為0.03ug/ml,308.19ug/ml,1.50ug/ml。3.(1)與對照組比較,不同濃度的L-asp作用Jurkat、K562、K562/ADM的G0/G1期均明顯升高,差異具有顯著性(P0.05)。(2)相同時間下不同濃度L-asp作用于Jurkat細胞、K562細胞和K562/ADM細胞,隨著濃度的增加G0/G1期細胞所占比例增加,差異有統(tǒng)計學(xué)意義(P0.01);相同濃度L-asp作用于Jurkat細胞、K562細胞和K562/ADM細胞不同時間,隨著時間的延長G0/G1期細胞所占比例增加,差異有統(tǒng)計學(xué)意義(P0.01)。(3)相同濃度L-asp作用相同時間時,K562細胞與K562/ADM細胞G0/G1期所占比例均有不同,K562/ADM細胞G0/G1期所占比例大于K562細胞,差別有統(tǒng)計學(xué)意義(P0.05)。(4)相同時間下L-asp對不同細胞G0/G1期細胞所占比例的影響,JurkatK562/ADMK562。結(jié)論L-asp對Jurkat、K562、K562/ADM細胞的增殖均有抑制作用,但不同白血病細胞對L-asp敏感性不同;L-asp可以將細胞阻滯在G0/G1期,對不同白血病細胞的阻滯作用也不同。目的探討在兒童急性淋巴細胞白血病治療過程中融合基因動態(tài)檢測的臨床意義,為臨床治療提供幫助。方法ALL患兒168例,融合基因陰性84例,融合基因陽性84例,其中包括TEL-AML1、EVI1、其他融合基因(BCR-ABL、E2A-PBX1、SIL-TAL1、MLL重排)、雙融合基因陽性(EVI1,E2A-PBX1雙表達;EVI1,TEL-AML1雙表達;EVI1,SIL-TAL1雙表達;TEL-AML1,MLL重排雙表達),在發(fā)病初期及治療過程中通過基因擴增技術(shù)檢測融合基因的變化和臨床多因素來分析臨床療效,根據(jù)卡方檢驗、Kaplan-Meier及COX比例風(fēng)險回歸模型進行分析。結(jié)果1.融合基因陽性ALL患兒對OS無顯著影響(?2=3.36,P0.05),對EFS有顯著影響(?2=14.61,P0.05);TEL-AML1患兒比基因陰性患兒、其他融合基因患兒EFS率顯著升高(?2=6.88、3.93,P0.05)。2.TEL-AML1、其他組融合基因持續(xù)轉(zhuǎn)陰患兒比融合基因未持續(xù)轉(zhuǎn)陰患兒3年EFS率顯著升高(?2=8.93、6.09,P0.05),EVI1、雙融合基因陽性患兒兩者之間無明顯差異(?2=1.53、0.67,P0.05)。3.融合基因1年內(nèi)是否轉(zhuǎn)陰對患兒3年EFS率無明顯意義(?2=0.82,2.75,0.01,0.67,P0.05)。4.初發(fā)ALL融合基因陽性且基因未持續(xù)轉(zhuǎn)陰患兒年齡10歲,免疫分型為T型,臨床危險度為高危,誘導(dǎo)緩解治療第15天、第33天MRD≥10-2患兒3年EFS率顯著降低,差異有統(tǒng)計學(xué)意義(?2=4.54~7.97,P0.05)。5.初發(fā)ALL融合基因陽性且未持續(xù)轉(zhuǎn)陰患兒,年齡10歲(95%CI 2.06~25.90,P0.05)、免疫分型是T細胞(95%CI 1.01~22.08,P0.05)是影響預(yù)后的危險因素。結(jié)論融合基因動態(tài)觀察對于ALL患兒預(yù)后有重要指導(dǎo)意義,有助于實施個體化治療。
[Abstract]:Objective to investigate the L-asparaginase (L-asp) on Jurkat, K562, inhibitory effect on cell cycle and proliferation of K562/ADM in three kinds of leukemia cells in vitro. Methods in vitro Jurkat, K562, K562/ADM three cells, logarithmic phase and intervention of.Jurkat cells with different concentrations of L-asp L-asp with good cell growth (0.02U/ml, 0.1 U/ml 0.5, U/ml, 2.5 U/ml, 12.5 U/ml), K562 and K562/ADM cells with different concentrations of L-asp (0.1 U/ml, 1 U/ml, 10 U/ml, 100 U/ml, 1000 U/ml) were 24h, 48h, 72h, inhibition of cell proliferation by CCK-8 assay, cell cycle by flow cytometry. Results (1. 1) the same time under different concentrations of L-asp in Jurkat, K562 cells and K562/ADM cells, with the increase of the concentration of cell proliferation inhibition rate increased, the difference was statistically significant (P0.01); the same concentration of L-asp acted on Jurkat cells, K562 cells and K562/ADM cells Time, with time prolonging cell proliferation inhibition rate increased, the difference was statistically significant (P0.01). (2) the same concentration of L-asp the same time, K562 cells and K562/ADM cell inhibition rate compared to L-asp 24h in addition to 0.1U/ml, the inhibition rate of 48h (P0.05), there was no significant difference in other concentration time, inhibition L-asp on K562/ADM cells was higher than that of K562 cells, the difference was statistically significant (P0.05). (3) at the same time the inhibition rate of L-asp effect on the value of different leukemia cells JurkatK562/ADMK562.2.24h, Jurkat, K562, K562/ADM IC50 cells were 0.62ug/ml, 766.34ug/ml, 16.10ug/ml; 48h Jurkat, K562, K562/ADM respectively in IC50 cells 0.11ug/ml, 399.55ug/ml, 6.17ug/ml; 72h Jurkat, K562 K562/ADM, IC50 cells were 0.03ug/ml, 308.19ug/ml, 1.50ug/ml.3. (1) compared with the control group, L-asp Jurkat, different concentrations of K562, K562/ ADM G0/G1 phase were significantly increased, the difference was significant (P0.05). (2) the same time under different concentrations of L-asp in Jurkat cells, K562 cells and K562/ADM cells, with the increase of the concentration of the proportion of G0/G1 phase cells increased, the difference was statistically significant (P0.01); the same concentration of L-asp acted on Jurkat cells at different time, K562 cells and K562/ADM cells, with the extension of time the proportion of G0/G1 phase cells increased, the difference was statistically significant (P0.01). (3) the same concentration of L-asp the same time, K562 cells and K562/ADM cell proportion of G0/G1 phase were K562/ADM, the proportion of G0/G1 phase cells than K562 cells. The difference was statistically significant (P0.05). (4) effect at the same time the proportion of L-asp cells in different cell stage G0/G1, JurkatK562/ADMK562. L-asp Jurkat K562, in conclusion, inhibited the proliferation of K562/ADM cells significantly, but not With different sensitivity of leukemic cells to L-asp; L-asp can arrest cells in G0/G1 phase, the blocking effect of different leukemia cells are different. Objective to investigate the clinical significance of dynamic detection of fusion gene in children with acute lymphoblastic leukemia treated in the process, provide help for clinical treatment. Methods 168 patients with ALL fusion gene, 84 cases were negative, fusion the gene was positive in 84 cases, including TEL-AML1, EVI1 and other fusion genes (BCR-ABL, E2A-PBX1, SIL-TAL1, MLL, fusion gene rearrangement) double positive (EVI1, E2A-PBX1, EVI1, double double expression; expression; TEL-AML1 EVI1, SIL-TAL1 TEL-AML1, double expression; MLL rearrangement, double expression) in the early stage of the disease and the treatment process by the changes of the fusion gene amplification gene technology detection and clinical factors to analyze the clinical curative effect, according to the chi square test, Kaplan-Meier and COX proportional hazards regression model were analyzed. Results of the 1. fusion gene Positive ALL patients had no significant effect on OS (? 2=3.36, P0.05), had a significant influence on EFS (? 2=14.61, P0.05); children with TEL-AML1 negative patients was significantly higher than other genes, the fusion gene in children with EFS rate (2=6.88,3.93,.2.TEL-AML1? P0.05), the other group sustained negative fusion gene fusion gene in children than for negative the 3 year rate increased significantly in children with EFS (? 2=8.93,6.09, P0.05, EVI1), and dual fusion between gene positive group had no significant difference (? 2=1.53,0.67, P0.05).3. fusion gene in 1 years is negative for children 3 year EFS rate had no obvious significance (? 2=0.82,2.75,0.01,0.67, P0.05).4. primary ALL fusion gene positive and the gene did not continue the negative children 10 years of age, the immune type T, the clinical risk for high-risk, remission induction therapy for fifteenth days, 3 year EFS rate thirty-third days MRD = 10-2 were significantly decreased, the difference was statistically significant (? 2=4.54~7.97, P0.05).5. primary ALL melt Gene positive and negative for children 10 years of age (95%CI, 2.06~25.90, P0.05), the immunophenotype of T cells (95%CI 1.01~22.08 P0.05) is the risk factors influencing the prognosis. Conclusion the dynamic observation of fusion gene have important guiding significance for the prognosis of ALL, contribute to the implementation of individualized treatment.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.7

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