门冬酰胺酶对不同白血病细胞增殖抑制及儿童ALL融合基因动态检测的研究

发布时间：2018-02-12 18:23

本文关键词： L-asp 增殖抑制细胞周期白血病融合基因　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨门冬酰胺酶(L-asp)对Jurkat、K562、K562/ADM三种白血病细胞的体外增殖抑制作用及细胞周期的影响。方法体外培养Jurkat、K562、K562/ADM三种细胞,取对数期且生长良好的细胞用L-asp进行干预。Jurkat细胞用不同浓度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细胞用不同浓度L-asp(0.1 U/ml、1 U/ml、10 U/ml、100 U/ml、1000 U/ml)分别处理24h、48h、72h,细胞增殖抑制率采用CCK-8法检测,细胞周期采用流式细胞仪检测。结果1.(1)相同时间下不同浓度L-asp作用于Jurkat、K562细胞和K562/ADM细胞,随着浓度的增加细胞增殖抑制率增加,差异有统计学意义(P0.01);相同浓度L-asp作用于Jurkat细胞、K562细胞和K562/ADM细胞不同时间,随着时间的延长细胞增殖抑制率增加,差异有统计学意义(P0.01)。(2)相同浓度L-asp作用相同时间时,K562细胞与K562/ADM细胞抑制率相比,除0.1U/ml L-asp作用24h、48h抑制率无明显差别(P0.05),在其他浓度时间作用下,L-asp对K562/ADM细胞的抑制率大于K562细胞,差异有统计学意义(P0.05)。(3)相同时间下L-asp对不同白血病细胞增值抑制率的作用,JurkatK562/ADMK562。2.24h时Jurkat、K562、K562/ADM细胞的IC50分别为0.62ug/ml,766.34ug/ml,16.10ug/ml;48h时Jurkat、K562、K562/ADM细胞的IC50分别为0.11ug/ml,399.55ug/ml,6.17ug/ml;72h时Jurkat、K562、K562/ADM细胞的IC50分别为0.03ug/ml,308.19ug/ml,1.50ug/ml。3.(1)与对照组比较,不同浓度的L-asp作用Jurkat、K562、K562/ADM的G0/G1期均明显升高,差异具有显著性(P0.05)。(2)相同时间下不同浓度L-asp作用于Jurkat细胞、K562细胞和K562/ADM细胞,随着浓度的增加G0/G1期细胞所占比例增加,差异有统计学意义(P0.01);相同浓度L-asp作用于Jurkat细胞、K562细胞和K562/ADM细胞不同时间,随着时间的延长G0/G1期细胞所占比例增加,差异有统计学意义(P0.01)。(3)相同浓度L-asp作用相同时间时,K562细胞与K562/ADM细胞G0/G1期所占比例均有不同,K562/ADM细胞G0/G1期所占比例大于K562细胞,差别有统计学意义(P0.05)。(4)相同时间下L-asp对不同细胞G0/G1期细胞所占比例的影响,JurkatK562/ADMK562。结论L-asp对Jurkat、K562、K562/ADM细胞的增殖均有抑制作用,但不同白血病细胞对L-asp敏感性不同;L-asp可以将细胞阻滞在G0/G1期,对不同白血病细胞的阻滞作用也不同。目的探讨在儿童急性淋巴细胞白血病治疗过程中融合基因动态检测的临床意义,为临床治疗提供帮助。方法ALL患儿168例,融合基因阴性84例,融合基因阳性84例,其中包括TEL-AML1、EVI1、其他融合基因(BCR-ABL、E2A-PBX1、SIL-TAL1、MLL重排)、双融合基因阳性(EVI1,E2A-PBX1双表达;EVI1,TEL-AML1双表达;EVI1,SIL-TAL1双表达;TEL-AML1,MLL重排双表达),在发病初期及治疗过程中通过基因扩增技术检测融合基因的变化和临床多因素来分析临床疗效,根据卡方检验、Kaplan-Meier及COX比例风险回归模型进行分析。结果1.融合基因阳性ALL患儿对OS无显著影响(?2=3.36,P0.05),对EFS有显著影响(?2=14.61,P0.05);TEL-AML1患儿比基因阴性患儿、其他融合基因患儿EFS率显著升高(?2=6.88、3.93,P0.05)。2.TEL-AML1、其他组融合基因持续转阴患儿比融合基因未持续转阴患儿3年EFS率显著升高(?2=8.93、6.09,P0.05),EVI1、双融合基因阳性患儿两者之间无明显差异(?2=1.53、0.67,P0.05)。3.融合基因1年内是否转阴对患儿3年EFS率无明显意义(?2=0.82,2.75,0.01,0.67,P0.05)。4.初发ALL融合基因阳性且基因未持续转阴患儿年龄10岁,免疫分型为T型,临床危险度为高危,诱导缓解治疗第15天、第33天MRD≥10-2患儿3年EFS率显著降低,差异有统计学意义(?2=4.54~7.97,P0.05)。5.初发ALL融合基因阳性且未持续转阴患儿,年龄10岁(95%CI 2.06~25.90,P0.05)、免疫分型是T细胞(95%CI 1.01~22.08,P0.05)是影响预后的危险因素。结论融合基因动态观察对于ALL患儿预后有重要指导意义,有助于实施个体化治疗。
[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.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7

【参考文献】