地西他滨联合三氧化二砷对NB4细胞株增殖和凋亡作用及对DAPK基因影响的实验研究
摘要
研究背景
近年来，通过维甲酸联合亚砷酸诱导分化，急性早幼粒细胞白血病(APL)的预后已大为改观，APL单用全反式维甲酸(ATRA)治疗，近期完全缓解率可达85%以上，但仍有部分患者复发，尽管其治疗前景仍保持乐观，但复发患者有较高的早期死亡和较短的2次缓解期。造血干细胞的移植是目前治疗恶性血液病的最佳方法，但移植期间移植相关并发症较多，且移植费用较高，大多数患者无法承受。目前研究发现大多数急性髓系白血病（AML）患者中存在2种或2种以上基因异常甲基化，由于DNA甲基化是一种可逆转的基因修饰过程,故在肿瘤或癌前病变中通过去甲基化处理则可以恢复基因表达,从而达到防治肿瘤的目的，因此,DNA的去甲基化作用将为肿瘤治疗提供新思路。DAPK基因是抑癌基因，亦是启动凋亡的正性调控因子，具有促进凋亡的作用，其启动子甲基化可能是血液系统肿瘤发生的十分重要的基因表达调控机制。地西他滨(DAC)是一种去甲基化制剂，对耐药和复发的白血病患者有一定的疗效，研究显示，DAC在治疗骨髓增生异常综合征（MDS）及一些实体瘤方面有着良好的疗效，我们用不同浓度的DAC、As2O3及两药联合处理NB4细胞，采用MTT法以及流式细胞术检测细胞凋亡，RT-PCR法检测DAPKmRNA的表达情况，旨在为临床应用DAC联合As2O3提供理论依据。
方法
1.四甲基偶氮唑蓝（MTT）法检测地西他滨及三氧化二砷对细胞株的增殖抑制作用；
2.流式细胞仪检测细胞凋亡率；
3.RT-PCR法检测DAPKmRNA的表达。
结果
1.MTT试验结果显示，不同浓度的地西他滨及三氧化二砷作用不同时间点后均具有一定的抑制作用；在同一时间点两单药组对细胞的生长抑制作用呈剂量依赖性（P＜0.05）,同一浓度地西他滨在72h时间点抑制作用达高峰。本实验中选取地西他滨浓度为1.0和2.0μmol/L、三氧化二砷0.5和1.0μmol/L两个工作浓度进行实验。
2.流式细胞仪结果显示，对照组经48h培养无明显凋亡（图3a），而各药物处理组均可见细胞凋亡，与对照组相比凋亡率明显增高，DAC 2μmol/L作用NB4细胞48h后凋亡率为5.0%（图3b），As2O3 1μmol/L作用NB4细胞48h后凋亡率为5.8%（图3c），联合组凋亡率增加为17.3%（图3d），联合用药组与各单药组比较，凋亡率显著增加。
3. RT-PCR结果显示，NB4细胞株低表达或者不表达DAPMmRNA,两药作用后其表达恢复，并且随着药物浓度的提高其表达增加。
结论
1.地西他滨对NB4细胞有增殖抑制和诱导凋亡的作用；
2.地西他滨发挥作用具有剂量依赖性；
3. DAC联合As2O3对NB4细胞增殖抑制及诱导凋亡有协同作用；
4.地西他滨预处理细胞后，可以增加As2O3对NB4细胞株的敏感性，其机制可能与恢复或增加DAPK的表达有关。
Background
In recent years, by arsenious acid and retinoic acid induced differentiation, Acutepromyelocytic leukemia(APL) treatment has improved considerably,APL treatmentwith retinoic acid alone,near complete remission rate above 85%,but still relapse.Although the outlook remains optimistic about its treatment of recurrence is still,However, patients with recurrence had higher early mortality and shorter remission 2.Hematopoietic stem cell transplantation is the best way to treat hematologicmalignancies,However, many transplant-related complications during the transplant,need to further explore ways to reduce its related complications, and the high cost oftransplantation, most patients can not afford. Recent studies showed that the majorityof acute myeloid leukemia (AML) patients, there is more than 2 or 2 aberrantmethylation, Because DNA methylation is a reversible process of geneticmodification, it is cancer or precancerous lesions in the demethylation treatment by,you can restore gene expression, so as to achieve the purpose of cancerprevention,Thus, DNA methylation to go to provide new ideas for cancer treatment.DAPK gene is a tumor suppressor gene, is also a positive start apoptosis regulatoryfactors, can promote apoptosis, the promoter methylation of tumor blood system maybe a very important mechanism of gene expression. DAC is a demethylating agent, ondrug resistance and relapse of leukemia patients have a certain effect.Study, DAC inthe treatment of myelodysplastic syndrome (MDS) and some solid tumors has a goodeffect. We used different concentrations of DAC, As2O3 and the two drugcombination treatment NB4 cells by MTT method and flow cytometry to detectapoptosis, RT-PCR detected the expression of DAPKmRNA to coast for the clinicalapplication of DAC to provide the theoretical joint As2O3 Basis
Methods
1.Cells proliferation was analyzed by MTT assay
2.Cell apoptosis rate was examined by floe cytometry(FCM);
3.The expression of DAPKmRNA was detected by RT- PCR .
Result
1. MTT results showed that different concentrations of decitabine and As2O3 atdifferent time have a certain degree of inhibition to the NB4 cell.At the same timepoint decitabine and As2O3 on cell growth inhibition in a dose-dependent manner(P＜0.05),the same concentration of decitabine effect reached the peak at the 72h. In thisexperiment, select decitabine1.0and 2.0μmol/L As2O3 0.5 and 1.0μmol/Lconcentration to research.
2. FCM analysis showed that the control group no significant apoptosis by 48htraining (Figure 3a).And the drug treatment group apoptosis can be seen,comparedwith the control group the tate of apoptosis was increased. At the 48h reach the peak5.0% (Figure 3b)after2.0μmol/L DAC achieves and 5.8%(Figure 3c) after 1.0μmol/LAs2O3 achieves,the combination group increased 17.3%(Figure 3d),than the singledrug group increased rate of apoptosis.
3. RT-PCR test results showed, NB4 cells has a low expression or did notexperession of DAPKmRNA, and it maybe re-experssion after the role of the twodrugs. With the increase of drug concentration, its expression increased.
ConclusiConclusion
1. Decitabine on NB4 cell proliferation and induce apoptosis in adose-dependent;
2. DAC Joint As2O3 on NB4 cell proliferation and a synergistic induction ofapoptosis
3. DAC on NB4 cell proliferation and induced apoptosis, one possiblemechanism is to restore or increase the expression of DAPK gene
近年来，通过维甲酸联合亚砷酸诱导分化，急性早幼粒细胞白血病(APL)的预后已大为改观，APL单用全反式维甲酸(ATRA)治疗，近期完全缓解率可达85%以上，但仍有部分患者复发，尽管其治疗前景仍保持乐观，但复发患者有较高的早期死亡和较短的2次缓解期。造血干细胞的移植是目前治疗恶性血液病的最佳方法，但移植期间移植相关并发症较多，且移植费用较高，大多数患者无法承受。目前研究发现大多数急性髓系白血病（AML）患者中存在2种或2种以上基因异常甲基化，由于DNA甲基化是一种可逆转的基因修饰过程,故在肿瘤或癌前病变中通过去甲基化处理则可以恢复基因表达,从而达到防治肿瘤的目的，因此,DNA的去甲基化作用将为肿瘤治疗提供新思路。DAPK基因是抑癌基因，亦是启动凋亡的正性调控因子，具有促进凋亡的作用，其启动子甲基化可能是血液系统肿瘤发生的十分重要的基因表达调控机制。地西他滨(DAC)是一种去甲基化制剂，对耐药和复发的白血病患者有一定的疗效，研究显示，DAC在治疗骨髓增生异常综合征（MDS）及一些实体瘤方面有着良好的疗效，我们用不同浓度的DAC、As2O3及两药联合处理NB4细胞，采用MTT法以及流式细胞术检测细胞凋亡，RT-PCR法检测DAPKmRNA的表达情况，旨在为临床应用DAC联合As2O3提供理论依据。
方法
1.四甲基偶氮唑蓝（MTT）法检测地西他滨及三氧化二砷对细胞株的增殖抑制作用；
2.流式细胞仪检测细胞凋亡率；
3.RT-PCR法检测DAPKmRNA的表达。
结果
1.MTT试验结果显示，不同浓度的地西他滨及三氧化二砷作用不同时间点后均具有一定的抑制作用；在同一时间点两单药组对细胞的生长抑制作用呈剂量依赖性（P＜0.05）,同一浓度地西他滨在72h时间点抑制作用达高峰。本实验中选取地西他滨浓度为1.0和2.0μmol/L、三氧化二砷0.5和1.0μmol/L两个工作浓度进行实验。
2.流式细胞仪结果显示，对照组经48h培养无明显凋亡（图3a），而各药物处理组均可见细胞凋亡，与对照组相比凋亡率明显增高，DAC 2μmol/L作用NB4细胞48h后凋亡率为5.0%（图3b），As2O3 1μmol/L作用NB4细胞48h后凋亡率为5.8%（图3c），联合组凋亡率增加为17.3%（图3d），联合用药组与各单药组比较，凋亡率显著增加。
3. RT-PCR结果显示，NB4细胞株低表达或者不表达DAPMmRNA,两药作用后其表达恢复，并且随着药物浓度的提高其表达增加。
结论
1.地西他滨对NB4细胞有增殖抑制和诱导凋亡的作用；
2.地西他滨发挥作用具有剂量依赖性；
3. DAC联合As2O3对NB4细胞增殖抑制及诱导凋亡有协同作用；
4.地西他滨预处理细胞后，可以增加As2O3对NB4细胞株的敏感性，其机制可能与恢复或增加DAPK的表达有关。
Background
In recent years, by arsenious acid and retinoic acid induced differentiation, Acutepromyelocytic leukemia(APL) treatment has improved considerably,APL treatmentwith retinoic acid alone,near complete remission rate above 85%,but still relapse.Although the outlook remains optimistic about its treatment of recurrence is still,However, patients with recurrence had higher early mortality and shorter remission 2.Hematopoietic stem cell transplantation is the best way to treat hematologicmalignancies,However, many transplant-related complications during the transplant,need to further explore ways to reduce its related complications, and the high cost oftransplantation, most patients can not afford. Recent studies showed that the majorityof acute myeloid leukemia (AML) patients, there is more than 2 or 2 aberrantmethylation, Because DNA methylation is a reversible process of geneticmodification, it is cancer or precancerous lesions in the demethylation treatment by,you can restore gene expression, so as to achieve the purpose of cancerprevention,Thus, DNA methylation to go to provide new ideas for cancer treatment.DAPK gene is a tumor suppressor gene, is also a positive start apoptosis regulatoryfactors, can promote apoptosis, the promoter methylation of tumor blood system maybe a very important mechanism of gene expression. DAC is a demethylating agent, ondrug resistance and relapse of leukemia patients have a certain effect.Study, DAC inthe treatment of myelodysplastic syndrome (MDS) and some solid tumors has a goodeffect. We used different concentrations of DAC, As2O3 and the two drugcombination treatment NB4 cells by MTT method and flow cytometry to detectapoptosis, RT-PCR detected the expression of DAPKmRNA to coast for the clinicalapplication of DAC to provide the theoretical joint As2O3 Basis
Methods
1.Cells proliferation was analyzed by MTT assay
2.Cell apoptosis rate was examined by floe cytometry(FCM);
3.The expression of DAPKmRNA was detected by RT- PCR .
Result
1. MTT results showed that different concentrations of decitabine and As2O3 atdifferent time have a certain degree of inhibition to the NB4 cell.At the same timepoint decitabine and As2O3 on cell growth inhibition in a dose-dependent manner(P＜0.05),the same concentration of decitabine effect reached the peak at the 72h. In thisexperiment, select decitabine1.0and 2.0μmol/L As2O3 0.5 and 1.0μmol/Lconcentration to research.
2. FCM analysis showed that the control group no significant apoptosis by 48htraining (Figure 3a).And the drug treatment group apoptosis can be seen,comparedwith the control group the tate of apoptosis was increased. At the 48h reach the peak5.0% (Figure 3b)after2.0μmol/L DAC achieves and 5.8%(Figure 3c) after 1.0μmol/LAs2O3 achieves,the combination group increased 17.3%(Figure 3d),than the singledrug group increased rate of apoptosis.
3. RT-PCR test results showed, NB4 cells has a low expression or did notexperession of DAPKmRNA, and it maybe re-experssion after the role of the twodrugs. With the increase of drug concentration, its expression increased.
ConclusiConclusion
1. Decitabine on NB4 cell proliferation and induce apoptosis in adose-dependent;
2. DAC Joint As2O3 on NB4 cell proliferation and a synergistic induction ofapoptosis
3. DAC on NB4 cell proliferation and induced apoptosis, one possiblemechanism is to restore or increase the expression of DAPK gene
引文
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