RKIP过表达对儿童白血病CCRF-CEM细胞系凋亡的影响

目的观察Raf激酶抑制蛋白（RKIP）对儿童白血病细胞系CCRF-CEM凋亡的影响。方法以RT-PCR方法从HepG2细胞总RNA中克隆RKIP基因CDS区,并亚克隆至pCDNA3.0质粒,构建RKIP重组质粒载体pCD-NA3.0-RKIP,以电穿孔法转染pCDNA3.0-RKIP与对照质粒pCDNA3.0至儿童白血病细胞系CCRF-CEM,建立过表达RKIP的细胞系。Western blot法检测转染48 h后细胞RKIP与磷酸化ERK的表达水平。转染72 h后,用Annex-in-V与PI双标、流式细胞仪检测细胞凋亡比例。结果成功构建携带RKIP的重组质粒载体pCDNA3.0-RKIP,通过电穿孔转染,在CCRF-CEM细胞成功高表达。与对照细胞相比,RKIP过表达CCRF-CEM细胞磷酸化ERK水平降低,凋亡比例显著增高（P〈0.01）。结论 RKIP过表达诱导CCRF-CEM细胞凋亡,可能机制是通过抑制Ras-ERK信号通路。     

左旋门冬酰胺酶致低血糖反应3例

左旋门冬酰胺酶(L-Asparaginase)是治疗急性淋巴细胞白血病及实体瘤的常用药物，特别是对急性淋巴细胞白血病疗效甚佳，其副作用常见有食欲减退，恶心，呕吐，腹泻，头痛，头晕，低蛋白血症，血脂过高或过低，氮质血症和肝功能损害，部分患者有骨髓抑制，凝血障碍，有的患者有脱发，蛋白尿，胰腺炎，高血糖等，还有的出现过敏反应，致过敏性休克，但出现低血糖反应临床少有报道。我院在应用左旋门冬酰胺酶治疗急性淋巴细胞白血病过程中，曾有3例患者出现低血糖反应，现报告如下。     

高三尖杉酯碱联合survivin抑制剂YM155对人白血病K562细胞系增殖和凋亡的影响

目的:探讨高三尖杉酯碱(HHT)联合survivin抑制剂YM155对人白血病K562细胞系增殖抑制和诱导凋亡的作用及机制。方法:CCK法检测细胞增殖抑制率,依据中效原理及CalcuSyn软件评价HHT和YM155的联合治疗效果。设空白对照组、HHT组、YM155组和联合用药组,采用Annexin V/PI双染法以流式细胞仪检测细胞凋亡率,Western blot法检测survivin蛋白变化,以特异性荧光底物检测caspase-3、-8活性变化。结果:HHT和YM155单药均以浓度依赖方式抑制K562细胞生长,二者联合应用具有化疗协同作用(CI1)。联合用药组K562细胞凋亡率明显高于单独用药组,且survivin蛋白表达显著下调(均P0.05)。HHT单药及联合用药组诱导caspase-3、-8活化,而YM155单药未引起caspase-3、-8活性变化。结论:HHT和YM155联合应用于K562细胞具有化疗协同作用,通过下调survivin诱导K562细胞凋亡。     

多克隆抗胸腺细胞球蛋白对白血病细胞增殖抑制及凋亡诱导作用

目的:探讨多克隆兔抗人胸腺细胞球蛋白(ATG)对白血病细胞的生长抑制以及诱导凋亡作用。方法:体外培养Jurkat、Raji、HL-60、NB4、K562、U937细胞株以及15例患者原代白血病细胞。用50、100、150、200、250μg/mL终浓度的ATG在不同时间分别作用于以上细胞,采用细胞计数试剂盒(CCK-8)比色法检测细胞增殖抑制率,应用流式细胞术测定细胞凋亡率。结果:ATG对Jurkat、Raji、HL-60细胞有明显的增殖抑制以及诱导凋亡作用,且高浓度ATG抑制作用更强。ATG对NB4、K562、U937细胞增殖抑制与诱导凋亡作用均不明显。15例初发白血病患者中7例的原代细胞经ATG作用后明显凋亡,凋亡率均>30%。结论:ATG具有广谱抗白血病作用,尤其是对淋巴细胞白血病作用较强,为其在异基因造血干细胞移植中的应用提供了实验依据。     

左旋门冬酰胺酶治疗急性淋巴细胞性白血病并发糖尿病的护理分析

目的探究急性淋巴细胞性白血病并发糖尿病应用左旋门冬酰胺酶的临床效果及护理。方法选取该院2018年9月—2020年4月收治急性淋巴细胞性白血病并发糖尿病患者190例,按数字法分为对照组和研究组,各95例,两组患者均给予左旋门冬酰胺敏进行治疗,对照组实施常规糖尿病护理干预,研究组在对照护理基础上加以综合护理干预,对比两组患者血糖相关指标、不良反应发生率以及患者满意程度。结果研究组FPG、2 hPG以及HbA1c血糖相关指标均较对照组有所改善,差异有统计学意义(t=5.220、6.478、5.058,P0.001);研究组不良反应发生率例数2.11%(2/95),低于对照组不良反应发生率9.47%(9/95),差异有统计学意义(χ~2=4.728,P0.05);研究组患者的护理满意度(97.89%)高于对照组护理满意度(90.52%),差异有统计学意义(χ~2=4.728,P=0.030)。结论综合护理干预联合左旋门冬酰胺酶用于急性淋巴细胞性白血病并糖尿病患者,有助于改善血糖水平,减少不良反应,提高患者对护理工作满意程度。     

VAD-L-asp治疗难治/复发成人急性淋巴细胞白血病的观察与护理

近年来,我们设计了长春新碱(VCR)、阿霉素(ADM)及地塞米松(DEX)联合左旋门冬酰胺酶(L-asp)的方案(VAD-L-asp),治疗了18例难治/复发急性淋巴细胞白血病(ALL),经过密切观察和精心护理,取得了较高的完全缓解(CR)率.现报告如下.     

HCTB006联合5-FU对胃癌细胞系MKN28、7901的作用及其机制

目的:探讨肿瘤坏死因子相关诱导配体受体(DR5)的单克隆抗体HCTB006联合5-FU对人胃癌细胞系7901、MKN28的作用以及机制.方法:用ATPlite法检测HBCT006单药组、5-FU单药组及两药物合用对胃癌细胞存活率的影响,研究两者之间的关系;采用流式细胞技术检测胃癌细胞系7901以及MKN28表面DR5的表达水平;Westernblot检测上述3组用药后胃癌细胞内XIAP,caspase3的变化.结果:胃癌细胞系7901、MKN28对HCTB006不敏感;5-FU对二者的增殖抑制作用具有时间以及浓度依赖效应;联合用药组具有很好的协同抑制胃癌细胞系增殖的效果,且具有浓度依赖效应,与给药次序无关.流式细胞技术检测胃癌细胞系7901,MKN28表面死亡受体DR5的表达依次为:93.8%以及87.7%.免疫迹印结果表明,联合用药组可以引发胃癌细胞内凋亡抑制蛋白XIAP的降解,激活最终凋亡执行蛋白caspase3,引起细胞死亡.结论:HTB006与5-FU联合应用具有协同杀伤胃癌细胞的作用.胃癌细胞7901、MKN28对于HCTB006的敏感程度与细胞表面DR5的表达量不相关;联合用药作用机制可能与细胞内抑制凋亡蛋白XIAP降解有关.     

表皮生长因子酪氨酸抑制剂联合奥沙利铂对肝癌细胞Hep-G2作用

目的探讨表皮生长因子受体(epidermal growth factor receptor,EGFR)抑制剂易瑞沙联合奥沙利铂对人肝癌细胞株增殖和细胞凋亡的影响,为原发性肝癌分子靶向治疗和联合化疗提供临床依据。方法利用噻唑蓝(MTT)法测定奥沙利铂、易瑞沙对细胞增殖的抑制作用及两药联合时的细胞杀伤作用。重复试验3次,每次检测5孔细胞,将平均值作为最终结果,并用中效法则判定两药合用的效果。采用流式细胞仪测定易瑞沙单药应用于Hep-G2细胞,对细胞凋亡的影响。结果易瑞沙联合奥沙利铂增加化疗药对Hep-G2细胞的抑制作用,并呈时间剂量依赖性。易瑞沙单药作用于Hep-G2细胞,发生了细胞凋亡。结论易瑞沙与奥沙利铂的联合应用有望成为治疗肝癌的有效手段。     

益气活血软坚解毒含药血清诱导人肝癌细胞系Bel-7402细胞的凋亡

目的:研究益气活血软坚解毒(YHRJ)含药血清对人肝癌细胞系Bel-7402生长抑制及诱导凋亡作用.方法:将YHRJ含药血清作用于人肝癌细胞系Bel-7402细胞,应用MTT检测对肝癌细胞生长抑制作用,倒置显微镜、荧光显微镜、激光共聚焦扫描显微镜等影像学方法观察细胞形态学变化,以及PI染色单染、AnnexinV-PI双染后,流式细胞术检测对细胞周期影响及诱导细胞凋亡程度.结果:MTT法检测结果显示:YHRJ含药血清具有抑制Bel-7402肿瘤细胞生长作用(其中20%YHRJ等效剂量抑制率49.1%,与NS比,P<0.01);荧光显微镜及激光共聚焦显微镜可观察到典型的凋亡形态学变化.流式细胞术检测结果,细胞周期出现G0/G1期阻滞,并出现典型的凋亡峰,AnnexinV-PI双染法检测到早期及中晚期细胞凋亡.结论:YHRJ含药血清有抑制人肝癌细胞系Bel-7402细胞生长并有诱导细胞凋亡作用.     

L-门冬酰胺酶对人白血病细胞的核酸代谢和细胞周期的影响

作者在5例急性淋巴细胞性白血病患儿研究了L—门冬酰胺酶对白血病细胞的核酸代谢和细胞周期的影响。 L-门冬酰胺酶以200单位/公斤的剂量静脉滴注,在30分钟内滴完,然后在48小时内连续采取骨髓标本,测定白细胞层的容积、分裂指数以及利用氚化胸腺嘧啶核苷和氚化嘧啶核苷的放射自显影法分别测定去氧核糖核酸(DNA)和核糖核酸(RNA)的合成。给药后白细胞层的容积迅速下降,说明L-门冬酰胺酶对原始淋巴细胞有溶解作用。     

弓形虫ROP16蛋白在人白血病细胞THP-1表达及对其细胞增殖与凋亡的影响北大核心CSCD

目的探讨弓形虫Ⅱ型(Me49)ROP16蛋白在急性单核细胞白血病细胞株THP-1中的表达及其对THP-1细胞增殖与凋亡的影响。方法构建过表达ROP16(overExp-ROP16)和空载体(overExp-NC)慢病毒,通过转染THP-1细胞,72 h后观察荧光表达情况,采用PCR与Western blot验证ROP16在THP-1细胞中的表达。免疫荧光技术检测ROP16蛋白在THP-1细胞中的定位。CCK-8与流式细胞术检测细胞增殖及凋亡。采用qRT-PCR检测细胞凋亡相关因子Bcl-2、Bax、Caspase-3 mRNA相对表达水平。Western blot检测Bax、Bcl-2、Pro-Caspase-3和Cleaved-Caspase-3蛋白的表达变化。结果构建的过表达ROP16重组慢病毒转染到THP-1细胞后,可观察强荧光信号,ROP16蛋白在THP-1细胞中成功表达。免疫荧光结果表明ROP16蛋白定位于THP-1细胞核。CCK-8与流式细胞术证实ROP16蛋白抑制THP-1细胞的增殖并促进其凋亡(均P<0.05)。与对照组相比,过表达组促凋亡因子Bax、Caspase-3 mRNA高表达(P<0.05),抗凋亡因子Bcl-2 mRNA低表达(P<0.01),Bax与Cleaved-Caspase-3蛋白水平上调(P<0.01),Bcl-2与Pro-Caspase-3蛋白水平下调(P<0.01)。结论构建的慢病毒表达质粒overExp-ROP16可在急性单核细胞白血病细胞株THP-1中表达且通过入核调节凋亡蛋白Bcl-2、Bax、Caspase-3而抑制THP-1细胞增殖,促进其凋亡。     

Evaluation of glucocorticoid sensitivity in 697 pre-B acute lymphoblastic leukemia cells after overexpression or silencing of MAP kinase phosphatase-1

Purpose To determine the effect of modulating MAP kinase phosphatase-1 (MKP-1) expression levels on cell death induced by glucocorticoid (GC) or hydroxyurea (HU) treatment in the human pre-B acute lymphoblastic leukemia cell line 697.Methods Stable MKP-1 overexpressing transformants of the 697 pre-B acute lymphoblastic leukemia cell line were created and tested for sensitivity to the GC triamcinolone acetonide (TA) and HU, and compared to a control 697 cell line containing normal MKP-1 expression levels. Small interfering RNAs (siRNAs) were designed to inhibit MKP-1 expression and evaluated for their effect on GC-mediated cell death.Results MKP-1 overexpression caused a phenotype of partial resistance to HU-induced apoptosis but not to GC-induced apoptosis. Electroporation of siRNAs effectively silenced MKP-1 expression, and increased sensitivity to TA by 9.6±1.9%.Conclusions Because MKP-1 protects certain tumor cells from chemotherapy-induced apoptosis, its inhibition is being considered as a possible strategy for combination cancer therapy. However, this study suggests that while MKP-1 inhibition may improve the efficacy of DNA damaging agents, it may have only limited utility in combination with glucocorticoids.Grant support: DOE/BER ER63055 to E.W.     

ZAP-70 is highly expressed in most cases of childhood pre-B cell acute lymphoblastic leukemia

The tyrosine kinase ZAP-70 plays a critical role in signal transduction in T cells and NK cells but has limited expression in primary human B cells. ZAP-70 is, however, expressed in adult B cell chronic lymphocytic leukemia where it correlates with a poor prognosis. We wished to determine if ZAP-70 is also expressed in pediatric B cell malignancy. A quantitative PCR assay for ZAP-70 expression was established and ZAP-70 expression in a range of human B cell lines was compared with expression in the Jurkat T cell line. ZAP-70 expression was then determined in bone marrow lymphoblasts obtained from 12 patients with pre-B cell acute lymphoblastic leukemia (ALL). ZAP-70 expression was not detected in mature B cell lines but was detected in pre-B cell lines at a level comparable to that seen in T cells. ZAP-70 expression was strongly expressed in nine of the 12 cases of primary pre-B cell lymphoblastic leukemia. The T cell-associated protein kinase ZAP-70 is highly expressed in pre-B lineage cells and most cases of pre-B acute lymphoblastic leukemia. ZAP-70 expression may hold prognostic value for pre-B ALL and raises the prospect of a novel therapeutic target.     

Mometasone furoate inhibits growth of acute leukemia cells in childhood by regulating PI3K signaling pathway

Objectives: Acute lymphoblastic leukemia (ALL) is the most common cancer before the age of 15 years, seriously endangering the health of children. The main treatment for Childhood ALL was pharmacotherapy. But these drugs have many side effects and some of them could develop drug resistance quickly. Mometasone furoate (MF) is an efficient glucocorticoid for topical treatment of inflammation on the skin, lung and nose.

Methods: In this study, we investigated whether the MF had effects on ALL cells proliferation and migration.

Results: The CCK-8 proliferation test showed that the cell viability was the lowest at 25?nM MF treatment and the increased OD value was time-dependent. In transwell assay, the number of CCRF-CEM cells was reduced in MF treated group. We found the expression of anti-apoptotic protein bcl-2 decreased the expression of pro-apoptotic protein caspase3 and bax increased in CCRF-CEM cell line treated with MF. The expression of p-AKT, p-mTOR, p70S6?K, vascular endothelial growth factor and CyclinD1 were decreased in MF treated group.

Conclusion: This study reveals that MF can inhibit proliferation and invasion/migration and induce apoptosis in Childhood ALL cells, which may be regulated by Phosphatidylinositol 3-kinase signaling pathway. These results suggest MF may be a potential new drug target for clinical ALL treatment.     

Bufalin Induces the Apoptosis of Acute Promyelocytic Leukemia Cells via the Downregulation of Survivin Expression

Background and Aims: Bufalin is a cardiotonic steroid isolated from the Chinese toad venom preparation Chan'su and has been shown to induce leukemia cell differentiation and apoptosis under certain experimental conditions. However, the detailed mechanism by which bufalin induces the apoptosis of acute promyelocytic leukemia cells is largely unexplored. Methods: The acute promyelocytic leukemia cell line NB4 was treated with bufalin, then the proliferation was evaluated by cell viability assay and apoptosis was detected by flow cytometry analysis. In addition, NB4 cells were treated by MEK inhibitor PD98059 in combination with bufalin, and the expression of survivin and activation of caspase-3 were detected by Western blot analysis. Results: Bufalin inhibited the proliferation and induced the apoptosis of NB4 cells in a dose- and time-dependent manner. Moreover, bufalin synergized with PD98059 to inhibit the proliferation and induce the apoptosis of NB4 cells, which was associated with the downregulation of survivin expression and the upregulation of caspase-3 activation. Conclusions: Bufalin is a potential regimen to be used in combination with conventional chemotherapeutic drugs to improve acute promyelocytic leukemia therapy.     

Proteasome inhibitor bortezomib overcomes P-gp-mediated multidrug resistance in resistant leukemic cell lines

Introduction: To study the effect of bortezomib alone or in combination with daunorubicin (DNR) on an mdr1 single‐factor drug‐resistant leukemia cell line K562/MDR1, a multifactor‐resistant cell line K562/A02, a drug‐sensitive cell line K562, and primary cells from acute myeloid leukemia patients. Methods: The cell lines were exposed to bortezomib, DNR, and bortezomib plus DNR, and cell proliferation, cell cycle, apoptosis rate, and expression of MDR1/BCL2 were analyzed. Results: Bortezomib potently inhibited growth and increased the apoptosis rate in the cell lines. In K562/MDR1 and K562/A02, the calcium channel blocker verapamil reduced the 50% inhibitory concentration and apoptosis rate of DNR, a P‐gp protein substrate, but not of bortezomib. Bortezomib plus DNR had synergistic effect on antiproliferation (synergistic ratio > 1). Apoptosis was substantially more increased by the combination of two drugs than by bortezomib alone. Bortezomib arrested the cell cycles of three cell lines at the G2/M stage, decreased BCL2 mRNA expression, but did not affect MDR1 mRNA levels. The antiproliferative role of bortezomib was also confirmed in primary leukemia cells. Conclusion: Bortezomib is a promising potential therapy for acute leukemia, especially mdr1 drug‐resistant leukemia.     

Melatonin exerts differential actions on X-ray radiation-induced apoptosis in normal mice splenocytes and Jurkat leukemia cells

Abstract:  The ability of melatonin as a potent antioxidant was used as a rationale for testing its antiapoptotic ability in normal cells. Recently, melatonin was shown to possess proapoptotic action by increasing reactive oxygen species in certain cancer cells. The modification of radiation-induced apoptosis by melatonin and the expression of apoptosis-associated upstream regulators were studied in normal mice splenocytes and Jurkat T leukemia cells. C57BL/6 mice were exposed to a single whole body X-ray radiation dose of 2 Gy with or without 250 mg/kg melatonin pretreatment. The Jurkat cells were divided into four groups of control, 1 m m melatonin alone, 4 Gy irradiation-only and melatonin pretreatment before irradiation. The highest level of apoptosis in the normal splenic white pulp was detected by TUNEL assay at 8 hr after irradiation. At this time, the apoptotic index of irradiation-only and melatonin pretreatment groups were 35.6% and 20.7%, respectively. This reduced apoptosis by melatonin was associated with the increase of Bcl-2 expression and a reduction of Bax/Bcl-2 ratio through a relative decrease of p53 mRNA and protein. In the Jurkat cells treated with a combination of melatonin and radiation, both Annexin V-FITC(+)/PI(−) and Annexin V-FITC(+) cells were increased at 48 hr after irradiation when compared with irradiation-only or melatonin alone. The expressions of p53 between groups were well correlated with the results of Annexin V binding. The irradiation or melatonin did not influence the JNK1 expression in Jurkat cells. The present results suggest that melatonin enhances radiation-induced apoptosis in Jurkat leukemia cells, while reducing radiation-induced apoptosis in normal mice splenocytes. These differential effects on radiation-induced apoptosis by melatonin might involve the regulation of p53 expression.     

Inhibiting Polo-like kinase 1 causes growth reduction and apoptosis in pediatric acute lymphoblastic leukemia cells

This study investigated Polo-like kinase 1, a mitotic regulator often over-expressed in solid tumors and adult hematopoietic malignancies, as a potential new target in the treatment of pediatric acute lymphoblastic leukemia. Polo-like kinase 1 protein and Thr210 phosphorylation levels were higher in pediatric acute lymphoblastic leukemia (n=172) than in normal bone marrow mononuclear cells (n=10) (P<0.0001). High Polo-like kinase 1 protein phosphorylation, but not expression, was associated with a lower probability of event-free survival (P=0.042) and was a borderline significant prognostic factor (P=0.065) in a multivariate analysis including age and initial white blood cell count. Polo-like kinase 1 was necessary for leukemic cell survival, since short hairpin-mediated Polo-like kinase 1 knockdown in acute lymphoblastic leukemia cell lines inhibited cell proliferation by G2/M cell cycle arrest and induced apoptosis through caspase-3 and poly (ADP-ribose) polymerase cleavage. Primary patient cells with a high Polo-like kinase 1 protein expression were sensitive to the Polo-like kinase 1-specific inhibitor NMS-P937 in vitro, whereas cells with a low expression and normal bone marrow cells were resistant. This sensitivity was likely not caused by Polo-like kinase 1 mutations, since only one new mutation (Ser335Arg) was found by 454-sequencing of 38 pediatric acute lymphoblastic leukemia cases. This mutation did not affect Polo-like kinase 1 expression or NMS-P937 sensitivity. Together, these results indicate a pivotal role for Polo-like kinase 1 in pediatric acute lymphoblastic leukemia and show potential for Polo-like kinase 1-inhibiting drugs as an addition to current treatment strategies for cases expressing high Polo-like kinase 1 levels.     

肉苁蓉多糖诱导Jurkat细胞凋亡作用

目的 探讨肉苁蓉多糖(CDP)对人急性白血病细胞系Jurkat细胞凋亡的影响.方法 利用噻唑蓝(MTT)比色法检测CDP对细胞增殖的影响,Western印迹检测凋亡相关蛋白含半胱氨酸的天冬氨酸蛋白水解酶(caspase)-9和caspase-3的活化情况,同时检测CDP诱导细胞凋亡的相关信号分子.结果 CDP在1 mg...