3-MA对三氧化二砷诱导Jurkat细胞凋亡作用的影响

目的:探讨自噬特异性抑制剂3-甲基腺嘌呤(3-MA)对三氧化二砷(As2O3)诱导急性T细胞白血病系Jurkat细胞系Jurkat细胞凋亡的影响及机制。方法:XTT法检测三氧化二砷(As2O3)对急性T细胞白血病细胞系Jurkat细胞的增殖抑制作用。电镜下观察不同浓度As2O3作用Jurkat细胞24 h后细胞形态。免疫印迹和流式细胞术检测微管相关蛋白1轻链3B(LC-3B)蛋白的表达变化。AnnexinV-FITC/PI双染法流式细胞术检测3-MA对AS2O3诱导急性T细胞白血病系Jurkat细胞凋亡的影响。结果:As2O3可以抑制急性T淋巴细胞白血病细胞株Jurkat细胞的生长,这种作用呈剂量和时间依赖性。2.5、5、10μmol/L AS2O3作用Jurkat细胞24 h后在电镜下可观察到自噬、凋亡、坏死的不同形态,并且自噬体数目不断增多。5μmol/L As2O3处理Jurkat细胞0、24、48 h后,LC-3B的平均荧光强度相对倍数分别(3.1±0.2)倍、(4.6±0.31)倍、(34.2±4.5)倍,组间相比,差异有统计学意义(P<0.05),与生长抑制率一样呈现时间依赖性增强;免疫印迹也显示As2O3处理24 h和48 h后,LC-3B的蛋白表达逐渐增强;相对于As2O3组(33.4±9.1)%的生长抑制率,3-甲基腺嘌呤(3-MA)联合As2O3处理细胞后,48 h的生长抑制率为(60.6±8.3)%,差异明显,而LC-3B的表达明显降低。联合应用自噬抑制剂3-MA后Jurkat细胞的凋亡率(44.96±3.60)%,与单用As2O3组(2.94±0.26)%相比明显增加,差异有统计学意义。结论:自噬抑制剂3-MA可以增加As2O3引起的急性T细胞白血病细胞系Jurkat细胞的凋亡细胞百分数,其作用机制与诱导凋亡及抑制自噬密切相关。     

长链非编码RNA HOTAIR对急性淋巴细胞白血病细胞增殖与凋亡的影响

目的:研究长链非编码RNA HOTAIR调控糖皮质激素受体的表达对急性淋巴细胞白血病细胞增殖及凋亡的作用。方法:采用RT-qPCR法检测人正常骨髓基质细胞系HS-5及急性淋巴细胞白血病细胞系MOLT-4、CCRF-CEM和CEM-C1中HOTAIR和糖皮质激素受体的表达。用si RNA沉默CEM-C1细胞中HOTAIR的表达,CCK-8法检测si-HOTAIR对CEM-C1细胞活力的影响; Brd U法检测si-HOTAIR对CEM-C1细胞增殖的影响以及对地塞米松抑制CEM-C1细胞增殖的增效作用; Hoechst 33342染色法和caspase 3/7活性检测法研究si-HOTAIR对CEM-C1细胞凋亡的影响;并采用Western blot法检测糖皮质激素受体的蛋白表达水平。结果:人急性淋巴细胞白血病细胞系MOLT-4、CCRF-CEM和CEM-C1中HOTAIR的表达显著高于人正常骨髓基质HS-5细胞(P 0. 01)。在CEM-C1细胞中干扰HOTAIR表达后,细胞活力降低,细胞增殖被抑制并发生凋亡,地塞米松抑制CEM-C1增殖的作用被增强,糖皮质激素受体表达上调(P 0. 01)。结论:长链非编码RNA HOTAIR增强急性淋巴细胞白血病的活力,促进其增殖并抑制其凋亡,该作用可能与其抑制糖皮质激素受体的表达有关。     

Sirt1增强人慢性粒系白血病K562细胞耐药性

 目的 研究Ⅲ类去乙酰化酶Sirt1对人慢性粒系白血病K562细胞耐药性的影响及其机制。方法 分别将酶活性缺失突变型Sirt1（H363Y）和Sirt1 shRNA的表达质粒转染K562细胞后,用G418筛选出稳定表达酶活性缺失突变型Sirt1或 Sirt1 shRNA的K562细胞。用H2O2和 etoposide处理筛选出来的稳定细胞株,Western Blot检测凋亡标志物caspase3的剪切及Bax的表达,同时检测DNA损伤的标志物H2A.X磷酸化;在MCF-7和293A中过表达野生型Sirt1,检测H2O2和 etoposide处理后H2A.X的磷酸化。结果 在K562细胞中,酶活性缺失突变型Sirt1（H363Y）的表达和Sirt1的干扰均能促进H2O2和 etoposide诱导的caspase3的剪切及Bax的表达并同时显著抑制H2O2和 etoposide诱导的H2A.X磷酸化;在MCF-7和293A细胞中,野生型Sirt1的过表达能明显增强H2O2和 etoposide诱导的H2A.X的磷酸化。结论 Sirt1能保护K562细胞对抗DNA损伤药物诱导的凋亡,增强DNA损伤修复的信号。     

酪氨酸磷酸化抑制剂AG490抑制Jurkat T细胞增殖

目的:通过AG490对Jurkat T细胞活化、增殖、周期、凋亡及ICBP90蛋白表达的影响,探讨阻断JAK/STAT信号通路以抑制Jurkat T细胞生长的可能性及其初步机制。方法:以Jurkat T细胞为模型,应用双荧光抗体标记结合流式细胞仪检测AG490对Jurkat T细胞表面分子CD69和CD25表达的影响;利用噻唑蓝(MTT)比色法观察AG490对Jurkat T细胞增殖的影响;采用碘化丙锭(PI)染色检测AG490对Jur-kat T细胞周期的影响;应用Annexin V-FITC和PI双染色检测AG490对Jurkat T细胞凋亡的影响;Western blot检测AG490对Jurkat T细胞中ICBP90蛋白表达的影响以确定其与AG490抑制Jurkat T细胞增殖的关系。结果:随着AG490浓度从1 mmol/L增至30 mmol/L,细胞停滞于G0/G1期,阻止其进入S期和G2/M期,导致Jurkat T细胞ICBP90蛋白的表达显著降低;AG490对细胞的抑制作用于24 h最为明显,抑制率可达27.37%,呈剂量依赖关系;AG490不能明显抑制Jurkat T细胞的活化或促进其凋亡。结论:AG490能明显抑制Jurkat T细胞的生长,其抑制作用可能通过下调Jurkat T细胞ICBP90蛋白的表达与细胞周期阻滞有关,而不是通过促进细胞凋亡而实现。     

EGCG通过调控SIRT1-P53通路诱导人卵巢癌SKOV-3细胞凋亡

 目的: 研究表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)调控人卵巢癌SKOV-3细胞活力和凋亡的分子机制。方法: SKOV-3细胞给予EGCG(0~50 μmol/L)、SIRT1激动剂SRT1720(1 μmol/L)和SIRT1抑制剂EX527(1 μmol/L)处理后,用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,real-time PCR检测Bax和Bcl-2 mRNA的表达水平;采用SIRT1去乙酰化酶活性检测试剂盒检测SIRT1酶活性;使用Western blot法检测SIRT1、乙酰化P53和P53的蛋白表达变化。结果: 与正常对照组相比,单独给予EGCG或EX527处理之后SKOV-3细胞活力下降,凋亡率增加;SIRT1的酶活性和蛋白表达水平均明显下降;P53的乙酰化水平显著增加。与EGCG组相比,SRT1720预处理组的细胞活力上升,凋亡率下降,Bax/Bcl-2的相对比值及激活型caspase-3的蛋白水平明显下降,并且SIRT1的酶活性和蛋白表达水平显著增加,P53的乙酰化水平下降。结论: EGCG可通过调控SIRT1-P53通路抑制卵巢癌SKOV-3细胞活力并诱导其凋亡。     

miRNA-93在急性淋巴细胞白血病中的表达

目的:探讨微小RNA(miRNA)-93在急性淋巴细胞白血病中的表达情况及其对急性T细胞白血病细胞株Jurkat增殖的影响和潜在作用机制。方法:Real-time PCR技术检测急性白血病患者骨髓样本中miRNA-93的表达水平。转染miRNA-93 inhibitor下调Jurkat细胞中miRNA-93的表达,分别采用CCK-8法、Ed U法和流式细胞术检测细胞活力、增殖及周期,Western blot检测周期相关调控因子cyclin D1、细胞周期蛋白依赖性激酶4(CDK4)、p-Rb及P27的蛋白表达水平。结果:miRNA-93在急性白血病患者中呈现高表达,且在高危患者中表达水平最高;沉默miRNA-93后,Jurkat细胞的活力下降并出现明显的G1/S期阻滞,同时细胞中cyclin D1、CDK4、p-Rb的蛋白水平显著降低,而P27的蛋白水平显著升高。结论:miRNA-93在急性淋巴细胞白血病中表达显著升高;沉默miRNA-93可通过调控周期相关因子的表达抑制急性T细胞白血病细胞株Jurkat的增殖。     

RNAi沉默CD59对急性T系白血病Jurkat细胞株增殖的影响

目的运用RNAi慢病毒沉默CD59的表达,观察其对急性T淋巴细胞白血病Jurkat细胞株增殖、凋亡的影响。方法构建CD59 RNAi-EGFP融合蛋白慢病毒载体,转染急性T系白血病Jurkat细胞株,筛选出稳定转染的细胞系,空病毒组为阴性对照,未处理的Jurkat细胞系作为空白对照;荧光显微镜和流式细胞仪观察各组的细胞转染效率;ELISA检测各组细胞CD59的表达情况;RT-PCR检测各组CD59基因以及凋亡相关基因Bcl-2/Bax m RNA的表达;CCK8表达检测细胞增殖效率的改变;流式细胞术检测各组细胞凋亡情况。结果荧光显微镜和FCM观察转染效率在90%以上;ELISA结果显示实验组细胞CD59蛋白表达降低;RT-PCR结果显示实验组CD59、Bcl-2 m RNA表达水平降低(P0.05),Bax m RNA表达水平升高(P0.05);CCK8结果显示实验组细胞增殖效率明显降低(P0.05);流式细胞仪结果显示沉默CD59的表达能够促进细胞凋亡(P0.05)。结论沉默D59基因表达可抑制急性T系白血病Jurkat细胞株的增殖能力并诱导细胞凋亡,为临床急性T系白血病的治疗提供了新靶标、新思路。     

迷迭香酸通过调控PI3K 通路诱导急性T 细胞白血病Jurkat细胞自噬和细胞凋亡

目的:探究迷迭香酸(RA)对急性T 细胞白血病Jurkat 细胞存活的作用及机制。方法:将细胞随机分为Jurkat 组、RA (5 μmol/ L) 组、RA (10 μmol/ L) 组和RA (20 μmol/ L) 组,分别用0、5、10 和20 μmol/ L 的RA 处理细胞,CCK8 检测培养不同时间的细胞增殖倍数,克隆形成实验检测细胞增殖,流式检测细胞凋亡,免疫印迹检测Beclin1、P62、LC3、p-PI3K、PI3K、p-Akt、Akt 和mTOR 的表达,免疫荧光检测LC3 的表达。结果:RA 处理细胞4 d 后,RA (5、10、20 μmol/ L) 组细胞增殖倍数与Jurkat 组比较明显降低,存活细胞数明显减少,细胞凋亡率明显升高;同时与Jurkat 组比较,RA (5、10、20 μmol/ L) 组细胞Beclin1 表达水平和LC3Ⅰ/ LCⅡ的比值明显降低,P62 表达水平明显升高,LC3 阳性表达与Jurkat 组比较也明显减少;此外,RA (5、10、20 μmol/ L) 还能显著降低p-PI3K/ PI3K、p-Akt/ Akt 的比值和mTOR 的表达水平。结论:RA 可诱导急性T 淋巴白血病Jurkat 细胞自噬和细胞凋亡,作用机制可能与抑制PI3K/ Akt 通路激活有关。     

组蛋白去乙酰化酶抑制剂TSA诱导人乳腺癌细胞凋亡及自噬

目的 探讨组蛋白去乙酰化酶抑制剂曲古霉素(TSA)对人乳腺癌细胞凋亡及自噬的作用及其可能的分子机制。方法采用MTT法检测T47D细胞的增殖能力;流式细胞术检测细胞凋亡的变化;Western blot法检测凋亡及自噬相关蛋白的表达。结果 TSA对人乳腺癌细胞T47D具有增殖抑制作用(P0.05);TSA诱导T47D细胞发生凋亡,下调BCL-2/Bax比值,上调Caspase-3的表达(P0.05);同时自噬相关蛋白LC3B及Beclin-1的表达也明显增加(P0.05,P0.01)。结论 TSA体外能抑制乳腺癌细胞的增殖,其机制与诱导细胞凋亡和自噬作用有关。     

Cytogenetic characterization of a T-cell line, ATN-1, derived from adult T-cell leukemia cells

A T-cell line, ATN-1, was established by culturing peripheral blood mononuclear cells derived from a patient with adult T-cell leukemia/lymphoma (ATL/L). Identities of the patterns of chromosomal abnormalities, cell surface phenotypes, morphologic findings, rearrangement patterns of T-cell receptor beta chain gene, and an integration site of human T-cell leukemia virus I proviral genome indicated that ATN-1 was derived from original leukemic cells. Both ATN-1 and the original leukemic cells showed a variety of patterns of chromosomal abnormalities that include 3q-, 6q-, rearrangements involving 2q31, 7q11.2, 8q11, 8q24, 19p13.3, and also 14q11 and 14q32, where genes for the T-cell receptor alpha chain and the immunoglobulin heavy chain are located. Availability of a genuine ATL/L cell line with these chromosomal abnormalities may greatly facilitate the biologic analysis of ATL/L.     

Does the HBZ gene represent a new potential target for the treatment of adult T-cell leukemia?

Links between human T-cell leukemia virus type 1 and adult T-cell leukemia (ATL) were first suspected in 1980. Provirus integration has since been found in all ATL cells. Although the viral Tax protein is involved in the proliferation of the infected cells during the preleukemic stage, Tax expression is not systematically detected in primary leukemic cells. Recent studies found that the viral HBZ gene was always expressed in leukemic cells, suggesting its involvement in the progression of the infected cells toward malignancy. How could this new discovery be translated into possible new avenues for the prevention or treatment of ATL?     

白血病患儿骨髓间充质干细胞与白血病细胞株K562/AO2生长增殖及凋亡的相关性

背景:骨髓微环境是白血病的发病根源,微环境的改变对白血病细胞的生物学行为和性能有一定的影响。目的:探讨白血病患儿骨髓间充质干细胞对K562/AO2细胞生长增殖及凋亡的影响。方法:体外环境下进行K562/AO2细胞单独悬浮培养,K562/AO2细胞与白血病患儿骨髓间充质干细胞共培养。锥虫蓝染色计数活细胞数,描绘两组细胞生长曲线,PI单染法检测细胞周期,Annexin V/PI法检测细胞凋亡情况。结果与结论:(1)单独培养组前3 d细胞生长未出现明显改变,第4天开始细胞增殖速度明显加快,并于培养第6天达到最高峰。共培养组整体增殖曲线较为平缓,无明显的增殖高峰;(2)单独培养组的G0-G1期细胞比例显著低于共培养组,S期细胞比例显著高于共培养组(P<0.05)。两组G2-M期细胞比例差异无显著性意义;(3)单独培养组的细胞凋亡数显著高于共培养组(P<0.05);(4)结果表明,与白血病患儿骨髓间充质干细胞共培养后K562/AO2细胞生长受到抑制,细胞周期阻滞在G0-G1期,且会对K562/AO2细胞凋亡产生抵抗作用。     

黄芩苷通过影响miR-141上调Sirt1表达而抑制高糖诱导的小鼠肾小球系膜细胞凋亡

目的:探讨黄芩苷在高糖环境下对小鼠肾小球系膜细胞凋亡的影响,并从微小RNA-141(miR-141)/沉默信息调节因子1(Sirt1)通路深入阐释黄芩苷对糖尿病肾病的治疗机制。方法:高糖(25 mmol/L葡萄糖)培养小鼠肾小球系膜细胞系SV40-MES-13模拟构建糖尿病肾病细胞模型,并分为正常对照组、高糖组、黄芩苷组和高糖+黄芩苷组。q PCR检测miR-141的表达水平,Western blot检测Sirt1的表达水平。双萤光素酶实验检测Sirt1与miR-141的调控关系。流式细胞术检测细胞凋亡。结果:与正常对照组相比,高糖条件下培养的系膜细胞内miR-141表达水平明显上调,Sirt1蛋白表达水平明显下调,细胞凋亡水平显著增加(P0.01);与高糖组比,高糖+黄芩苷组的细胞凋亡和miR-141表达水平明显降低,Sirt1蛋白表达水平明显升高(P0.01)。敲减Sirt1表达可以逆转下调miR-141对系膜细胞细胞凋亡和细胞内Sirt1蛋白水平的作用。过表达miR-141可以逆转黄芩苷抑制高糖对Sirt1蛋白水平和细胞凋亡的作用;过表达miR-141对系膜细胞的作用可以进一步被Sirt1的过表达所逆转。结论:黄芩苷可以通过抑制miR-141过表达,促进Sirt1表达水平上升,最终缓解高糖诱导的小鼠肾小球系膜细胞凋亡,达到治疗糖尿病肾病的作用。     

Does the HBZ Gene Represent a New Potential Target for the Treatment of Adult T-Cell Leukemia?

Links between human T-cell leukemia virus type 1 and adult T-cell leukemia (ATL) were first suspected in 1980. Provirus integration has since been found in all ATL cells. Although the viral Tax protein is involved in the proliferation of the infected cells during the preleukemic stage, Tax expression is not systematically detected in primary leukemic cells. Recent studies found that the viral HBZ gene was always expressed in leukemic cells, suggesting its involvement in the progression of the infected cells toward malignancy. How could this new discovery be translated into possible new avenues for the prevention or treatment of ATL?     

NF-kappaB in pathogenesis and treatment of adult T-cell leukemia/lymphoma

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) induces aberrant nuclear factor-kappaB (NF-kappaB) activation. Although Tax is thought to play major roles in NF-kappaB activation, cells expressing Tax become a target of cytotoxic T cells. Accordingly, HTLV-1-infected cells lose Tax expression and acquire Tax-independent NF-kappaB activation. Blocking NF-kappaB not only induces apoptosis in adult T-cell leukemia/lymphoma (ATL) cells but also reduces the number of HTLV-1-infected cells in virus carriers. Therefore, because constitutively activated NF-kappaB appears to be the common biological basis shared between HTLV-1-infected untransformed cells and ATL cells, blocking NF-kappaB might be a potential strategy for treating and preventing ATL.     

Human T-cell lymphotropic virus type 1 infection of CD34+ hematopoietic progenitor cells induces cell cycle arrest by modulation of p21(cip1/waf1) and survivin

Human T-cell lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus and the etiologic agent of adult T-cell leukemia (ATL), an aggressive CD4(+) malignancy. HTLV-2 is highly homologous to HTLV-1; however, infection with HTLV-2 has not been associated with lymphoproliferative diseases. Although HTLV-1 infection of CD4(+) lymphocytes induces cellular replication and transformation, infection of CD34(+) human hematopoietic progenitor cells (HPCs) strikingly results in G(0)/G(1) cell cycle arrest and suppression of in vitro clonogenic colony formation by induction of expression of the cdk inhibitor p21(cip1/waf1) (p21) and concurrent repression of survivin. Immature CD34(+)/CD38(-) hematopoietic stem cells (HSCs) were more susceptible to alterations of p21 and survivin expression as a result of HTLV-1 infection, in contrast to more mature CD34(+)/CD38(+) HPCs. Knockdown of p21 expression in HTLV-1-infected CD34(+) HPCs partially abrogated cell cycle arrest. Notably, HTLV-2, an HTLV strain that is not associated with leukemogenesis, does not significantly modulate p21 and survivin expression and does not suppress hematopoiesis from CD34(+) HPCs in vitro. We speculate that the remarkable differences in the activities displayed by CD34(+) HPCs following infection with HTLV-1 or HTLV-2 suggest that HTLV-1 uniquely exploits cell cycle arrest mechanisms to establish a latent infection in hematopoietic progenitor/hematopoietic stem cells and initiates preleukemic events in these cells, which eventually results in the manifestation of ATL.     

Safety,efficacy, and pharmacokinetics/pharmacodynamics of daclizumab (anti-CD25) in patients with adult T-cell leukemia/lymphoma

Interleukin-2 receptor α chain (CD25) is overexpressed in human T-cell leukemia virus 1 associated adult T-cell leukemia/lymphoma (ATL). Daclizumab a humanized monoclonal antibody blocks IL-2 binding by recognizing the interleukin-2 receptor α chain (CD25). We conducted a phase I/II trial of daclizumab in 34 patients with ATL. Saturation of surface CD25 on circulating ATL cells was achieved at all doses; however saturation on ATL cells in lymph nodes required 8 mg/kg. Up to 8 mg/kg of daclizumab administered every 3 weeks was well tolerated. No responses were observed in 18 patients with acute or lymphoma ATL; however, 6 partial responses were observed in 16 chronic and smoldering ATL patients. The pharmacokinetics/pharmacodynamics of daclizumab suggest that high-dose daclizumab would be more effective than low-dose daclizumab in treatment of lymphoid malignancies and autoimmune diseases (e.g., multiple sclerosis) since high-dose daclizumab is required to saturate IL-2R alpha in extravascular sites.