视黄酸依赖反义cyclinD1 RNA表达载体的构建与鉴定

目的 构建人细胞周期素D1(CyclinD1)基因的视黄酸(Retinoic acid，RA)依赖反义RNA表达载体，为进一步在细胞内应用视黄酸诱导其表达从而发挥效应的肿瘤基因治疗奠定基础。方法 运用DNA重组技术构建含有RARE3-TK启动子的反义cyclin D1 RNA真核表达载体(pCI-neo／RARE3-TK／AScyclin D1)，经酶切、PCR以及DNA测序确认后，用脂质体方法转染HL-60细胞(人早幼粒白血病细胞系)，RA处理后，利用免疫组织化学方法检测细胞内靶基因cyclin D1的表达情况。结果 成功构建重组反义cyclin D1 RNA表达载体，与预期设计路线相符；检测DNA序列与原始片段互补，且方向相反。ATRA处理转染和未转染HL-60细胞后，cyclin D1的表达均有所下降，其中转染组细胞的表达量要明显低于未转染组。结论 本实验成功构建了视黄酸依赖反义cyclin D1 RNA表达载体；转染HL-60细胞后，反义cyclin D1的表达可受到RA的诱导调控。     

视黄酸依赖Fas/RARα融合基因转染HL-60细胞启动的凋亡效应研究

目的：检测视黄酸依赖Fas／RARα表达载体转染HL-60细胞启动的凋亡效应。方法：成功构建视黄酸依赖Fas／RARα表达载体，用脂质体转染HL-60白血病细胞，经四甲基偶氮唑盐[3-(4，5-dimethylthiaol-2-yl)-2，5-diphenyl tetrazolium bromide,MTT]、流式细胞、DNA梯形带和透射电镜等方法观测视黄酸处理后细胞凋亡效应的发生。结果：以一定剂量的视黄酸处理HL-60后，细胞增殖受抑，呈凋亡性变。结论：视黄酸及其依赖的融合基因表达可协同诱导HL-60细胞发生凋亡效应。     

二十碳五烯酸联合视黄酸对HL-60细胞增殖与

目的研究二十碳五烯酸(eicosapentaenoicacid,EPA)及其联合视黄酸(retinoicacid,RA)对白血病细胞增殖与分化功能的影响.方法采用MTT法测定细胞增殖功能,NBT还原实验鉴定细胞分化,高效液相色谱法分析细胞内RA代谢.结果与对照组相比,EPA组细胞增殖抑制率为24.38%,RA组为35.74%,EPA+RA组为42.75%;NBT还原实验,EPA+RA组OD值约为对照组的5.9倍,而RA组为2.6倍,EPA组为1.3倍;分析HL-60细胞及其培养介质中RA含量,发现EPA+RA组高于RA组.结论EPA可抑制HL-60细胞增殖和诱导其分化,与RA联合应用能明显增强HL-60细胞的增殖抑制及诱导分化效应,这种联合效应可能与EPA减缓HL-60细胞内RA的代谢相关.     

二十碳五烯酸联合视花酸对HL—60细胞增殖与分化功能的影响

目的 研究二十碳五烯酸（eicosapentaenoic acid,EPA）及其联合视黄酸（retinoic acid,RA）对白血病细胞增殖与分化功能的影响。方法 采用MTT法测定细胞增殖功能,NBT还原实验鉴定细胞分化,高效液相色谱法分析细胞内RA代谢。结果 与对照组相比,EPA组细胞增殖抑制率为24.38%,RA组为35.74%,EPA RA组为42.75%;NBT还原实验,EPA RA组OD值约为对照组的5.9倍,而RA组为2.6倍,EPA组为1.3倍;分析HL-60细胞及其培养介质中RA含量,发现EPA PA组高于RA组。结论 EPA可抑制HL-60细胞增殖和诱导其分化,与RA联合应用能明显增强HL-60细胞的增殖抑制及诱导分化效应,这种联合效应可能与EPA减缓HL-60细胞内RA的代谢相关。     

HL-60细胞ATRA诱导后MMP-9/TIMP-1表达的变化

为了检测HL-60细胞经过全反式维甲酸(ATRA)诱导后基质金属蛋白酶(MMP-9、MMP-2)及金属蛋白酶组织抑制剂1(TIMP-1)表达变化,并探讨其变化的意义。采用瑞氏染色观察细胞形态,WST1实验测定细胞增殖变化,NBT还原实验测定细胞分化状态,RT-PCR方法检测MMP-9、MMP-2、TIMP-1、VEGF的mRNA的表达。结果显示,HL-60细胞经ATRA作用24h后,随着细胞增殖降低与分化的发生,MMP-9mRNA表达增加,TIMP-1mRNA和VEGF表达降低,MMP-2mRNA未见明显表达。ATRA诱导HL-60细胞分化后MMP-9表达增加,而TIMP-1表达降低,MMP-9不促进HL-60细胞表达VEGF。     

Caspases在视黄酸诱导pRARE4-IFNα转染肿瘤细胞凋亡中的作用

目的探讨视黄酸(RA)及其诱导的外源性α-干扰素(IFNα)抗肿瘤作用与半胱氨酸蛋白酶(caspase)的关系。方法含视黄酸反应元件(RARE)的IFNα表达载体(pRARE4-IFNα)，转染肿瘤细胞并证实外源性IFNα基因的表达受RA的诱导后，分析了细胞增殖功能、细胞DNA梯形带，caspase-1、3基因表达，caspase-3活性以及caspase-3特异性抑制剂(DEVD-CHO)对RA和／或IFNα诱导细胞凋亡的抑制作用。结果对RA敏感的HL60细胞和caspase-3基因缺失的MCF-7细胞经RA处理后，增殖能力减弱，出现明显的DNA梯形带，caspase-1基因表达上调节，HL-60细胞caspase-3基因表达上调节，活性升高，DEVDCHO可拮抗RA引起的HL-60细胞caspase-3活性升高，并部分削弱RA引起的细胞增殖抑制和凋亡。结论Caspase-3在RA诱导的细胞凋亡中有重要作用，同时，RA可能通过其它途径诱导caspase-3基因缺失细胞的凋亡。     

视黄酸对pRARE4-IFNα表达载体转染细胞增殖与凋亡的影响及其机制研究

目的：探讨视黄酸（RA）及其诱导的外源性α－干扰素（TFNα）抗肿瘤的协同效应及其可能分子机制。方法：通过构建的含视黄酸反应元件（RARE）的IFNα表达载体（pRARE4-IFNα)，转染肿瘤细胞后，使外源性IFNα基因的表达受RA的诱导。运用MTT比色法，流式仪分析和RT－PCR等技术方法，观察RA对pRARE4-IFNα转染和未转染的HL－60和MCF－7细胞的体外增殖能力，细胞周期分布和细胞凋亡的影响，以及IRF－1，STAT1和Fas抗原表达的改变。结果：pRARE4-IFNα转染和未转染的HL－60和MCF－7细胞经RA处理后，生长减慢，细胞周期被阻滞在G1／G0期，细胞凋亡率增加，DNA片段化百分率升高，IRF－1和STAT1 mRNA水平明显增高，其中，以pARE4-IFNα转染细胞经RA处理后变化更明显，结论：RA及其诱导的外源性INFα具有协同抗肿瘤作用，上调节IRF－1和STAT1基因表面可能是其主要分子机制。     

黄芩苷对HL-60白血病细胞的诱导分化作用

目的： 观察中药单体黄芩苷(baicalin) 对人髓系白血病（AML）细胞株HL-60细胞的诱导分化作用。方法: 应用细胞形态学方法、细胞克隆形成试验、流式细胞术分析和NBT还原实验检测黄芩苷诱导HL-60细胞分化的能力。结果: 黄芩苷可诱导HL-60细胞向成熟阶段分化，低浓度黄芩苷可显著抑制HL-60细胞克隆的形成；HL-60细胞经黄芩苷处理后CD11b表达显著增高，CD33表达显著降低；NBT还原实验示黄芩苷处理组的分化成熟细胞阳性率明显高于未加药组。结论: 黄芩苷具有诱导HL-60白血病细胞向成熟粒细胞分化的作用。     

RARE介导的视黄酸可调控的IFN—α表达载体的构建与鉴定

目的构建一个含视黄酸反应元件 ( RARE)的 IFN- α表达载体 ,使其在真核细胞内的表达受视黄酸 ( RA)的诱导。方法运用 DNA重组技术构建含有 4个 RARE重复序列的 IFN- α真核表达载体 ( p RARE4 - IFN- α) ,经酶切、PCR鉴定和测序确认后 ,用脂质体转染法使其转染 HL- 60细胞 (人早幼粒白血病细胞系 )、Bcap- 3 7和 MCF- 7细胞 (人乳腺癌细胞系 ) ,RA处理后 ,RT- PCR分析 IFN- α m RNA水平 ,EL ISA检测培养细胞上清中 IFN- α含量。结果 p RARE4 - IFN- α转染的 HL- 60、Bcap- 3 7和 MCF- 7细胞 ,IFN- α分泌量为 16～ 2 4 ng/ ( m L· 10 6 )细胞 ,而经 RA处理 2 4 h,IFN- α表达水平可增加 8～ 13倍 ,但经 RA处理 2 4 h的细胞 ,换无 RA的新鲜培养基继续培养 2 4 h后 ,IFN- α表达水平又下降到 2 5～ 3 4 ng/ ( m L· 10 6 )细胞。结论带有 RARE的 IFN- α表达载体转染细胞后 ,外源性 IFN- α基因的表达受 RA的诱导。     

二十碳五烯酸联合视黄酸对HL-60细胞增殖与分化功能的影响

目的研究二十碳五烯酸 ( eicosapentaenoic acid,EPA)及其联合视黄酸 ( retinoic acid,RA)对白血病细胞增殖与分化功能的影响。方法采用 MTT法测定细胞增殖功能 ,NBT还原实验鉴定细胞分化 ,高效液相色谱法分析细胞内 RA代谢。结果与对照组相比 ,EPA组细胞增殖抑制率为 2 4 .3 8% ,RA组为 3 5 .74 % ,EPA+ RA组为 4 2 .75 % ;NBT还原实验 ,EPA+ RA组 OD值约为对照组的 5 .9倍 ,而 RA组为 2 .6倍 ,EPA组为 1.3倍 ;分析 HL- 60细胞及其培养介质中 RA含量 ,发现 EPA+ RA组高于 RA组。结论 EPA可抑制 HL- 60细胞增殖和诱导其分化 ,与 RA联合应用能明显增强 HL- 60细胞的增殖抑制及诱导分化效应 ,这种联合效应可能与 EPA减缓 HL - 60细胞内 RA的代谢相关。     

Differential cell fates induced by all-trans retinoic acid-treated HL-60 human leukemia cells

HL-60 human leukemia cells, differentiated into a neutrophil lineage by all-trans retinoic acid (ATRA) treatment, express three members of the carcinoembryonic antigen (CEA) gene family, CEA-related cell adhesion molecule 1 (CEACAM1; CD66a), CEACAM3 (CD66d), and CEACAM6 (CD66c). CD66d is a neutrophil lineage-specific marker, and CD66a and CD66c are found on epithelial and other cells. HL-60 cells continuously treated with ATRA underwent apoptosis, and cells transiently treated for 1 day underwent cell-cycle arrest, entered into senescence, and exhibited reduced apoptosis with CD66-positive cells accounting for the majority of live cells. CD66 antigens were also induced in NB4 leukemic cells upon continuous treatment with ATRA. NB4 cells underwent apoptosis with a higher frequency in transient versus continuous-treated cells (38% vs. 19% at Day 5), in contrast to HL-60 cells that underwent cell-cycle arrest and senescence when transiently treated with ATRA. CD66 antigens were not induced in transient, ATRA-treated NB4 cells compared with HL-60 cells. Cell-cycle arrest in HL-60 cells involved reduction in expression levels of p21, cyclins D and E, while Rb1 exhibited reduction in protein levels without changes in mRNA levels over the time course of ATRA treatment. Analysis of several proapoptotic proteins implicated the activation of calpain and cleavage of Bax in the intrinsic apoptotic pathway, similar to published studies about the apoptosis of neutrophils. CD1d expression was also induced by ATRA in HL-60 cells and ligation with anti-CD1d antibody-induced apoptosis. In contrast, CD1d-positive primary monocytes were protected from spontaneous apoptosis by CD1d ligation. These studies demonstrate distinct cell fates for ATRA-treated HL-60 cells that provide new insights into ATRA-induced cell differentiation.     

Study on the synergic effect of all-trans retinoic acid combined with IFN-alpha on the proliferation and differentiation of HL-60 cells]

AIM: To investigate the synergic effect of all-trans retinoic acid (ATRA) combined with IFN-alpha on the proliferation and differentiation of HL-60 cells. METHODS: HL-60 cell's differentiation and apoptosis were assessed by NBT reduction and TUNEL in situ apoptosis assay kit. CYP26 mRNA expression was detected by in situ hybridization assay kit. Metabolism of ATRA was measured by high performance liquid chromatography. RESULTS: Either IFN-alpha or ATRA induced HL-60 cell differentiation and apoptosis, which was enhanced when combining ATRA with IFN-alpha. The level of ATRA and the expression of CYP26 were higher in HL-60 cells treated with both ATRA and IFN-alpha than in the cells treated with ATRA alone. CONCLUSION: ATRA has remarkable synergic effect with IFN-alpha on HL-60 cells, probably because IFN-alpha inhibits CYP26 mRNA expression and thus reduces the metabolism of ATRA.     

维甲酸诱导HL-60细胞向粒细胞分化时蛋白激酶C的作用

RA诱导HL-60细胞向粒系分化时,伴随有PK-C活力的两次升高。第一次是细胞接触RA的第一天;第二次是接触RA的第四天。细胞形态学分化和NBT还原细胞生成的资料提示第一次PK-C活力升高可能是诱导细胞分化的关键步骤,后一次则可能是细胞诱导分化的结果。RA导致酶活力的变化主要在胞质组分。三氟哌嗪事先处理细胞,再经RA诱导则无PK-CS力的升高,细胞分化亦受抑制。对RA诱导HL-60细胞向粒系分化时PK-C的作用进行了讨论。     

中性粒细胞增强类风湿关节炎滑膜细胞产生基质金属蛋白酶及其细胞侵袭力

目的：研究中性粒细胞表面CD147分子对类风湿关节炎（RA）成纤维样滑膜细胞（FLS）产生基质金属蛋白酶（MMP）及细胞侵袭力的作用。方法：取自关节镜或骨科手术治疗的RA、骨关节炎（OA）患者的滑膜组织，体外分离培养FLS，通过流式细胞术（FCM）检测细胞表面分子及形态学方法鉴定。采用全反式维甲酸（ATRA）诱导HL-60细胞（人早幼粒白血病细胞株）诱导中性粒细胞分化，并用硝基蓝四氮唑（NBT）还原反应测定其分化程度。进而以FCM测定分化前后的HL-60细胞及FLS表面CD147的表达，并通过明胶酶谱和重组基质侵袭实验检测分化前后HL-60细胞对FLS MMP表达和细胞侵袭能力的作用以及CD147拮抗肽（AP-9）对这种MMP表达和细胞侵袭能力的抑制作用。结果：成功建立了FLS分离培养和HL-60细胞分化的实验体系。培养的FLS具有典型成纤维细胞的形态，均一的高度表达CD90（〉98％）而不表达CD14。分化后的HL-60细胞CD147表达上调，RAFLS细胞CD147表达水平低于分化前及分化后HL-60细胞（P〈0．05）。明胶酶谱试验显示：①与OAFLS相比，单培养的RAFLS表达较高水平的酶原及活化形式的MMP-2（P〈0．05）；②分化前／后的HL-60细胞分别与FLS共培养，酶原及活化形式的MMP-2和MMP-9的表达水平较这些细胞单独培养均显著升高（P〈0．05），且分化后的HL-60细胞与FLS共培养酶原及活化形式MMP-2表达水平较分化前HL-60细胞与FLS共培养明显升高（P〈0．05）；③AP-9显著抑制两共培养组中MMP-2和MMP-9的上调作用（P〈0．05）。侵袭实验显示：RAFLS侵袭能力高于OA FLS（P〈0．05）；分化前后的HL-60均可增加RAFLS的侵袭能力（P〈0．05），且分化后强于分化前（P〈0．05）；AP-9可抑制这一促进作用，降低FLS的侵袭能力（P〈0．05）。结论：中性粒细胞可能通过CD147促进RA患者滑膜FLS MMP-2、MMP-9的表达和活化，增强FLS的侵袭能力，这种促进作用可被CD147拈抗肽所抑制，将为RA关节软骨和骨损伤机制和治疗的进一步研究提供了重要的线索。     

Down-regulation of CD44 contributes to the differentiation of HL-60 cells induced by ATRA or HMBA

CD44 is highly expressed in human acute myeloid leukemia （AML） cells. Some experiments had shown that it was possible to reverse differentiation blockage in AML cells by CD44 ligation with specific antibodies, indicating that CD44 was closely related to the differentiation of leukemia cells. The differentiation of acute promyelocytic leukemia cell line HL-60 cells could be induced by all trans-retinoic acid （ATRA） and hexamethylene bisacetamide （HMBA）, but so far the mechanism was not demonstrated clearly. In the present study, we investigated whether ATRA or HMBA induced the growth arrest of HL-60 cells by down-regulating the expression of CD44. The results indicated that the proliferation of HL-60 cells was obviously inhibited and the differentiation was induced by both ATRA and HMBA. The decreased expression of CD44 and cyclin E mRNA, and the increased expression of p27 and p21 at mRNA levels were observed. Furthermore, there was a negative correlation between the expression of CD44 and p27. It was concluded that ATRA and HMBA played a role in the differentiation induction of HL-60 cells, which was mediated by the down-regulation of CD44, accompanied by down-regulation of cyclin E, and up-regulation of p27 and p21 mRNA. Cellular ＆ Molecular Immunology. 2007;4（1）：59-63.     

Changes in susceptibility to Fas-mediated apoptosis during differentiation of HL-60 cells

The Fas-mediated pathway has been implicated as an important cellular pathway mediating apoptosis in diverse cell types. We conducted studies to examine the susceptibility to Fas-mediated apoptosis of HL-60 cells treated with differentiation-inducing factors such as dimethyl sulfoxide (DMSO), retinoic acid (RA), and 1alpha, 25 dihydroxyvitamin D3 (VD3). Although the expression of Fas antigen (Ag) and its mRNA showed a marked increase in HL-60 cells with cell differentiation, that of Bcl-2 protein and its mRNA revealed the reverse. The expression of caspase proteins such as caspases-3 and -8 was also enhanced during cell differentiation. DNA fragmentation, annexin V binding, and caspase activities increased in differentiated HL-60 cells with the addition of anti-Fas Ag antibody. These findings were more clearly demonstrated in DMSO- or RA-induced neutrophil-like cells than in VD3-induced monocyte-like cells. Therefore, susceptibility to Fas-mediated apoptosis showed an increase with differentiation of HL-60 cells, especially in the neutrophil lineage. These results suggest that the difference of susceptibility to Fas-mediated apoptosis among cell populations depends on the expression of Fas Ag, Bcl-2, and caspases. Cell maturation and susceptibility to Fas-mediated apoptosis may be linked in hematopoietic cells.     

维甲酸诱导HL-60细胞分化过程中WT1基因表达及其异构体比例变化的研究

目的 探讨全反式维甲酸(ATRA)诱导HL-60细胞分化过程中WT1基因及其异构体表达水平及比例变化.方法 采用ATRA诱导HL-60细胞分化,以硝基四氮唑蓝(NBT)还原实验及细胞免疫标记CD11b检测判断细胞分化程度;即时荧光定量RT-PCR方法检测HL-60细胞在诱导分化过程中总WT1、WT1(17AA+)及WT1(KTS+)的表达,并计算出WT1(+/+)、WT1(+/-)、WT1(-/+)、WT1(-/-)四种异构体的比例.结果 ATRA诱导HL-60细胞分化过程中NBT阳性率及CD11b表达阳性率与诱导分化前(0 h)相比均显著增加(P<0.05,P<0.001).随着细胞分化,WT1表达水平由0 h的(4.17±2.21)× 10~(-3)降低至96 h的(7.53±2.30)×10~(-4);17AA+和KTS+两类异构体的比例也逐渐下降,17AA+异构体比例由0 h的0.60±0.05降到96 h的0.42±0.08(P<0.05).KTS+异构体比例由0.53±0.08降至96 h的0.41±0.04(P<0.05);四种异构体的比例变化表现不一致,WT1(+/+)比例由0 h的0.32±0.06降至96 h的0.17±0.03,而WT1(-/-)比例由0 h的0.19±0.04升至96 h的0.34±0.05,另外两类异构体比例变化差异无统计学意义.结论 ATRA诱导HL-60细胞分化过程中总WT1表达水平逐渐降低,分化前异构体以WT1(+/+)为主,而分化后以WT1(-/-)为主,提示WT1基因可能通过调节四种异构体比例而发挥抑制或促进分化的作用.     

Induction of differentiation of the human myeloid cell line, ML3, by tumour necrosis factor and interferon-gamma is accompanied by enhanced expression of the CD4 protein and messenger RNA.

Tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma) induce differentiation of human myeloid cell lines along the monocytic lineage. In this study we investigated the effects of TNF and IFN-gamma on the expression of the CD4 protein and messenger RNA (mRNA) in the two myeloid cell lines, ML3 and HL-60. We observed that CD4 antigen expression on ML3 cells is almost undetectable and that TNF and IFN-gamma induced CD4 antigen expression on these cells. HL-60 cells express surface CD4 antigen at high density and treatment with TNF and IFN-gamma caused a decrease of CD4 expression. We also investigated the expression of CD4 mRNA in ML3 and HL-60 cells. ML3 constitutively express, albeit at low levels, CD4 mRNA. TNF induced CD4 mRNA in ML3 cells and IFN-gamma synergistically potentiated the effect of TNF, thus indicating that the enhanced expression of the CD4 protein on ML3 cells is due, at least in part, to an enhanced accumulation of the CD4 mRNA. CD4 mRNA is constitutively expressed in HL-60 cells at high levels. TNF and IFN-gamma, alone or in combination, did not cause any significant change of CD4 mRNA expression in HL-60 cells, thus indicating that decrease of surface CD4, which accompanies differentiation with these cytokines, is likely due to alterations of the CD4 protein synthesis and/or transport to the plasma membrane. These results provide evidence that myeloid cell lines are heterogeneous in expression of CD4, and that in ML3 cells, which constitutively express low levels of CD4 mRNA and undetectable amounts of surface CD4, the predominant effect of the two cytokines is to induce both CD4 mRNA and protein.