人胎肝中两种抑制肿瘤细胞生长活性成分的分离鉴定及功能研究

胎儿组织中存在低分子肿瘤抑制物。胎肝细胞的甲醇－丙酮提取物中保留有大部分抑瘤活性。用体外液体培养条件下对人急性粒系白血病细胞系（ＨＬ－６０）的抑制作用为指标,跟踪分离过程。提取物经反相Ｃ１８中压液相色谱、Ｓｅｐｈａｄｅｘ ＬＨ－２０凝胶色谱及正相高效液相色谱分离得到两种活性物质,经ＮＭＲ和ＨＲＭＳ鉴定为７－酮基胆固醇（７－ｋｅｔｏｃｈｏｌｅｓｔｅｒｏｌ,７－ＫＣ）和７－β－羟基胆固醇（７－β－ｈｙ     

人胎肝中低分子抑瘤物的分离纯化、结构鉴定及其对肿瘤细胞生长的选择性抑制

在胎儿组织中存在一类低分子肿瘤抑制物,胎肝细胞的甲醇－丙酮提取物中保留有大部分抑瘤活性,用体外液体培养条件下对人急性粒系白血病细胞系（HL－60）的抑制作用为指标,跟踪指导分离过程,提取物经反相C18中压液相色谱,Sephadex LH-20凝胶色谱及氨基键合相高效液相色谱分离得到一纯活性物质。经NMR和MS鉴定为7－酮基胆固醇（7－ketocholesterol,7-KC)。体外琼脂培养条件下7－KC对小鼠WEHI－3和S－180细胞较对正常小鼠骨髓粒－巨噬系祖细胞有更强的抑制增殖及集落生成作用,7－KC对HL－60细胞增殖较对正常人骨髓CFU－GM有更强的抑制作用。     

脑肿瘤抑制因子的功能研究

本文证明了胎脑组织中存在一类可以抑制人白血病细胞系的生长的低分子天然抑癌物．这种抑瘤物抑制活性分布在小于１０ｋＤａ组分,经Ｓｅｐｈａｄｅｘ－Ｇ２５凝胶过滤可分离为单一组分,且具有广谱的抗肿瘤效应．体外可抑制人白血病、肝癌、胃癌细胞系和小鼠粒、单核和淋巴系白血病细胞,而对人骨髓ＣＦＵ－ＧＭ和小鼠骨髓ＣＦＵ—ＧＭ的抑制作用较弱。     

低分子抑瘤物净化白血病细胞的实验研究及临床应用

胎儿肝脏中存在一类小分子量（分子量〈１０ｋＤ）的肿瘤抑制物,其在体外对ＨＬ－６０等多种白血病细胞系具有明显的选择性抑制作用,对急性白血病患者骨髓的白血病祖细胞也具有这种选择性抑制效果;分离纯化获得两种天然低分子抑瘤物（７－ＫＣ和７－β－ＨＣ）和一种外源性低分子抑瘤物（ＤＢＰ）,研究证实其抑制白血病细胞生长的作用机制是诱导白血病细胞凋亡;将其应用于白血病及恶性淋巴瘤自体骨髓移植时的体外净化,完成１２     

肿瘤细胞中端粒酶催化亚基hTERT基因启动子结合因子的检测

为了研究端粒酶催化亚基ｈＴＥＲＴ基因在癌变细胞中组成型表达的调控机制,采用凝胶阻滞电泳（ＥＭＳＡ）实验方法检测人粒系白血病细胞ＨＬ－６０、人红系白血病细胞Ｋ５６２、人肺癌细胞Ａ５４９、人肝癌细胞ＨｅｐＧ２及正常人肺成纤维细胞２ＢＳ等体外传代的肿瘤和正常二倍体细胞核提取物中与ｈＴＥＲＴ启动子核心序列结合的核因子活性。结果在４种实验肿瘤细胞中均可检测出与ｈＴＥＲＴ基因启动子结合的核因子活性,而正常二倍     

重组人粒系集落刺激因子（rhG—CSF）对白血病细胞HL—60生长 …

本文研究了ｒｈＧ－ＣＳＦ对人白血病细胞系ＨＬ－６０的作用。结果表明：ｒｈＧ－ＣＳＦ能够显著抑制ＨＬ－６０细胞生长和Ｃ－ｍｙｃ基因的表达,降低＾３Ｈ－ＴｄＲ的摄入。在含ｒｈＧ－ＣＳＦ的培养液中经过２－５天的培养,部分ＨＬ－６０细胞具备ＮＢＴ还原能力。这或许说明ｒｈＧ－ＣＳＦ能导致ＨＬ－６０细胞向成熟方向分化的结果。     

几种MK－CSA活性物质对小鼠CFU－MK体外形成的影响

几种ＭＫＣＳＡ活性物质对小鼠ＣＦＵＭＫ体外形成的影响黄成龙王亦流１柳晓兰邱丽玲张卿西毛秉智（军事医学科学院放射医学研究所,北京１００８５０;１海军总医院）自从１９７５年Ｍｅｔｃａｌｆ及１９７９年Ｖａｉｎｃｈｅｎｋｅｒ等人分别建立起小鼠和人类骨髓巨...     

用NSF─60细胞MTT比色法检测重组人粒系集落刺激因子的生物活性

利用Ｇ－ＣＳＦ依赖型细胞株Ｎ８Ｆ－６０,尝试将ＭＴＴ比色法应用于基因工程产品重组人粒系集落刺激因子（ｒｈＧ－ＣＳＦ）的活性检测。多次实验探索出最适的细胞浓度和培养时间,细胞数为低于１×１０￣４／孔,培养时间为３０─４８ｈ。在宽的线性范围内检测Ｇ－ＣＳＦ样品的生物活性,结果经传统的ＣＦＵ－Ｇ方法验证可靠,且较后者更灵敏、简便、省时、省力,并可定性、定量地检测样品活性。特别适用于大量样品的同时检测。另外,此方法可避免接触放射性。     

人胚胎脑组织中低分子肿瘤抑制物的实验研究

人胚胎脑组织提取液可以抑制人白血病细胞系的生长。分析表明:肿瘤抑制活性主要分布在小于10kDa组分,经Sephadex-G25凝胶过滤可初步分离为单一组分。这种低分子肿瘤抑制物具有广谱的抗肿瘤效应,在体外可以抑制人白血病、肝癌、胃癌细胞系和小鼠粒、单核和淋巴系白血病细胞,但对人骨髓CFU-GM和小鼠骨髓CFU-GM的抑制作用较弱,说明人胎脑低分子肿瘤抑制物对肿瘤细胞具有一定的选择性抑制作用。     

骨髓高增殖潜能集落形成细胞（HPP—CFC）分化产生巨核细胞的研究

本研究用正常和５－ＦＵ处理的小鼠骨髓细胞,作血浆凝块培养,对一种称作ＨＰＰ－ｍＣＦＵ－ＭＫ的ＨＰＰ－ＣＦＣ亚型细胞集落进行体外追踪。发现ＨＰＰ－ｍＣＦＵ－ＭＫ不但能形成符合ＨＰＰ－ＣＦＣ标准的巨大集落,而且能产生大量的巨核细胞。该巨大集落的生长,依赖添加再生障碍性贫血猪血清（ＡＡＳ）或合用三种以上的基因重组造血生长因子而增强,在体外不被ＴＧＦ－β１和ＰＦ４抑制。原代培养１２天所得到的ＨＰＰ－ｍＣＦ     

新生牛肝中低分子抑瘤物影响白血病细胞生长的实验研究

新生牛肝经匀浆、离心和分级超滤后,得到分子量≤1.0kDa的、耐热的成分——新生牛肝抑瘤物(new-bom calfliver suppressor,BLS-1)。对BLS-1的生物学活性进行了检测,结果表明在体外液体和半固体琼脂培养条件下,它对人和小鼠白血病细胞系(如HL-60、L833 和 P388 等)的生长均有明显的抑制作用,并存在着一定的量效关系;与同样条件下BLS-1对人和小鼠正常粒-巨噬系祖细胞的抑制作用相比较,BLS-1对白血病细胞生长的抑制作用具有较强的选择性。进一步观察了BLS-1对5例急性非淋巴细胞白血病病人骨髓的原代白血病细胞集落(leukemic blast progenitor,L-CFU)生成的影响,结果表明,当BLS-1的浓度达到500μg/ml时,L-CFU的生成受到明显抑制。上述研究结果提示,在新生牛肝中可能存在类似于人胎儿肝脏中的一类低分子天然抑瘤物。     

人胎肝细胞分泌的低分子抑瘤物对白血病细胞的抑制作用

本工作证明了在胎儿组织中存在一类低分子天然肿瘤抑制物,它是胎儿组织细胞生成和分泌的,对瘤株细胞和原代白血病细胞有选择性抑制作用。初发期或复发期急性非淋巴细胞白血病患者的骨髓在体外液体培养条件下与肝细胞上清及其甲醇提取物共同孵育4d,可使所有病例的骨髓AML—CFU降低到不可检出的程度。因而,低分子天然抑瘤物是天然肿瘤免疫中的一个组成部分,它在肿瘤的诊断和治疗中有着深远的意义。     

胎儿肝脏中一种抑制HL—60细胞生长的因子初步研究

胎儿肝脏中存在着两类抑制HL-60细胞生长的抑制物,一类是精氨酸酶,它是一类非特异性的细胞毒剂,在我们的实验条件下,不仅对HL-60细胞,而且对正常人骨髓CFU-GM也具有相似的抑制细胞生长的毒性作用。此外,还存在着一类较小分子的抑制物,它对HL-60细胞生长的抑制作用明显高于对人骨髓CFU-GM的作用,因此,在一定程度上,这是一类对HL-60细胞生长具有选择性作用的抑制物。     

Evading apoptosis by calcitriol-differentiated human leukemic HL-60 cells is not mediated by changes in CD95 receptor system but by increased sensitivity of these cells to insulin

Previous studies revealed that 1,25-dihydroxyvitamin D(3) (calcitriol)-induced differentiation of human promyelocytic leukemia cells leads to an increased resistance of the cells to apoptosis-inducing agents. However many attempts were made to explain it, the mechanism underlying this effect still remains unclear. Our results suggest that the acquired resistance to apoptosis-inducing agents in HL-60 cells is not mediated by the CD95 receptor/ligand system. The expression of CD95 on the surface of HL-60 cells is very low and does not change during the calcitriol-induced differentiation of HL-60 cells. Studies presented here provide a strong indication that this receptor is unable to transmit the death signal in either differentiated or undifferentiated HL-60 cells. We therefore asked if evading apoptosis by differentiated human leukemia HL-60 cells may be caused by their increased sensitivity to growth factors contained in fetal calf serum. This study demonstrates that HL-60 promyelocytic leukemia cells, differentiated by exposure to calcitriol, undergo apoptosis in serum-free conditions. As low as 1% of fetal calf serum is enough to prevent cell death of differentiated HL-60 cells. The ability of 1% fetal calf serum to prevent apoptosis can be blocked by the specific inhibitor of phosphatidylinositol 3-kinase, LY294002. We then tried to find out which component of fetal calf serum may be able to prevent serum-free cell death of differentiated cells. It appeared that serum-free cell death of differentiated HL-60 cells is reversed by addition of 10 microM insulin to the culture medium. The antiapoptotic activity of insulin can be inhibited by LY294002. Moreover, insulin increases the viability of differentiated, but not of undifferentiated, HL-60 cells.     

7-Ketocholesterol

7-Ketocholesterol is a major oxidation product of cholesterol found in human atherosclerotic plaque and is more atherogenic than cholesterol in some animal studies. 7-Ketocholesterol can inhibit cholesterol 7 alpha-hydroxylase, the rate-limiting step in bile acid biosynthesis, as well as strongly inhibiting HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. It has even been suggested that 7-ketocholesterol is formed enzymically as an endogenous regulator of cholesterol biosynthesis. However, when tested as a pharmacological cholesterol-lowering agent, inhibition of HMG-CoA reductase was rapidly overcome and the 7-ketocholesterol metabolised. In vitro, 7-ketocholesterol has wide-ranging and potent effects, most of which have the potential to contribute to atherosclerosis. For example, 7-ketocholesterol can be cytotoxic and can induce apoptosis in vascular cells. These effects, either individually or more likely, in combination, all implicate 7-ketocholesterol in the initiation and development of atherosclerosis, but further work is needed to establish whether or not its role is a direct causal one.     

Antileukemic effect of coral-prostanoids clavulones from the stolonifer Clavularia viridis on human myeloid leukemia (HL-60) cells

To elucidate the biological activities of coral-prostanoids, clavulones, discovered from the Japanese stolonifer Clavularia viridis, we examined the effect of clavulone on the cell growth of human cancer (human promyelocytic leukemia (HL-60) cells and HeLa cells) and normal (Chang liver cells and lung fibroblasts) cells in vitro. Clavulone showed strong antiproliferative and cytotoxic activities in the human cells and it had some selectivity to leukemic (HL-60) cells over other HeLa cells or normal cells on the basis of the IC50 values and cytotoxic effect of the cells. The IC50 value of clavulone in the HL-60 cells was about 0.4 microM (0.2 micrograms/ml). Over 1.0 microM (0.5 micrograms/ml), clavulone showed a significant cytotoxic activity on the HL-60 cells. The data on DNA synthesis and flow cytometric analysis revealed that clavulone arrests the cells in the G1-phase and inhibits the cell growth of HL-60 cells by inhibiting S-phase DNA synthesis. These results suggest that clavulone has a potent antileukemic effect on HL-60 cells.     

Effects of recombinant human PLD2 on proliferation and apoptosis of HL-60 cells

In the present study, we investigated the role of recombinant human phospholipase D2 (rhPLD2) on proliferation and apoptosis in human leukemia HL-60 cells which induced by camptothecin. Our research demonstrated that various concentrations of rhPLD2 inhibit the growth of HL-60 cells in a dose-dependent manner, and rhPLD2 plus camptothecin can produce a synergistic effect on growth inhibition of HL-60 cells in vitro. So, we conclude that rhPLD2 alone cannot induce apoptosis in HL-60 cells, but it can potentiate the apoptosis of HL-60 cells induced by camptothecin. Similarly, we show that both rhPLD2 and standard PLD were able to enhance camptothecin-induced apoptosis of HL-60 cells.     

Combination effects of digalloylresveratrol with arabinofuranosylcytosine and difluorodeoxycytidine in human leukemia and pancreatic cancer cells

Digalloylresveratrol (DIG) is a newly synthesized agent aimed to combine the biological effects of the natural compounds, gallic acid and resveratrol, which both are free radical scavengers exhibiting anticancer activity. In this study, we investigated the effects of DIG on the growth of human HL-60 leukemia cells and on the colony formation of human BxPC-3 and PANC-1 pancreatic cancer cells. DIG was applied alone and in combination with arabinofuranosylcytosine (Ara-C) or difluorodeoxycytidine (dFdC), depending on the cell line employed. All IC(50) values observed were in the low micromolar range rendering DIG a promising antitumor compound in vitro. Considering the combination experiments, DIG yielded additive effects with Ara-C in HL-60 cells and-to a lesser extent-with dFdC in BxPC-3 and PANC-1 cells. Owing to our results, DIG may be further investigated in vitro and in animals.     

氟苯达唑对人急性髓系白血病HL-60细胞的增殖抑制作用研究

目的:研究氟苯达唑对人急性髓系白血病HL-60细胞增殖的抑制作用,明确氟苯达唑对HL-60细胞周期,凋亡发生的作用机制。方法:噻唑蓝法(MTT)检测氟苯达唑对人急性髓系白血病HL-60细胞的生长抑制作用,流式细胞术检测氟苯达唑对HL-60细胞周期,DNA片段化的影响,免疫印迹法检测Caspase, Raf, Bcl-2家族蛋白表达。结果:氟苯达唑抑制人急性髓系白血病HL-60细胞生长,HL-60细胞G2/M期增加,与阴性对照组相比,在一定的剂量和时间内,差别具有显著统计学意义;DNA片段化上升,0.25,0.5,1μM组与对照组相比差别具有显著统计学意义,促使Cleaved PARP,Cleaved-caspase 3,Cleaved-caspase 9蛋白表达量趋势增加;Bag-1和Bcl-2蛋白表达量降低;b-raf,c-raf磷酸化蛋白表达水平逐渐降低。结论:氟苯达唑通过诱导HL-60细胞阻滞于G2/M期,增加DNA片段化水平,激活Caspase, Raf, Bcl-2家族介导的凋亡相关通路抑制人急性髓系白血病HL-60细胞增殖,诱导人急性髓系白血病HL-60细胞发生凋亡而发挥抗肿瘤作用。