黄芪不同提取物对K562细胞向红系分化的诱导作用研究

目的探讨黄芪不同提取物对K562细胞向红系分化的影响。方法用超声醇提水提法从黄芪根中获取富含黄芪总苷(AST)的醇溶部分和富含黄芪多糖(APS)的水溶部分并分别作用于K562细胞,联苯胺染色法观察K562细胞向红系分化的情况,MTT法测定细胞活性。以丁酸钠(BA)处理的K562细胞作为阳性对照。结果以不同浓度(1、2、4、8mg/ml)APS作用于K562细胞后显示其有诱导后者向红系分化的作用,以4mg/ml为最适浓度,而AST诱导作用不明显。4mg/ml APS、AST及0.5mmol/L BA作用后K562细胞的联苯胺染色阳性率(BZ%)峰值分别为13.2%、2.9%及17.5%。以4mg/ml APS作用于K562细胞24、48、72h后,其BZ%显著低于同时点BA组(P<0.01),但72、96h的联苯胺染色阳性细胞总数明显高于BA组(P<0.01)。MTT测定结果显示,APS、AST对K562细胞的生长无明显抑制作用,而BA对细胞生长有明显抑制作用(P<0.01)。结论黄芪可诱导K562细胞向红系分化,其有效成分在富含多糖的水溶部分(APS),以4mg/ml浓度的诱导作用最强。黄芪提取物对K562细胞的生长无明显抑制作用。     

低强度He—Ne激光照射对K562细胞的抑制作用

目的：观察不同量密度的低强度He-Ne激光对K562细胞的生物学作用。方法：以能量密度为1．89、4．74和9．47J/cm^2的He-Ne激光照射K562细胞,检测细胞活性：以能量密度为0．41、1．02和2．03J／cm^2的He-Ne激光照射K562细胞,测定超氧化物歧化酶（SOD）活性。结果：中、高能量密度He-Ne激光照射显著抑制K562细胞生长,升高细胞内SOD活性,而低能量密度激光照射对细胞无明显影响。结论：较高能量密度的He-Ne激光对K562细胞的生长有抑制作用,其机制可能与胞内SOD活性增高有关。     

OD-PTD融合蛋白对K562细胞的作用

目的研究OD-PTD融合蛋白对K562细胞的作用。方法应用细胞计数的方法研究OD-PTD融合蛋白对K562细胞的生长曲线的影响;用MTT研究OD-PTD融合蛋白对K562细胞的细胞毒作用;用流式细胞仪技术研究OD-PTD融合蛋白对K562细胞周期的影响。结果OD-PTD融合蛋白能显著抑制细胞的生长(P<0.01);并且对K562细胞具有明显的细胞毒作用(P<0.01)。OD-PTD融合蛋白作用于K562细胞后,细胞周期变化明显,细胞从G0/G1期到S期发生明显阻滞(P<0.05)。结论OD-PTD融合蛋白对K562细胞有明显的抑制作用,为进一步探讨慢性粒细胞白血病新的治疗手段提供了实验依据。     

无活性放线菌野生株抗肿瘤活性突变株的自发突变抗性筛选和化学诱变抗性筛选

目的由无活性放线菌野生株转化获取活性突变株,为药源活性产物研究拓展新菌株资源。方法以海洋来源无活性放线菌野生株L35-1为出发菌株,通过自发突变抗性筛选以及化学诱变结合抗性筛选,筛选获得抗性突变株。采用MTT法检测突变株发酵样品对K562细胞的抑制活性。结果通过自发突变抗性筛选,得到新霉素抗性突变株114株、链霉素抗性突变株68株,其中7株新霉素抗性突变株和3株链霉素抗性突变株样品对K562细胞有抑制作用,在100μg/ml浓度下抑制率大于20%。通过化学诱变结合抗性筛选,得到硫酸二乙酯诱变链霉素抗性突变株41株、硫酸二乙酯诱变新霉素抗性突变株32株、亚硝基胍诱变新霉素抗性突变株46株,其中1株硫酸二乙酯诱变链霉素抗性突变株和1株亚硝基胍诱变新霉素抗性突变株有抗肿瘤活性,100μg/ml样品对K562细胞的抑制率大于20%。结论上述结果初步表明,无活性放线菌野生株可通过抗性筛选转化为活性突变株,因此有可能成为筛选获取药源活性新菌株的重要原始资源。     

PB231诱导K562细胞凋亡的初步研究

 目的研究PB231诱导K 562细胞凋亡作用,探讨PB231的抗肿瘤机制.方法采用荧光显微镜观察细胞形态学变化,MTT法测定PB231对K 562细胞生长抑制作用,琼脂糖凝胶电泳检测细胞DNA裂解.结果K562细胞经PB31处理后,在倒置光显微镜下可见细胞变形和凋亡小体形成;荧光显微镜下可见染色质凝集.MTT法检测其IC50值为5×10-5M.琼脂糖凝胶电泳呈典型的"DNA Ladder".结论PB231可诱导K562细胞发生凋亡.这可能是其抑制K562细胞生长的作用机理之一.     

人参总皂甙对K562细胞凋亡相关基因XIAP和Survivin表达的影响

目的探讨人参总皂甙（TSPG）对K562细胞凋亡相关基因XIAP和Survivin表达的影响。方法采用四唑盐比色试验（MTT法）检测TSPG对K562细胞增殖的影响;Hoechst33342/PI荧光染色法观察K562细胞的凋亡;逆转录-聚合酶链反应（RT-PCR）检测K562细胞XIAP和Survivin基因的mRNA的表达。结果TSPG在低浓度时有促进细胞增殖的作用（P〉0.05）,高浓度时逐渐出现抑制作用（P〈0.05）;荧光技术显示不同浓度TSPG培养K562细胞后24和48小时后凋亡细胞数目明显增多（P〈0.01）;TSPG下调K562细胞XIAP和Survivin基因的表达（P〈0.01）。结论TSPG通过下调K562细胞XIAP和Survivin基因的表达从而诱导K562细胞凋亡。     

角燕提取物抑制肝癌生长的实验研究

目的：观察深海鱼类角燕提取物对肝癌的抑制作用。方法：采用盐溶液抽提，有机溶剂分级沉淀，分子筛层析等步骤，从海洋鱼类一角燕的药用部位获得角燕提取物。选用QGY7721人肝癌细胞体外实验模型，MTT法观察角燕提取物在体外对肝癌细胞生长的抑制作用；选用小鼠HAC肝癌模型，评价角燕提取物在体内对肝癌生长的抑制作用。结果：角燕提取物显著抑制QGY7721人肝癌细胞的生长，呈明显的剂量依赖关系，但对人肺成纤维细胞（HLF）的生长无明显影响。角燕提取物在剂量为0.25-1.00g/kg的范围内对小鼠HAC肝癌的生长均具有显著的抑制作用，并呈较好的剂量依赖关系；在剂量为1.00g/kg地，对小鼠HAC肝癌的抑瘤率为55．50％。结论：角燕提取物在体内和体外均能有效地抑制肝癌的生长。     

髓细胞白血病β—catenin表达及与外源性Wnt5a的作用关系

目的调查β-catenin在髓细胞白血病标本及细胞株中的表达,明确外源性Wnt5a与K562细胞β-catenin表达的关系。方法首先以RT-PCR和免疫组化检测β-catenin在髓细胞白血病标本及细胞株中的表达,再以Wnt5a条件培养液处理K562细胞,然后用Westernblot检测β-catenin表达变化。结果β-catenin在髓细胞系白血病标本及细胞株中有普遍表达,但外源性Wnt5a不能明显下调K562细胞β-catenin表达。结论外源性Wnt5a对K562细胞Wnt／β-catenin信号传导途径无明显抑制作用。     

瑞香狼香水提物小鼠药物血清诱导K562细胞凋亡

目的从细胞凋亡角度探讨瑞香狼毒(SCL)抗肿瘤作用机理.方法用SCL小鼠药物血清处理培养的人白血病K562细胞,MTT比色法观察对细胞生长的抑制作用,荧光显微镜观察凋亡细胞的形态变化,流式细胞仪观察DNA改变.结果SCL(5～20)g*kg-1小鼠药物血清显著抑制K562细胞增殖,明显诱导K562细胞凋亡的形态学改变和DNA变化.凋亡细胞率与小鼠服用SCL水提物的剂量呈正相关.结论SCL水提物可诱导肿瘤细胞凋亡.     

黄芪的含药血清诱导K562细胞表达^Gγ-mRNA和合成HbF

目的研究黄芪对K562细胞的^Gγ-珠蛋白基因表达的作用。方法以K562细胞为体外模型，用联苯胺染色方法来确定黄芪的含药血清诱导K562细胞向红系分化的作用；用RT—PCR方法研究黄芪的含药血清诱导K562细胞^Gγ-珠蛋白mRNA的表达；用Western Blot方法研究黄芪的含药血清诱导K562细胞HbF的合成。结果黄芪的含药血清作用于K562细胞5d后联苯胺染色阳性率达19．8％；同时伴有^Gγ-珠蛋白mRNA的表达的增加（最高可达3．6倍）和HbF的合成的增加，与丁酸钠组比较，黄芪组诱导K562细胞^Gγ-珠蛋白基因mRNA的表达可以持续3～5d，而丁酸钠组只有1d。结论黄芪有望成为一种有效的γ蛋白基因诱导剂。     

海洋放线菌和真菌野生株及其突变株的分离培养与抗肿瘤活性筛选

目的:从海泥样品中分离培养并筛选获取野生型与突变型药源微生物抗肿瘤活性菌株.方法:通过单菌落挑选与划线培养,分离纯化野生菌株;利用核糖体工程抗性筛选技术.筛选获得抗生素抗性突变株;用K562细胞,采用MTT法测试发酵样品的抗肿瘤活性.结果:从天津塘沽驴驹河渤海湾潮间带海泥样品中分离得到放线菌126株、真菌29株,其中,在100μg/ml样品浓度下抑制率大于20%的放线菌17株、真菌3株,在1000μg/ml样品浓度下抑制率小于5.5%的无活性放线菌19株.以1000μg/ml样品浓度下抑制率为4.8%的无活性放线菌野生株L8-5x为原始茼,经抗性筛选获得链霉素抗性突变株78株,其中在100μg/ml样品浓度下抑制率大于25%的活性突变株2株.结论:所获野生型与突变型抗肿瘤活性菌株为后续新药筛选提供了药源微生物活性菌株.由无活性菌株转化获取活性突变株的研究结果表明,已分离得到的大量无活性菌株也是可转化获取药源活性突变株的很好资源.     

A 700 MHz 1H-NMR study reveals apoptosis-like behavior in human K562 erythroleukemic cells exposed to a 50 Hz sinusoidal magnetic field

PURPOSE: To study cell damage and possible apoptosis in K562 human erythroleukemic cells exposed for 2 h to an extremely low frequency (ELF) 50 Hz sinusoidal magnetic field with a magnetic induction of either 1 or 5 mT using high resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. MATERIALS AND METHODS: One-dimensional 1H-NMR spectra were obtained on whole K562 cells and perchloric acid extracts of these cells. In addition, two-dimensional 1H-NMR spectra were also acquired. Cell damage was examined by lactate dehydrogenase release and changes in cell growth were monitored by growth curve analyses, bromodeoxyuridine incorporation and Ki67 antigen localization. Cell death (necrosis and apoptosis) were also studied by using the chromatin dye Hoechst 33258. RESULTS: The variations in numerous metabolites observed with 1H-NMR reveal apoptosis-like behavior in response of K562 cells to ELF fields. CONCLUSION: 1H-NMR can be extremely useful in studying the effects of ELF fields on cells. In particular, the variations in metabolites which suggest apoptosis-like behavior occur when the cells are not identifiable as apoptotic by more traditional techniques.     

激光激发5-氨基乙酰丙酸己酯对人红白血病耐药/非耐药细胞株的杀伤效应研究

目的探讨5-氨基乙酰丙酸己酯-光动力疗法(He-ALA-PDT)对人红白血病细胞株K562及其耐阿霉素细胞株K562/ADM的杀伤效应。方法实验设4组:PDT组(加光敏剂并接受光照)、激光组(不加光敏剂只接受光照)、暗毒性组(只加光敏剂而不接受光照)、正常对照组(不加光敏剂且不接受光照)。用不同浓度(0.025、0.1、0.4、1.6mmol/L)的He-ALA避光孵育细胞,给予不同功率密度(4.5、9、18、36J/cm2)的630nm波长激光照射后,继续培养12h,观察细胞形态学变化,以CCK8法测定细胞存活率,采用甲基纤维素培养体系分析白血病细胞克隆形成能力的变化。结果暗毒性组、激光组均未对细胞产生明显杀伤作用,而PDT组能明显杀伤白血病细胞,且细胞存活率随He-ALA浓度和激光功率密度的增加而降低,且在相同条件下,K562细胞的存活率显著低于K562/ADM细胞(P<0.05)。PDT组的集落形成率明显低于正常对照组(P<0.001),且与K562/ADM细胞相比,K562细胞的集落形成率降低程度更为显著(P<0.001)。结论He-ALA-PDT可杀伤K562、K562/ADM细胞,并显著降低白血病细胞的克隆形成能力;K562/ADM细胞对PDT的反应不如K562细胞敏感。     

A 700 MHz 1H-NMR study reveals apoptosis-like behavior in human K562 erythroleukemic cells exposed to a 50 Hz sinusoidal magnetic field

Purpose: To study cell damage and possible apoptosis in K562 human erythroleukemic cells exposed for 2?h to an extremely low frequency (ELF) 50 Hz sinusoidal magnetic field with a magnetic induction of either 1 or 5 mT using high resolution proton nuclear magnetic resonance (¹H-NMR) spectroscopy.

Materials and methods: One-dimensional ¹H-NMR spectra were obtained on whole K562 cells and perchloric acid extracts of these cells. In addition, two-dimensional ¹H-NMR spectra were also acquired. Cell damage was examined by lactate dehydrogenase release and changes in cell growth were monitored by growth curve analyses, bromodeoxyuridine incorporation and Ki67 antigen localization. Cell death (necrosis and apoptosis) were also studied by using the chromatin dye Hoechst 33258.

Results: The variations in numerous metabolites observed with ¹H-NMR reveal apoptosis-like behavior in response of K562 cells to ELF fields.

Conclusion: ¹H-NMR can be extremely useful in studying the effects of ELF fields on cells. In particular, the variations in metabolites which suggest apoptosis-like behavior occur when the cells are not identifiable as apoptotic by more traditional techniques.     

青蒿素及其衍生物体外抗肿瘤活性研究

目的研究青蒿素及其衍生物体外抗肿瘤活性。方法采用四甲基偶氮唑盐法测定青蒿素、双氢青蒿素、蒿甲醚和青蒿琥酯体外抑制人肺癌细胞、人胃癌细胞、人结肠癌细胞、人红白血病细胞和人肝癌细胞的活性。结果青蒿素、双氢青蒿素、蒿甲醚和青蒿琥酯对5株肿瘤细胞有选择性的抑制作用。结论青蒿素及其衍生物在体外对不同的肿瘤细胞有抑制活性。     

N-ras基因在K562细胞中的表达研究

目的探讨N-ras基因突变及表达水平与慢性粒细胞白血病发病的关系。方法以慢粒细胞株K562细胞为研究对象，采用RT-PCR和直接测序的方法对N-ras的表达水平及突变进行检测。结果RT-PCR结果示N-ras基因在K562细胞中表达升高，直接测序结果表明N-ras基因在K562细胞中不存在突变。结论N-ras基因在K562细胞中高表达，而其高表达机制与突变无关。     

Loss of mitochondrial membrane potential and caspase activation enhance apoptosis in irradiated K562 cells treated with herbimycin A

PURPOSE: We previously reported that herbimycin A (HMA) alters the mode of cell death of K562 cells induced by radiation and enhanced their radiosensitivity. In the present study, we explored the apoptosis-inducing activity of HMA and the fundamental mechanism via which it regulates radiation-induced cell death. MATERIALS AND METHODS: Chronic myelogenous leukemia (CML) cell line K562 was used. For X-irradiation and drug treatment, cells were plated at approximately 2x10(5) cells/ml. Exponentially growing cells were treated with 10 Gy of X-ray using a 6-MeV X-ray machine at a dose rate of 200-300 cGy/min. The cells were treated with 0.25 microM HMA immediately after irradiation and HMA remained for the entire culture period. The modes of cell death were discriminated by morphological changes, analysis of cell cycle, analysis of the mitochondrial events, and the expression of apoptosis-related proteins. RESULTS: Our data demonstrates that radiation induced a significant time-dependent increase of cell death and failed to sustain a prolonged G2 arrest in K562 cells. Radiation-induced cell death caused the accumulation of cyclinB1 and weak nuclear fragmentation, suggesting a mitotic catastrophe. This mitotic catastrophe was dependent upon the mitochondrial permeability transition pore (PTP) opening and was independent of caspase-3. In contrast, K562 cells treated with radiation and HMA had an accelerated cell death and induced a p53-independent apoptosis. This apoptotic pathway was dependent upon an initial hyperpolarization of the mitochondrial inner membrane, following the release of cytochrome c and subsequent caspase-3 activation. CONCLUSIONS: Two mechanisms of radiation-induced cell death in K562 cells, mitotic catastrophe and apoptosis, are regulated through distinct pathways, mitochondria and caspase-independent and -dependent, respectively. The findings of this study may provide new insights into improving the efficiency of radiotherapy in CML patients.     

反义bcr／ab1重组逆转录病毒表达载体的构建及其转染对K562细胞系的影响

用逆转录聚合酶链反应技术从Ｋ５６２细胞系中扩增出ｂｃｒ／ａｂ１融合区２５３ｂｐＤＮＡ片段，经反复连接与克隆，得到含同方向串联的２、３和４个拷贝的该融合基因片段的克隆载体ｐＵＣｓ。这些片段分别反向连接到逆转录病毒表达载体ｐＤＯＲｎｅｏ。ＤＮＡ序列分析、限制性内切酶分析和菌落原位杂交证实：不同拷贝数的ｂｃｒ／ａｂ１融合区基因片段已反向插入ｐＤＯＲｎｅｏ。利用脂质体介导，将重组质粒导入Ｋ５６２细胞系后，观察到转染细胞的增殖明显被抑制     

海洋来源放线菌无活性野生株的链霉素抗性抗肿瘤活性突变株新产代谢产物研究

目的研究阐明无活性放线菌野生株L35-1来源硫酸二乙酯（DES）诱变链霉素抗性抗肿瘤活性突变株D2s4-1新产代谢产物及其抗肿瘤活性。方法组合利用活性跟踪与微量预试先导-放大实验制备的实验模式,通过与原始菌样品直接对照,在快速确定差异活性斑点基础上,组合利用各种色谱技术,分离纯化突变株新产代谢产物。根据理化性质和波谱数据鉴定化合物结构。采用MTT法测定抗肿瘤活性。结果从突变株D2s4-1发酵物中分离鉴定了6个该突变株新产代谢产物,即1,9-二甲酯吩嗪（1）、2-羟基苯乙酰胺（2）、2-吡咯甲酸（3）、大豆黄素（4）、环（4-羟基-脯氨酸-亮氨酸）二肽（5）和尿嘧啶（6）。其中1、3、5和6有一定抗肿瘤活性,浓度为100μg/ml时对K562细胞的抑制率分别为33.3%、16.3%、33.3%和21.1%。结论阐明了抗肿瘤活性突变株D2s4-1的6个新产物,其中4个为活性产物。化合物1为新天然产物,其抗肿瘤活性亦属首次筛选发现。用化学诱变组合抗性筛选技术从无活性放线菌野生株转化获取的活性突变株可供筛选新产活性产物,从而拓展放线菌药源活性新菌株资源。