人参皂苷Rg1、Rb1和Re对人慢性粒细胞白血病细胞株K562增殖的影响

目前已发现30余种人参皂苷单体,不同的人参皂苷单体的药理作用及机制各异。本实验通过研究人参皂苷单体Rg1、Rb1和Re对K562细胞增殖的影响,探讨其抗肿瘤作用及机制。取对数生长期K562细胞,分为阴性对照组、不同浓度的Rg1组、Rb1组、Re组,培养24h、48h、72h,以噻唑蓝（MTT）比色法和台盼蓝活细胞计数法测定不同浓度的Rg1、Rb1、     

bcr／abl反义RNA片段对Ph＋人白血病细胞系分化的影响

为探讨Ph 白血病细胞中bcr/ab1表达的降低对细胞分化的影响,本文利用脂质体介导的方法将表达bcr/ab1融合区反义RNA片段的重组质粒导入K562和BV173细胞系,以Southern和Northern杂交以及筑巢式逆转录酶-聚合酶链反应技术证实外源DNA已在靶细胞内整合与表达,且bcr/ab1反义RNA片段的表达使内源性bcr/ab1 mRNA表达水平降低。未观察到表达bcr/ab1反义RNA片段的K562细胞出现粒单系或红系分化,重组质粒转染前后BV173细胞表面的CD10和CD34抗原以及K562细胞表面的CD13和CD33抗原表达无明显变化,提示bcr/ab1反义RNA片段抑制增殖的同时未引发分化与成熟。     

人参皂甙Rg1对体外人胃癌细胞增殖的抑制作用及机制

本研究用不同浓度人参皂甙Rg1作用人胃癌BGC-823细胞24 h、48 h和72 h,采用MTT法、流式细胞术及半定量RT-PCR检测GS-Rg1对胃癌细胞的增殖抑制作用、细胞周期分布时相和p16~(INK4a)、p21~(WAF1)表达水平的影响,以探讨人参皂甙Rg1对人胃癌BGC-823细胞增殖的抑制作用及机制。结果表明,随着作用时间和浓度的增加,人参皂甙Rg1对胃癌细胞增殖抑制作用逐渐增强(P<0.05),G_0/G_1期细胞比例增加,G_2/S期细胞比例下降,p16~(INK4a)、p21~(WAF1)基因水平上调。上述结果提示人参皂甙Rg1能抑制体外培养的胃癌BGC-823细胞增殖,其机制可能与上调肿瘤细胞内细胞周期蛋白依赖激酶抑制因子p16~(INK4a)及p21~(WAF1)mRNA的表达有关。     

人参皂甙Rg1对脑缺血再灌注大鼠脑组织Bax和Bcl-2蛋白表达的影响

目的探讨人参皂甙Rg1对脑缺血再灌注大鼠脑组织Bcl-2和Bax表达的影响及其意义。方法分别给大鼠腹腔注射人参皂甙Rg1 10、20、40 mg/kg,采用大脑中动脉闭塞方法建立脑缺血再灌注模型,观察大鼠脑缺血再灌注后不同时间段（2 h、24 h）神经功能缺损评分;应用免疫组化法检测脑组织缺血再灌注24h后Bcl-2、Bax的表达。结果人参皂甙Rg1组大鼠脑缺血后各时间点神经功能缺损评分显著低于单纯缺血再灌注组（P〈0.05）;与单纯缺血再灌注组相比,人参皂甙Rg1各组Bcl-2表达显著增高,Bax表达显著降低,Bcl-2/Bax比值显著上调（P〈0.05）。结论人参皂甙Rg1防治大鼠脑缺血再灌注损伤的机制可能与促进脑组织Bcl-2表达、抑制Bax表达有关,且以高剂量效果较好。     

人参皂苷Rg1对APP/PS小鼠脑组织内Aβ沉积及行为学的影响

阿尔茨海默症(Alzheimer disease, AD)是一种常见神经退行性疾病,它的主要病理学特征为老年斑、基底前脑胆碱能神经元退变及神经纤维缠结。β淀粉样蛋白(amyloidβ-protein, Aβ)是老年斑的主要成分,过量的Aβ的产生和聚集与AD的发病有着密切的关系。而人参皂甙Rg1是人参促智的主要有效成分之一,对Aβ诱导的神经元的损害具有保护作用。本实验研究了人参皂苷Rg1对APP/PS转基因AD模型小鼠脑组织内APP代谢过程中相关蛋白的表达变化、小鼠行为学的影响。实验发现人参皂甙Rg1处理小鼠在Morris水迷宫实验中定位航行实验游泳路程逐日明显递减,且游泳路程较对照组明显缩短;皮质区老年斑数目较对照组显著减少(p0.05),且Aβ沉积面积相比对照组更小;海马齿状回区与皮质区MAP-2着色阳性神经元较对照组明显增多(p0.05),皮质区可见呈束状排列的神经纤维着色。结果提示人参皂苷Rg1能够显著改善AD模型小鼠的学习记忆能力,其作用机制可能与减少Aβ产生或抑制Aβ沉积、促进神经纤维的生长以及修复,从而减轻AD引起的脑组织受损程度有关。     

人参皂甙Rb1对阿尔茨海默病大鼠海马结构β-淀粉样蛋白表达的影响

目的观察人参皂甙Rb1对阿尔茨海默病（AD）模型大鼠学习记忆能力及海马结构β-淀粉样蛋白表达的影响。方法动物分3组：对照组、模型组及治疗组,用D半乳糖联合三氯化铝建立AD大鼠模型,治疗组在造模后给予人参皂甙Rb1腹腔注射4周;采用Morris水迷宫测试大鼠的空间学习记忆能力,用免疫组织化学方法观察海马结构β-淀粉样蛋白的表达。结果与对照组相比,模型组大鼠各时间段的逃避潜伏期均显著延长（P〈0．01）,海马CA1、CA3区及齿状回β-淀粉样蛋白表达的阳性细胞数明显增多（P〈0．01）;治疗组大鼠的逃避潜伏期较模型组明显缩短（P〈0．01）,海马CA1、CA3区及齿状回的β-淀粉样蛋白阳性细胞数显著减少（P〈0．01）。结论人参皂甙Rb1对AD模型大鼠学习记忆损害具有明显改善作用,其机制可能与人参皂甙Rb1减少海马结构β-淀粉样蛋白的表达有关。     

Rb1、Rg1对肾缺血／再灌注血清诱导HK-2细胞凋亡中Bcl-2、Bax表达的影响

目的：探讨人参皂甙Rb1、Rg1在肾缺血／再灌注血清诱导HK-2细胞凋亡中对Bol-2、Bax表达的影响。方法：制备家兔肾缺血／再灌注血清（SIR）和对照组血清（SC）用于HK-2细胞培养,TUNEL法检测细胞凋亡。实验分组：对照组、缺血／再灌注组、Rb1干预组、Rg1干预组,培养24h后免疫细胞化学法检测Bcl-2、Bax的表达。结果：与缺血／再灌注组比较,Rb1干预组和Rg1干预组Bax的表达明显下降（P〈0．01）,Bcl-2／Bax比值增大。结论：人参皂甙Rb1、Rg1对肾缺血／再灌注血清诱导HK-2细胞凋亡具有保护作用。     

人参皂甙Rb1,Rg1,Re和Rh1对细胞脱氢酶活性的影响

应用显微分光光度术,定量地分析了人参皂甙Rb_1、Rg_1、Re、Rh_1对人胚肺成纤维细胞(2BS)和HeLa细胞脱氢酶活性的影响。结果表明,4种单体皂甙增加了高代龄2BS细胞内乳酸脱氢酶(LDH),琥珀酸脱氢酶(SDH),葡萄糖-6-磷酸脱氢酶(G-6-PDH)和丙酮酸脱氢酶(PVO)的活性,降低了HeLa细胞内这几种酶的活性。     

人参皂苷Rg1对脑缺血再灌注大鼠Caspase-3蛋白表达的影响

目的探讨人参皂苷Rg1对脑缺血再灌注大鼠脑组织半胱氨酸天冬氨酸酶3(Caspase-3)表达的影响。方法将大鼠随机分为假手术组、模型组、人参皂苷Rg110、20、40mg/kg组、尼莫地平组,每组10只。采用线栓法栓塞大脑中动脉2h制作大鼠脑缺血再灌注模型;观察再灌注22h后神经功能缺损评分;应用免疫组化、免疫印迹法检测大脑皮层缺血半暗带Caspase-3的表达。结果(1)假手术组、模型组、人参皂苷Rg110、20、40mg/kg组和尼莫地平组神经功能缺损评分分别为0、2.8±0.9、2.1±0.9、1.5±0.7、1.3±1.1、1.5±0.7,差异有统计学意义(P0.05)。人参皂苷Rg120、40mg/kg组与模型组比较,差异有统计学意义(P0.05);人参皂苷Rg110mg/kg组与尼莫地平组比较,差异有统计学意义(P0.05);人参皂苷Rg120、40mg/kg组与尼莫地平组比较,差异均无统计学意义(P0.05)。(2)免疫组化和免疫印迹结果显示各组大鼠皮层缺血半暗带均有Caspase-3的表达,其中假手术组仅有少量表达,模型组表达最多。与模型组比较,人参皂苷Rg1各剂量组及尼莫地平组Caspase-3表达量减少,差异有统计学意义(P0.05);与尼莫地平组比较,人参皂苷Rg110mg/kg组的Caspase-3表达量显著增高,40mg/kg组显著降低(P0.05),而20mg/kg组则差异无统计学意义(P0.05)。结论人参皂苷Rg1防治脑缺血再灌注的机制与抑制脑组织Caspase-3表达有关,且以高剂量效果较好。     

人参皂甙Rg1与1,6-二磷酸果糖配伍抗疲劳作用的研究

观察人参皂甙Rg1与1,6-二磷酸果糖配伍的抗疲劳效果,寻找最佳配伍剂量。根据雄性清洁级昆明种小鼠按体重随机分为安静对照组、运动对照组、人参皂甙Rg1对照组及4个配伍组。通过析因实验设计分析人参皂甙Rg1与1,6-二磷酸果糖提高运动耐力的效果及两者的交互作用。结果不同剂量人参皂甙Rg1对小鼠力竭游泳时间影响有显著差异(P<0.05)。各配伍组力竭游泳时间均有显著延长,C组延长最显著;与人参皂甙Rg1对照组相比,各配伍组力竭游泳时间均显著降低(P<0.01)。MDA含量A组显著低于运动对照组和D组,与安静对照组相比,各组均升高。SOD/MDA比值A、C组显著高于运动对照组,C组显著高于人参皂甙Rg1对照组,与安静对照组相比,各组均有下降趋势,D组下降最显著(P<0.01)。乳酸脱氢酶C、D组显著低于运动对照组和人参皂甙Rg1对照组(P<0.05)。与运动对照组及人参皂甙Rg1对照组相比,配伍在一定程度上减少心肌、骨骼肌细胞线粒体和其他细胞超微结构损伤。结论:与FDP配伍未能延长小鼠力竭游泳时间,配伍可减轻耐力运动对小鼠心肌和骨骼肌的细胞损害,一定程度上保护线粒体的呼吸功能,缓解细胞缺氧损伤。     

Rapid detection of chimeric bcr/abl mRNAs in acute lymphoblastic and chronic myeloid leukemia by the polymerase chain reaction

We have developed a rapid method for the detection of bcr/abl mRNAs, the products of the BCR/ABL fusion genes. The method is based on the polymerase-chain-reaction (PCR). Through the use of additional internal primers it is possible to detect directly a single Ph1-positive cell among 10(5) unaffected cells thus omitting time-consuming blotting procedures. The whole analytical procedure starting from RNA isolation to agarose gel electrophoresis including two rounds of PCR can be performed in less than six hours.     

Activation of tyrosinase kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins.

Chronic myelogenous leukemia and one type of acute lymphoblastic leukemia are characterized by a 9;22 chronosome translocation in which 5' sequences of the bcr gene become fused to the c-abl proto-oncogene. The resulting chimeric genes encode bcr/abl fusion proteins which have deregulated tyrosine kinase activity and appear to play an important role in induction of these leukemias. A series of bcr/abl genes were constructed in which nested deletions of the bcr gene were fused to the c-abl gene. The fusion proteins encoded by these genes were assayed for autophosphorylation in vivo and for differences in subcellular localization. Our results demonstrate that bcr sequences activate two functions of c-abl; the tyrosine kinase activity and a previously undescribed microfilament-binding function. Two regions of bcr which activate these functions to different degrees have been mapped: amino acids 1 to 63 were strongly activating and amino acids 64 to 509 were weakly activating. The tyrosine kinase and microfilament-binding functions were not interdependent, as a kinase defective bcr/abl mutant still associated with actin filaments and a bcr/abl mutant lacking actin association still had deregulated kinase activity. Modification of actin filament functions by the bcr/abl tyrosine kinase may be an important event in leukemogenesis.     

Targeting the kinase activity of the BCR-ABL fusion protein in patients with chronic myeloid leukemia

Imatinib mesylate is a major advance in the therapy of patients with chronic myelogenous leukemia (CML). Imatinib mesylate binds to the inactive conformation of BCR-ABL tyrosine kinase suppressing the Philadelphia chromosome positive clone in CML. Clinical studies have yielded impressive results in all phases of CML. With higher rates of complete cytogenetic response with imatinib, molecular monitoring of disease is now advisable in assessing response and determining prognosis. Emergence of resistance to imatinib may be manifest at the hematologic, cytogenetic, or molecular levels in patients who remain in chronic phase, or may be evidenced by the development of more advanced CML phases. Resistance and eventual clinical failure of imatinib occurs in most patients with blastic phase disease. Resistance may occur at the level of Bcr-Abl, with reduction or loss of imatinib effectiveness as a kinase inhibitor, or, despite retention of its inhibitory ability, with changes in the ability to deliver an effective dose at the cellular level, and/or, the leukemia becoming less dependent on Bcr-Abl. The various mechanisms underlying these differing, non-mutually exclusive, mechanisms of resistance must be understood to develop corresponding therapeutic remedies. We review the current data on imatinib in CML, the criteria for diagnosis of imatinib resistance, and the mechanisms that underlie such resistance in CML.     

人参皂甙Rg1对昆明小鼠早胚体外发育的影响

目的观察人参皂甙Rg1对昆明(kunming,KM)小鼠早胚体外发育的影响,为改善小鼠早胚体外培养体系奠定实验基础。方法以空白M16培养液为对照组,M16中添加浓度为5μmol/L、10μmol/L、20μmol/L Rg1为实验组,收集KM小鼠1-细胞胚进行体外连续培养,计数各组发育至2-、4-细胞胚、桑葚胚和囊胚等各个阶段的数目,比较各组发育至不同阶段的比率。结果添加Rg1实验组发育到桑葚胚和囊胚的比率明显高于对照组,其中以添加浓度为10μmol/L的Rg1实验组的效果最显著,其桑葚胚发育率为59.79%,而对照组只有19.17%(P<0.01)。10μmol/L的Rg1实验组囊胚发育率为17.82%,也明显高于对照组1.87%(P<0.01)。结论 Rg1可提高KM小鼠1-细胞胚体外发育到桑葚胚及囊胚的比率,以10μmol/L浓度的Rg1效果最显著。     

Bcr3/abl2和VEGF基因反义寡核苷酸联用增强对K562细胞的生长抑制作用

目的:研究BCRABL和VEGF反义寡核苷酸联用对K562细胞株的作用及其相互作用的影响。方法:设计针对bcr3/abl2和VEGF的反义寡核苷酸(ASODNs),应用脂质体Oligofectamine作为转染载体。在转染后72h进行台盼蓝染色细胞计数;建立裸鼠K562移植瘤动物模型,瘤内注射ASODNs,观察肿瘤体积生长变化,组织学检测肿瘤血管密度和肿瘤细胞凋亡情况。结果:转染后72h,各实验组与空白组相比,细胞增殖抑制率分别为13.47%(ASOB3/A2组),12.79%(ASOVEGF组)和41.55%(半量联合治疗组)。经过4次治疗后,与对照组相比,肿瘤生长抑制率分别为23.18%(ASOB3/A2组),17.28%(ASOVEGF组)和57.83%(半量联合治疗组)。联合治疗组肿瘤生长速率显著低于单一治疗组,伴随明显的肿瘤细胞凋亡增加和肿瘤血管密度减少。结论:双基因反义寡核苷酸联合应用协同抑制K562细胞增殖,抗肿瘤作用明显优于单一治疗组,可为CML基因治疗提供一项新策略。     

Aberrant expressions of leptin and adiponectin receptor isoforms in chronic myeloid leukemia patients

Background

Leptin and adiponectin receptors mediate the role of leptin in stimulating the growth of leukemic cells and the protective function of adiponectin undertaken in several malignancies such as leukemia. In this study, we investigated the involvement of the expression of leptin and adiponectin receptors in chronic myeloid leukemia (CML) pathogenesis.

Methods

The expression of leptin receptor isoforms, OB-Rt, OB-Ra, and OB-Rb, and the expression of adiponectin receptors, AdipoR1 and AdipoR2, were measured as mRNA levels in two CML cell lines (K562 and Meg-01) and 20 CML patients and 24 healthy controls by using RT-PCR.

Results

OB-Rt and OB-Ra isoforms expression of the leptin receptors were found to be significantly lower in Meg-01 cell lines than K562 cells. All leptin receptors were downregulated in CML patients and more particularly OB-Rb level was found to be undetectably low in normal PBMC as well as in CML patients. AdipoR1 expression level was higher in Meg-01 than in K562, whereas AdipoR2 level was found to be unchanged in both cell lines. Interestingly, while AdipoR1 expression increased in CML patients, AdipoR2 decreased. Moreover, imatinib therapy did not affect both leptin and adiponectin isoform expressions.

Conclusion

While the decrease in leptin receptor levels in CML patients was confirmed, the increase in AdipoR1 levels and relevant decrease in AdipoR2 levels depicted their possible involvement in CML pathogenesis. This suggests different functions of adiponectin receptors in CML development.     

Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.     

Purification of human granulocyte catalase in chronic myeloid leukemia.

Human granulocyte catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6) was purified from chronic myeloid leukemia cells. The purification procedure included heat precipitation, ammonium sulphate fractionation, DEAE-Sephadex chromatography, gel chromatography on Sephadex G-200 and isoelectric focusing with an approximate yield of 30% and a 1000-fold purification. The molecular weight of the subunit obtained by sodium dodecyl sulphate electrophoresis was 65 800. So20,w was 11.6 +/- 0.24. The pH-optimum was 6.6-6.7 and the spectrum showed a major peak at 405 nm and shoulders at 500, 540 and 625 nm typical for catalase. The electrophoretic mobility was towards the anode at pH 8.6 and identical to normal granulocyte and erythrocyte catalase. These three species of catalase gave the reaction of identity on immunodiffusion and crossed immunoelectrophoresis. The content of catalase and its activity of isolated granulocytes were approximately identical in normal and chronic myeloid leukemia granulocytes while the specific activity of leukemic catalase was higher than normal. No difference in catalase content was found between mature and immature leukemic granulocytes.