长春新碱载体红细胞天然免疫活性及其分子变化研究

目的:研究长春新碱(VCR)载体红细胞的天然免疫黏附活性变化,并从分子水平进行深入探讨。方法:改良低渗预膨胀法制备VCR载体红细胞。采用新鲜血天然免疫功能快速测定法和荧光标记流式细胞分析的方法,检测载体制备过程中洗涤、预膨胀和载药各阶段红细胞的天然免疫黏附活性及相关表面分子的变化情况。结果:低渗预膨胀处理的肿瘤患者红细胞,天然免疫黏附能力[黏附率(39.20±18.14)%]明显高于其他对照组(P<0.05)。健康人红细胞载药前后天然免疫黏附能力[黏附率(48.30±13.52)%和(51.20±11.24)%],均明显高于肿瘤患者(P<0.05),但VCR载药对红细胞的天然免疫黏附活性影响不明显。洗涤、低渗预膨胀和载药处理后,红细胞表面CD35、CD44、CD55、CD59等分子有不同程度的下降,CCR4分子呈上升趋势。结论:载药处理对红细胞天然免疫黏附活性无明显影响。     

吴茱萸碱对S180荷瘤小鼠抑瘤作用及血常规的影响

目的研究吴茱萸碱对小鼠肉瘤S180细胞肿瘤模型生长的抑制作用及对外周血常规的影响。方法小鼠左后肢皮下接种S180。接种后将小鼠按体重随机分为3组:模型组(0.5%羧甲基纤维素钠,剂量10 ml/kg)、阳性对照组(给予环磷酰胺,剂量50 mg/kg)、吴茱萸碱组(给予吴茱萸碱,剂量50 mg/kg)。每组小鼠10只。给药结束后,观察抑瘤率,检测血常规。结果吴茱萸碱对荷瘤小鼠的肿瘤生长具有较明显的抑制作用,50 mg/kg吴茱萸碱可使抑瘤率达到43.6%左右。吴茱萸碱(剂量50 mg/kg)能升高S180荷瘤小鼠外周血的白细胞(P<0.05),红细胞和血小板的影响不大。结论吴茱萸碱对小鼠在体肿瘤S180具有一定的抑制作用,吴茱萸碱作为抗肿瘤药有较大的开发潜力;吴茱萸碱能升高S180荷瘤小鼠外周血的白细胞数量,可能是其抗肿瘤的作用机制之一。     

梅花点舌丹与IL-2联用对荷瘤小鼠红细胞免疫功能的影响

据研究报道 [1～ 3] ,梅花点舌丹等中药与免疫调节剂联合应用有抑瘤作用。现将白细胞介素 -2 (IL-2 )与梅花点舌丹联合应用对小鼠 S1 80 肉瘤的抑瘤作用及其红细胞免疫功能的影响进行研究 ,以便为肿瘤的临床治疗提供实验依据。1　材料与方法1 .1　实验材料　 1动物 :昆明小鼠 80只 ,雄性 ,体重2 0～ 2 2 g,济宁医学院实验动物中心提供。2瘤株 :小鼠 S1 80 肉瘤 ,山东省医学科学院药物所提供。3药物 :IL-2 ,梅花点舌丹 ,环磷酰胺。1 .2　实验方法　 1荷瘤小鼠模型的建立及治疗 :80只小鼠均在右前肢皮下接种经小鼠腹腔传种 7天的小鼠 S1 80…     

长春新碱和环磷酰胺周期序贯联合对慢性移植物抗宿主病狼疮样小鼠肾脏病变的疗效研究

目的 通过周期序贯联合免疫抑制剂(长春新碱、环磷酰胺)治疗慢性移植物抗宿主病(cGVHD)狼疮样小鼠肾脏病变的实验研究,以阐明序贯联合用药对小鼠肾脏病变的治疗效果显著,且不良反应较小.方法 选取cGVHD狼疮样小鼠模型30只,随机分为健康对照组、长春新碱间歇给药组、环磷酰胺隔日给药组、环磷酰胺间歇给药组、长春新碱+环磷酰胺联合间隔给药组.经18周治疗后观察并检测.采用单因素方差分析和重复测量的方差分析进行统计学处理.结果 ①诱导造模后6周尿蛋白定量(24 h)达(5.02±0.88) mg,抗双链DNA(dsDNA)抗体阳性,肾脏病理呈狼疮肾炎Ⅳ型表现,模型成功.②环磷酰胺隔日组[(0.48±0.32)mg ]、长春新碱+环磷酰胺联合组[(0.37±0.30)mg]对造模小鼠尿蛋白定量(24h)的改变最大,环磷酰胺间歇给药组、环磷酰胺隔日给药组与长春新碱+环磷酰胺联合给药组对造模小鼠尿单核细胞趋化蛋白(MCP)-1值、尿转化生长因子(TGF)-β1值的降低幅度较大;而不同给药方案对血清MCP-1、TGF-β1的改变差异无统计学意义(P＞0.05).环磷酰胺隔日给药组、长春新碱+环磷酰胺联合给药组造模小鼠肾脏MCP-1、TGF-β1表达在肾小球、肾小管中的阳性数最低,与对照组、长春新碱间歇给药组及环磷酰胺间歇给药组相比差异有统计学意义(P＜0.01).③长春新碱和环磷酰胺周期联合对造模小鼠血丙氨酸转氨酶、血肌酐、血抗dsDNA抗体、尿蛋白定量(24 h)、尿MCP-1、尿TGF-β1有交互作用(P＜0.05).结论 ①序贯周期联合长春新碱和环磷酰胺联合治疗有交互作用,在降低尿蛋白定量(24 h)、血清肌酐,减少肾脏MCP-1、TGF-β1的表达和排泌等方面有明显作用,与环磷酰胺隔日用药组相当,优于长春新碱间歇给药组、环磷酰胺间歇给药组.②长春新碱+环磷酰胺联合间歇给药与单用药比较不良反应并没有增加.环磷酰胺隔日给药组的不良反应明显大于其他各组,表现为体质量不增、肝酶增高.     

肺结核病人红细胞免疫功能初步研究

检测50例不同程度肺结核病人和32例健康人红细胞受体花环率及红细胞免疫复合物花环率的结果表明,其下降率与病情轻重有关,稳定性肺结核高于轻度及重症活动性肺结核。提示红细胞免疫参与肺结核病情有关,对于判断疗效及预后有参考价值。     

CIK细胞对S180荷瘤小鼠的抑瘤作用及机制

目前，细胞因子诱导的杀伤细胞(CIK Cell)作为新一代抗肿瘤过继细胞免疫治疗的首选方案，正成为人们研究的热点。CIK细胞是人外周血单个核细胞(PBMC)在体外经多种细胞因子刺激后获得的一种异质细胞，具有增殖速度快、杀瘤活性高、杀瘤谱广、对正常骨髓造血影响轻微等优点。近年来，     

复配自由基清除剂对移植性小鼠S180肉瘤抑制作用的研究

目的　探讨复配自由基清除剂（ Ｍ  Ｆ Ｒ Ｓ）对小鼠移植性 Ｓ１ ８０ 抑制作用的量效关系，确定其在抗癌作用中的效果。方法　采用灌胃不同浓度的 Ｍ  Ｆ Ｒ Ｓ 对小鼠 Ｓ１ ８０ 肉瘤进行肿瘤抑制作用的研究。结果　高、中、低 ３ 个剂量对 Ｓ１ ８０ 肉瘤的抑制率分别为 ５９７３％ 、５２５７％ 、４１３３％ ，表现出较为明显的剂量效应关系；三个剂量组分别与其相应未给药对照组比较，差别均有显著或非常显著性意义（ Ｐ＜ ００５ 或 Ｐ ＜ ００１）。结论　 Ｍ  Ｆ Ｒ Ｓ 作为一种含中药的复配自由基清除剂抗癌药物，在对 Ｓ１ ８ ０肉瘤的抑制作用具有一定的效果，在肿瘤药物学上具有一定的应用前景。     

红细胞分布与心力衰竭严重程度的关系研究

目的探讨慢性心力衰竭患者红细胞分布宽度与心力衰竭严重程度的关系。方法收集近4年来在我院就医的心力衰竭患者共300例,根据多种标准进行分组比较,再经临床统计学分析得出结论。结果红细胞分布宽度每增加1%,慢性心力衰竭患者死亡危险率增加1.7倍。结论心力衰竭的严重程度与红细胞分布宽度有明显相关性,红细胞分布宽度增高使心力衰竭患者的死亡危险性增高。     

红细胞分布宽度和冠心病的关系

红细胞分布宽度（RDW）与炎症、冠状动脉病变的严重程度、冠状动脉的不稳定性有一定的相关性,能够预测冠心病（CHD）的预后。RDW的检测可能成为一种经济、简便易行用于CHD危险分层的方法。     

红细胞分布宽度对冠心病患者预后的价值研究

目的探讨红细胞分布宽度(RDW)对冠心病(CAD)患者预后的价值。方法收集本院2008年3月—2010年5月155例因CAD住院的患者,并进行冠状动脉造影以明确诊断。根据入院24h的首次RDW,按照其中位数(13.7%)将患者分为两组:A组(RDW≤13.7%,n=78)和B组(RDW>13.7%,n=77),比较两组患者住院期间及出院1年内发生再次心肌梗死、新发心力衰竭、心源性死亡等不良心血管事件的差异。结果 B组发生再次心肌梗死、新发心力衰竭、心源性死亡发生率均高于A组,差异有统计学意义(P<0.05)。结论 RDW对CAD预后有一定价值,RDW水平较高的CAD患者预后较差。     

Direct effect of insulin on 45calcium uptake in human erythrocytes

Abstract. Objective. High blood pressure is prevalent in obesity and non-insulin dependent diabetes mellitus: both conditions, with insulin resistance and essential hypertension, have been associated with increasing intra-erythrocytic levels of calcium ions. We tested the hypothesis of whether insulin itself might be responsible for the abnormal red cell cytosolic free calcium. Design. The ionic effects of insulin were studied on the kinetics of 45calcium uptake in vitro in normal human erythrocytes. Setting. The study was performed in the outpatient clinic of a central hospital. Subjects. Sixteen healthy, normotensive individuals with normal body mass index were recruited for the study. Main outcome measures. Blood from eight individuals was used for time-dependent studies of ⁴⁵calcium uptake in erythrocytes and blood from another eight individuals was used for dose-dependent studies of insulin effect. Results. The rate of ⁴⁵calcium influx in red blood cells has two components, a fast component (0–10 min), which measures the initial rate of ⁴⁵calcium influx, and a slow component (10–60 min) probably reflecting a relatively large backflux of calcium (calcium efflux), which accordingly determines an apparent low rate of ⁴⁵calcium influx between 10–60 min. The uptake was linear with time between 10–120 min regardless of insulin being present or not. Insulin at a concentration of 120 mUL^-1 significantly decreased the ⁴⁵calcium uptake in a timedependent fashion between 10–120 min. The uptake was 508 (±59) at 60 min in the presence of insulin vs. a control value of 529 (±59) pmol mL red blood cells^-1 (P < 0.001). The corresponding figures at 120 min were 742 (±log) and 767 (±127), respectively (P = 0.02). Inconsistent results were obtained on ⁴⁵calcium uptake at 60 min by varying insulin concentrations from 40–640 mUL^-1 and a dual effect of insulin on ⁴⁵calcium uptake could not be excluded, one at a fairly low concentration of insulin (40–120 mUL^-1) and another at a high concentration (160–640 mUL^-1). Conclusion. The data indicate a direct role of insulin in the transport process of calcium into normal human erythrocytes.     

Bio-content of Telferia occidentalis and their effect on methemoglobin formation in sickled erythrocytes

ObjectiveTo perform phytochemical and mineral analyses on leaves, stem and seeds of Telferia occidentalis (T. occidentalis), and examine the inhibition of methemoglobin build-up in sickled erythrocytes.MethodsThe phytochemical evaluation was carried out by qualitative and quantitative analyses, whereas mineral elements were quantitatively analyzed. The effect of T. occidentalis on methemoglobin formation in sickled erythrocytes was examined using the ratio of ferric ion (Fe²⁺) to ferrous ion (Fe³⁺) concentration, as index.ResultsThe phytochemical evaluation showed the presence of total phenolics, cyanogenic glycosides, flavonoids and alkaloids. Mineral analysis revealed potassium, magnesium, sodium, iron and zinc. Extract concentrations (0.2%–0.8% w/v) of leaves, seeds and stem of T. occidentalis have shown the ability to inhibit the formation of methemoglobin in sickled erythrocytes. The methanolic leaves extract showed the highest effect at 0.8% w/v.ConclusionsThese results suggest that T. occidentalis has the capacity to mop-up methemoglobin in sickled erythrocytes, and may therefore enhance oxygen-hemoglobin binding and transport in sickle cell disease patients.     

Hemoglobin aggregation and pseudosickling in vitro of hemoglobin setif-containing erythrocytes

Erythrocytes from individuals heterozygous for hemoglobin Setif (α⁹⁴ Asp→Tyr) sickle in vitro without deoxygenation when incubated in chloride buffer due to hemoglobin aggregation. We now report quantitative studies of hemoglobin polymerization and deformability in these cells. Hemoglobin polymer gradually increased in intact cells during a 24 h incubation period at 24°C. After 24 hr, about 80% of the cells in 290 mosm sodium chloride buffer contained polymer which appeared as short rods compared to >99% containing polymer at 450 mOsm. Similar proportions of cells were morphologically sickled. Deformability of erythrocytes with 40% hemoglobin Setif incubated in 290 mOsm buffer at 37°C decreased to 80% of normal by 210 min but in 450 mOsm decreased to 50% after only 30 min as measured by the ektacytometer. However, at 4°C deformability remained normal even in 450 mOsm buffer. The solubility of gelled hemolysate containing 40% hemoglobin Setif was 24 g/dl and 21 g/dl at 290 and 459 mOsm buffer respectively. The gel persisted at 4°C with a solubility of 25 g/dl, but melted when dialyzed into sodium phosphate or potassium phosphate buffer. These data suggest that hemoglobin polymerization, reduced deformability, and sickling of hemoglobin Setif-containing erythrocytes are related to reduced hemoglobin solubility. The rate and extent of intracellular polymerization in vitro are considerably reduced (as in the case of sickle trait) compared with erythrocytes from individuals with sickle cell anemia. Hence, the slower kinetics of hemoglobin aggregation in hemoglobin Setif-containing cells provide an alternate system for studying hemoglobin aggregation in hemoglobin Setif-containing cells provide an alternate system for studying hemoglobin polymerization and abnormal rhelogy.     

In vitro and in vivo studies with human carrier erythrocytes loaded with polyethylene glycol-conjugated and native adenosine deaminase

Polyethylene glycol-conjugated adenosine deaminase (pegademase) is used for enzyme replacement therapy for patients with severe combined immunodeficiency caused by adenosine deaminase deficiency. The entrapment of pegademase within human energy-replete carrier erythrocytes using a hypo-osmotic dialysis procedure was investigated with the objective of prolonging the in vivo circulatory half-life of the enzyme and maintaining therapeutic blood levels. Native unmodified adenosine deaminase (ADA) was similarly studied. The efficiency of pegademase entrapment was low (9%) whereas the entrapment of native unmodified ADA was substantial (50%), suggesting that the polyethylene glycol side-chains were impeding intracellular entrapment. The biochemical characteristics and the osmotic fragility of these carrier erythrocytes were not adversely affected by the entrapment of either pegademase or native ADA. In vivo survival studies of pegademase-loaded 51Cr-labelled carrier erythrocytes in an ADA-deficient adult patient showed a mean cell half-life of 16 d. Carrier erythrocyte-entrapped pegademase and native ADA had in vivo half-lives of 20 and 12.5 d, respectively, demonstrating that entrapment prolongs the half-life over that of plasma pegademase, which has a circulating half-life of 3-6 d. These results provide the basis for a more extensive clinical evaluation of carrier erythrocyte-entrapped native adenosine deaminase therapy.     

Perfluorocarbon compounds: effects on the rheological properties of sickle erythrocytes in vitro

The effects of oxygenated perfluorotributylamine (Fluosol-43) on the rheological properties of sickle (HbSS) erythrocytes have been determined by means of microviscometry and positive pressure cell filtration. Incubation of deoxygenated sickled erythrocytes (pO2 congruent to 30 mmHg) with oxygenated Fluosol-43 reduced the percentage of sickled erythrocytes from about 63 to 33%. Deoxygenation of 40% suspension of sickle erythrocytes in autologous plasma increased the viscosity by about 160% at shear rate of 1.15 sec-1. Incubation of the deoxygenated sickled erythrocytes with oxygenated Fluosol-43 significantly reduced the viscosity at the low shear rates. Filtration of 0.2% suspension of deoxygenated sickle erythrocytes through capillary-sized Nuclepore filters showed high resistance at low flow rates. Oxygenated Fluosol-43 increased the deformability of HbSS erythrocytes and thereby reduced the resistance at flow rates less than 1 ml/min. These data suggest that perfluorocarbons may be useful in reducing the propensity of hemoglobin S polymerization and sickling and thereby prevent tissue infarction in vaso-occlusive crisis. Therefore, the concept of examining the potential application of perfluorochemicals for alleviating severe vaso-occlusive events may be useful.     

Epidermal growth factor binding sites on human erythrocytes in donors with different ABO blood groups.

Analysis of epidermal growth factor (125J-EGF) binding to human red cells revealed the presence of two classes of binding sites with apparent equilibrium dissociation constants (app.Kd) in the 10(-10)-10(-9) M and in the 10(-8) M range, respectively. The number of binding sites/cell ranged between 600 and 2,400 for the high-affinity binding site and between 7,200 and 23,000 for the low-affinity site. No differences were seen in the apparent Kd values for both types of binding sites between red cells obtained from donors with different ABO-blood groups. An increase in the number of high affinity EGF binding sites was observed in donors with blood group A1-erythrocytes as compared to red cells taken from donors with blood groups O and B.     

Impaired echinocytic transformation of ankyrin- and spectrin-deficient erythrocytes in mice

The membrane skeleton of the red blood cell plays an important role in the determination of cell deformability and cell shape. Under various in vitro conditions, red blood cells undergo an echinocytic or stomatocytic shape transformation. The mechanism of this fundamental process is not well understood. We have studied the red cell shape transformation in normoblastic anemia mice (nb/nb) and spherocytic anemia mice (sph/sph), which are deficient in ankyrin and spectrin, respectively. We found that both ankyrin-deficient cells (nb/nb) and spectrin-deficient cells (sph/sph) have a reduced capacity to undergo echinocytic transformation with various echinocytogenic treatments, that is, incubation with sodium salicylate (40 and 120 mM), calcium loading (50 microM A23187 + 2.2 mM Ca2+), or metabolic depletion (24 hr at 37 degrees C). These results suggest that the functional integrity of the membrane skeleton is essential for the maintenance and transformation of the red cell shape.     

Aging changes of riboflavin concentration and glutathione reductase activity in erythrocytes

Aged erythrocytes obtained by fractionation using gradient centrifugation with Dextran 40, showed lower glutathione reductase activity and riboflavin content than young cells. However, both young and old cells displayed almost the same increase in enzyme activity upon addition of flavin adenine dinucleotide to the test hemolysate in vitro. The lipid peroxide content in the cell membranes showed no consistent changes with aging.     

The effect of BCNU and adriamycin on normal and G6PD deficient erythrocytes

In cell free systems Adriamycin induces oxygen radicals. We have shown previously that Adriamycin generates peroxide in human erythrocytes. BCNU, by inhibiting glutathione reductase, interferes with the major erythrocyte pathway to degrade peroxide. In this investigation we looked at interactions of these drugs with normal and abnormal erythrocytes. In G6PD-deficient erythrocytes Adriamycin posed a significant oxidant stress as demonstrated by hexose monophosphate shunt (HMPS) activity, hydrogen peroxide (H2O2) production, and glutathione depletion. At similar molar concentrations Adriamycin was a stronger oxidant than acetylphenylhydrazine. BCNU=treated normal erythrocytes showed an enhanced susceptibility to oxidant stress as demonstrated by a lack of HMPS response to H2O2 and glutathione depletion during incubations with Adriamycin. The HMPS shunt of BCNU treated RBC was intact as shown by their nearly normal response to methylene blue stimulation. These BCNU studies also demonstrated the inability of H2O2 to react directly with NADPH. In conclusion Adriamycin poses a potent oxident stress to G6PD-deficient erythrocytes. BCNU promotes enhanced susceptibility of normal RBC to oxidant stress and BCNU can act as a probe to define drug interactions with the HMPS. These studies add to a growing body of evidence postulating the importance of oxygen radicals in the therapeutic and/or effects of Adriamycin.