联合海藻糖和二甲基亚砜进行冻干前负载的人红细胞冻干效果研究

探讨人红细胞在冻干前经添加低浓度二甲基亚砜(DMSO)海藻糖负载缓冲液负载后,是否会提高红细胞的冻干效果. 实验组:为6%DMSO 800 mmol/L海藻糖.对照组:冻干前负载缓冲液为等渗PBS.取等量压积红细胞(6份)分别重悬于两组负载缓冲液,37℃孵育8 h后离心去除上清,加入冻干保护剂混匀后程序冻干,15%PVP溶液37℃三步复水.分别检测并比较两组冻干红细胞复水后血红蛋白回收率、复水红细胞变形性、再水化后4℃放置时间对冻干红细胞再水化稳定性的影响及冻干红细胞长期保存的稳定性.结果表明:冻干红细胞再水化后,实验组红细胞血红蛋白回收率为(63.019±4.901)%,对照组为(28.787±7.514)%,两组之间存在显著差异(P＜0.01);冻干红细胞再水化后,两组冻干红细胞的综合变形指数均有明显下降,实验组红细胞综合变形指数为(18.133±2.392)%,对照组为(8.017±1.934)%,两组之间存在显著差异(P＜0.01);两组冻干红细胞复水后4℃保存4 h、6 h、8h后,相对即刻再水化血红蛋白回收率的差值,经配对t检验显示有显著统计学差异(P＜0.05).复水后4℃保存12 h、24 h后,相对即刻再水化血红蛋白回收率的差值,经配对t检验显示无统计学差异;两组冻干红细胞4℃保存6个月前后复水后血红蛋白回收率的差值,经配对t检验显示两者之间存在显著差异(P＜0.05). 结论:红细胞冻干前经添加6% DMSO的海藻糖负载缓冲液处理后,可提高冻干后红细胞血红蛋白回收率、改善复水红细胞变形性,提高复水后4℃保存的稳定性和冻干红细胞长期保存的稳定性.     

糖的浓度和种类对人血小板冷冻干燥保存的影响

以海藻糖、蔗糖、麦芽糖、乳糖、葡萄糖作为冻干血小板的保护剂,研究糖的浓度和种类对血小板冻干保存的影响.结果表明,冻干血小板的保存效果随海藻糖浓度的增加而提高,当海藻糖质量分数增加到20%时,非活化血小板的恢复率达到85.7%;对于质量分数均为20%不同种类的糖保护剂,非活化血小板的恢复率都达到70%以上,海藻糖略优于其他几种糖,但它们之间没有显著性差异.     

保存前白细胞滤除对红细胞悬液质量和保存的影响

为研究保存前白细胞滤除对于红细胞悬液质量的影响,用一次性使用去白细胞采血袋(规格200mL)采集健康无偿献血者全血30袋,采集后6h内进行白细胞滤除。通过离心将全血制备为去白细胞红细胞悬液,4℃保存。收集过滤前后全血血样,分别检测过滤前后红细胞计数(RBC),白细胞计数(WBC),游离血红蛋白(FHb),乳酸脱氢酶(LDH),K+,及全血容积变化。计算红细胞回收率和白细胞去除率。取10袋在4℃保存35d后,检测FHb,LDH及K+,并与相同环境保存35d后的未去白红细胞悬液进行比较。结果在保存前进行去白细胞滤除的红细胞悬液其白细胞残留量为(0.72±0.46)×106个/U,白细胞去除率为(97.59±1.47)%,红细胞回收率(87.74±2.42)%。过滤前后全血的血样FHb指标统计学差异无显著性(P>0.05),滤后LDH明显低于滤前(P<0.01)。滤后K+则高于滤前(P<0.01)。保存35d后,去白红细胞悬液K+与未去白组统计学差异无显著性(P>0.05),去白组FHb略高于未去白组(P<0.05),去白组LDH明显低于未去白组(P<0.01)。说明保存前白细胞滤除过程可能会对红细胞造成轻微损伤。...     

HA纳米颗粒对HepG2细胞冷冻干燥效果的影响

针对纳米颗粒对细胞冷冻干燥的影响进行研究。将不同质量浓度的羟基磷灰石(HA)纳米颗粒(质量浓度分别为0,0.1,0.3,0.5,0.7,1,5 g/L)添加到冻干保护剂中,研究其对HepG2细胞冷冻干燥效果的影响,结果显示:当保护剂中纳米颗粒质量浓度为0.5 g/L时,细胞的冻干效果最好,复水后细胞的回收率为37.39%,存活率为62.02%(P0.05);但是当纳米颗粒质量浓度高于0.5 g/L,对细胞具有较大损伤,显著降低细胞的24 h贴壁率。研究表明:添加适当浓度的纳米颗粒可以保护细胞,若纳米颗粒浓度过高,则会起到相反的作用;HA纳米颗粒冻干保护剂作用于冷冻、升华过程,在复水过程不起作用。     

红细胞保存期对冻干前海藻糖负载量的影响研究

探讨冻干前红细胞保存期对海藻糖负载量的影响,确定红细胞负载海藻糖的最佳条件。在37℃条件下,4℃保存0、24、48和72 h的红细胞在海藻糖浓度800 mmol/L的负载缓冲液中孵育7 h后,检测胞内海藻糖浓度和胞外外游离血红蛋白浓度。经相同条件孵育后,4组红细胞对海藻糖的摄取量基本相同,分别为(49.747±2.253)、(50.316±0.612)、(49.768±2.179)和(49.013±1.991)mmol/L,各组之间无统计学差异。4℃保存24、48和72 h的红细胞相对于新鲜红细胞,3组负载红细胞胞外游离血红蛋白明显增加,分别高达(8.473±2.138)g/L(P0.05)、(12.697±1.787)g/L(P0.01)和(14.036±2.796)g/L(P0.01)。说明冻干前红细胞4℃保存期不会影响海藻糖负载量,红细胞负载海藻糖的主要影响因素仍为负载温度、负载时间和负载缓冲液中海藻糖浓度,若固定孵育条件,红细胞对海藻糖的胞内负载量基本保持不变。但随保存期的延长,红细胞在高渗环境中孵育更易损伤,溶血率明显增加。所以,为达到更好的海藻糖负载效果,应尽量采用新鲜红细胞进行处理。     

不同冷冻保护剂对鸡胚胎干细胞冻存与复苏的影响

通过计算复苏后细胞存活率以及观察细胞生长状态.比较了二甲基亚砜(DMSO)、乙二醇(EG)和甘油3种冷冻保护剂对鸡ES细胞的冷冻保存效果.结果 显示:1)当DMSO的浓度为5%、10%和15%时.鸡ES细胞复苏后的存活率分别为73.57%、85.47%和78.90%;当EG浓度为5%,10%和15%时.复苏后细胞存活率分别为68%、74.30%和67.70%:当甘油浓度为5%、10%,和15%时细咆的复苏存活率分别为31.20%、51.40%和50.60%.在相同浓度下.以DMSO保护效果最佳.EG次之.甘油最差,同一种保护剂.以10%浓度的保存效果最好;2)以DMSO为冷冻保护剂.复苏后的鸡ES原代培养在饲养层上可以较好的生长.并形成集落.当浓度为10%时AKP阳性克隆数最高为23.5个,上述结果提示.以10%的DMsO作为鸡ES细胞的冷冻保护剂效果最佳.     

载入海藻糖红细胞冷冻干燥保存的实验研究

探讨载入海藻糖对红细胞冷冻干燥保存的效果.利用硫酸蒽酮比色法测量细胞内海藻糖的含量,对载入海藻糖的红细胞进行了冷冻干燥保存.结果表明,细胞载入海藻糖的最高浓度为35mmol/L,经冷冻干燥后的细胞回收率与血红蛋白回收率提高非常明显,分别为冷冻干燥前的59.78±4.51%和57.49±3.69%;渗透脆性与新鲜红细胞无显著差异.载入海藻糖对红细胞的冷冻干燥保存效果有密切作用.     

人参蛋白真空冷冻干燥技术工艺研究

目的 通过真空冻干保护剂的筛选和冻干曲线的优化,建立人参蛋白冻干的最佳工艺.方法 以外观、色泽、再分散性为指标,考察不同种类和浓度的冻干保护剂对人参蛋白样本的影响,筛选出最佳保护剂;采用电阻法检测其共熔点,在此基础上,以T-SOD酶活力为指标,优化样本,解析干燥程序,以建立人参蛋白样本的冻干曲线.结果 加入5%甘露醇的冻干保护剂,人参蛋白冻干样本外观、形态、再分散性较好;通过检测确定人参蛋白样本的共熔点为-4℃;经过优化后建立的冻干曲线为-40℃预冷2h后,6h内升温至-10℃,随后2h升温至-4℃,维持10h,然后1h内升温至5℃,再1h升温至15℃,维持2h.结论 通过对人参蛋白样本冻干保护剂和冻干曲线的优化,确定了人参蛋白样本最佳的冻干工艺.     

松材线虫低温冷冻保存研究

为满足大量松材线虫虫株长期保存的需要,对其低温冷冻保存技术进行了研究。试验比较了不同种类及浓度冷冻保护剂对松材线虫在低温冷冻保存时的保护效果,以及冷冻线虫繁殖力与致病力的变化。结果表明:使用15%甘油和4%DMSO作为冷冻保护剂,经-80℃保存1 d后,松材线虫存活率分别为(39.4±6.6)%和(37.5±21.8)%,且冻存10 d、1个月后均没有显著性下降。与未冷冻的线虫相比,冷冻线虫的繁殖力与致病力均未发生改变。     

电渗透用于强化海藻糖载入红细胞的实验研究

海藻糖是一种有效的冻干保护剂,实验采用电渗透的方法强化海藻糖载入红细胞,在不同电压、脉宽、频率和细胞外海藻糖浓度的条件下对红细胞进行电渗透.实验结果表明,当电压300 V、脉宽1 ms、频率4 次/min、细胞外海藻糖浓度800 mmol/L时,细胞内海藻糖浓度达到(63.68±2.14)mmol/L.电渗透后红细胞形态正常,细胞膜的渗透脆性略有降低,电渗透致溶血率为17%,ATP酶活性与新鲜红细胞无显著差异.     

红细胞低温保存中海藻糖的加载方法

在红细胞低温保存中,海藻糖被认为是能够替代甘油的生物相容性保护剂,而海藻糖难以进入红细胞内提供保护.归纳了使海藻糖加载至红细胞内的各种方法,包括孵育法、渗透休克法、脂质体法等,详细介绍了每种方法的原理、载入效果及优缺点.最后得出温和且不生成膜孔的加载方案具有一定的优越性,也可以尝试多种方法联用以提高加载效率和保护效果,...     

Estimating Fatty Acid Composition of Infant Buccal Mucosal Cells by Capillary Gas Chromatography

Long chain polyunsaturated fatty adds, i. e., docosahexaenoic acid （DHA or C22 ： 6n - 3）, amchidonic acid （AA or C20 ： 4n -6） have been identified as essential fatty acids and play an important role in growth and development of infants. Measurement of fatty acid composition is usually by collection of blood, but to obtain blood in infants is difficult. Nowadays, the fatty acid composition can be estimated by collecting buccal mucosal cells, which can avoid repeated blood sampling. The of this paper is to compare the fatty acid composition of cheek cells with that of plasma and red blood cells （RBCs）. In this study, twenty-seven infants were enrolled, and buccal mucosal cells and blood samples were obtained from these infants of the same time. Fatty acid composition of buccal mucosal cells, plasma and RBCs were measured by capillary gas chromatography. The results show that the contents of AA and DHA in the buccal macosal cells are correlated well with that in the plasma [r=0.36 （P=0.042） and r =0.38 （P =0.033）, respectively]. The ratio of AA to DHA is 1.32% in buccal mucosal cells, 1.60% in plasma and 1.55% in RBCs and there are no significant differences among groups （P = 0.134）. It shows that the fatty acid composition in buccal macosal cells can reflect the fat nutrition status in infants and can be detected by capillary gas chromatography. Estimating fatty acid composition of buceal mucosal cells in infants by capillary gas chromatography is feasible, and because of its noninvasiveness, it can be suitable for nutrition research in infants.     

环境温度对人红细胞膜形态和表面电荷的即时影响

利用显微图像分析技术和相分析电泳光散射技术,在无损、在位、实时的情况下,观察和测量了不同环境温度下活态人红细胞的形态、大小和表面电荷密度、Zeta电位的即时变化情况．结果发现随着环境温度的升高,人红细胞的大小逐渐减小,细胞形状指数增大,红细胞从双凹圆盘形向棘形转变,同时红细胞的表面电荷密度和Zeta电位的负值逐渐增大．表明人红细胞膜的形态结构、组成等与环境渝摩密切相关．     

Determination of cell volume during equilibrium freezing process

A new type electronic particle counter (EPC, Multisizer^TM 3, Beckman Coulter Inc., USA) was used to determine the volumes of human red blood cells (RBCs) in NaCl solutions of different osmolalities. The thermodynamics model describing cell response during freezing process was used to simulate the volume change of RBC in 0.9% NaCl solution during equilibrium freezing process. It was assumed that the effect of temperature on cell volume can be neglected compared to that of osmolality, then by using the phase diagram for the binary system sodium chloride/water, the osmolalities of the NaCl solution under different sub-zero temperatures can be obtained (converted from mass concentration), then the calculated values of RBC volumes can be validated by the experiments.     

大鼠外周血单个核细胞保存方法研究

通过观察大鼠外周血单个核细胞（PBMCs）的形态,了解不同保存方法和不同保存时期对PBMCs活性和细胞周期分布的影响,建立其短期保存方法．用密度梯度离心法获得大鼠外周血单个核细胞,分别在RPMI1640培养液中4℃保存和冷冻保护剂中－80℃保存,并分别在第1－4天和第7天,14天,21天,28天,35天用台盼蓝染色法测定细胞活力,用流式细胞仪检测细胞周期．结果分离获得的PBMCs成活率为97．69％±1．59％,基本处于G0／G1期．PBMCs置4℃保存4d后,存活率在60．84％±2．75％;置－80℃保存35d后,存活率在78．54％±2．97％,PBMCs基本处于G0／G1期．最后得出－80℃低温保存方法对PBMCs活性和细胞周期检测有一定影响,但其活性率接近80％,是一种简便易行短期保存PBMCs的方法的结论．     

A voltage-dependent channel involved in nutrient uptake by red blood cells infected with the malaria parasite

Growth of the malaria parasite in human red blood cells (RBCs) is accompanied by an increased uptake of many solutes including anions, sugars, purines, amino acids and organic cations. Although the pharmacological properties and selectivity of this uptake suggest that a chloride channel is involved, the precise mechanism has not been identified. Moreover, the location of this uptake in the infected RBC is unknown because tracer studies are complicated by possible uptake through fluid-phase pinocytosis or membranous ducts. Here we have studied the permeability of infected RBCs using the whole-cell voltage-clamp method. With this method, uninfected RBCs had ohmic whole-cell conductances of less than 100 pS, consistent with their low tracer permeabilities. In contrast, trophozoite-infected RBCs exhibited voltage-dependent, non-saturating currents that were 150-fold larger, predominantly carried by anions and abruptly abolished by channel blockers. Patch-clamp measurements and spectral analysis confirmed that a small (< 10 pS) ion channel on the infected RBC surface, present at about 1,000 copies per cell, is responsible for these currents. Because its pharmacological properties and substrate selectivities match those seen with tracer studies, this channel accounts for the increased uptake of small solutes in infected RBCs. The surface location of this new channel and its permeability to organic solutes needed for parasite growth indicate that it may have a primary role in a sequential diffusive pathway for parasite nutrient acquisition.     

Cultivation of the liver forms of Plasmodium vivax in human hepatocytes

The blood schizogonic cycle of human malaria parasites has thus far been the most exhaustively studied phase of parasite development. However, before entering red blood cells (RBCs), the parasite undergoes its first multiplication not in blood, but in hepatic cells. These hepatic stages were the last to be discovered and only a few studies have been performed in humans and other primates. Despite recent advances, in vivo studies have limitations and other approaches such as cultures of these liver forms may be necessary to investigate their chemosensitivity and their biochemical or immunological properties. Recently, sporozoites of species of rodent malaria have been made to infect cultured cell lines or primary hepatocyte cultures. We report here that the complete cycle of the human malaria parasite Plasmodium vivax can be obtained in primary cultures of human hepatocytes up to release of merozoites able to penetrate RBCs.     

桢楠种子脱水过程中的生理响应

[目的]通过对桢楠种子脱水过程中的生理响应进行研究,探讨恰当的脱水处理方式,为桢楠种子的长期保存提供必要的理论和技术支持.[方法]采用硅胶脱水的方法对桢楠种子进行脱水处理,当失水率在0、3％、6％、9％、12％、15％和18％时进行各项生理指标的测定,研究其脱水耐性.[结果]桢楠种子初始含水率为37.74％,初始生活力...     

Quantifying cell binding kinetics mediated by surface-bound blood type B antigen to immobilized antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance （SPR）-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound bio- molecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 bio- sensor. Human red blood cells （RBCs） presenting blood group B antigen and CM5 chip bearing immo- bilized anti-B monoclonal antibody （mAb） were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of sus- pending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.