冷冻干燥法对人精子DNA的影响

目的探讨冷冻干燥法用于人类精子保存的安全性。方法取健康志愿者合格精液40份，平均分为4组，其中3组分别加入不同的冻干保护剂（ETBS；ETBS＋海藻糖：ETBS＋海藻糖＋蛋黄）后给予冷冻干燥处理，在4℃冰箱中保存3周；1组作为新鲜精液对照组。对4组标本分别以原位缺口末端标记（TUNEL）法和彗星试验进行DNA断裂精子百分率检测。结果ETBS、ETBS＋海藻糖、ETBS＋海藻糖＋蛋黄组和新鲜精液组DNA断裂精子百分率以TUNEL法检测，分别为（6．39±1．46）％、（5．75±1．29）％、（5．20±1．38）％、（4．94±1．86）％；以彗星试验检测，分别为（6．48±1．58）％、（5．83±1．48）％、（5．28±1．42）％、（5．12±1．65）％。冷冻干燥保存后的各组与新鲜精液相比较，DNA断裂精子百分率差异均无统计学意义（P〉0．05）。结论以ETBS或ETBS加海藻糖、蛋黄为保护剂的冷冻干燥法对人精子DNA无明显损伤，可有效地保护人精子的DNA。     

肝癌细胞Hep-G_2冻结过程中冻干保护剂热物性和效果研究

细胞冷冻干燥分为冻结和干燥两个过程,其中冻结过程中冰晶对细胞造成的机械损伤和溶质损伤不容忽视,而使用冻干保护剂可以减少此过程中细胞受到的伤害。为了探究冻干保护剂冻结过程中对细胞的保护机制以及优化冻干保护剂配方,本实验以肝癌细胞Hep-G2为对象,利用差示扫描量热仪(DSC)探究不同冻干保护剂在冻结过程中的热物性和未冻水份额;通过细胞冻存实验,检测使用不同保护剂的肝癌细胞Hep-G2的台酚蓝染色存活率和24 h贴壁率,结果表明配方为40%(W/V)聚乙烯吡咯烷酮K30(PVP)+10%(V/V)丙三醇+15%(V/V)胎牛血清(FBS)+20%(W/V)海藻糖的保护剂对细胞冻结过程的保护效果最佳,细胞24 h贴壁率可以达到44.56%±2.73%。因此,研究表明该冻干保护剂配方在冷冻过程中可以有效保护细胞。     

改善卵母细胞冷冻损伤的研究进展

卵母细胞的冷冻保存是女性生育力保存最具前景的技术。但由于卵母细胞特殊的生理结构,极易在冷冻过程中受到损伤。因此改进冷冻方法,添加冷冻保护剂(包括细胞骨架稳定剂和抗氧化剂)可以改善冷冻损伤,增加发育潜能,同时对于冷冻技术的运用有着重要意义。     

不同保护剂对大鼠肾脏冻融法脱细胞的影响

目的:利用不同保护剂对大鼠肾脏进行预处理,辅助冻融法脱细胞获得脱细胞支架,优化冻融脱细胞工艺。方法:采用不同保护剂预处理大鼠肾脏,放入-20℃环境下进行冷冻,12 h后进行37℃水浴复温,然后利用Triton X-100灌注24 h、PBS灌注2 h。最后通过CT三维重构、染色切片、蛋白质定量以及力学性能分析等手段评估所得脱细胞支架。结果:未添加保护剂组血管网络损伤较大,洗脱效果一般。添加不同保护剂组的血管网络也均存在一定的损伤,但其中10%DMSO和5%海藻糖组血管网络保留得较为完整。但10%DMSO组DNA等物质残留过多,5%海藻糖组洗脱细胞效果良好。结论:加载保护剂能够对冻融法脱细胞起到一定的促进作用,且能保护大鼠肾脏内部血管网络等结构,但不同保护剂对冻融法脱细胞的作用效果不同。     

不同冷冻保护剂对低温冰箱转液氮阶梯降温冷冻保存造血干细胞效果的影响

背景：非程序降温-80 ℃低温冰箱保存方便快捷,程序降温-196 ℃液氮保存可靠长久,将两者合二为一简化流程已成功用于临床。 目的：观察不同冷冻保护剂对-80 ℃低温冰箱转液氮阶梯降温冷冻保存造血干细胞效果的影响。 方法：分设10%二甲亚砜组、5%二甲亚砜联合3%羟乙基淀粉组、5%二甲亚砜联合0.25 mol/L海藻糖组、5%二甲亚砜联合3%羟乙基淀粉及0.25 mol/L海藻糖组。采用  -80 ℃低温冰箱转液氮阶梯降温法对单采外周造血干细胞进行冷冻保存,通过透射电镜观察细胞超微结构变化,流式细胞仪观察Annexin-V、PI、Caspase-3水平。 结果与结论：4组冷冻保存细胞的存活率、凋亡率和死亡率差异均无显著性意义(P > 0.05)。透射电镜下各组细胞超微结构变化差异不明显。单个核细胞群落冷冻保存后存活率在90%以上,含成熟细胞较多的CD45⁺细胞群落凋亡发生率可达50%左右。造血干祖细胞群落中,早期细胞较晚期细胞更能耐受冷冻损伤。提示在基础冷冻保护剂二甲亚砜的基础上,加入羟乙基淀粉和海藻糖并未显示出对冷冻保存效果的增强作用。     

乙酰左旋肉碱对人类精子冷冻前后顶体完整性及超微结构的保护作用探讨

目的 研究乙酰左旋肉碱对人精子冷冻前后顶体完整性及超微结构的保护作用.方法 将18例患者的精液标本液化及PureSperm梯度离心处理后分为2组,分别为A组(对照)和B组(7.5 mmol/L乙酰左旋肉碱),液氮中冷冻2周,比较冻融前后各组精子存活率、 活力、 头部和尾部超微结构损伤比例和顶体完整性.结果 精子冷冻后存活率和活力均较冷冻前明显下降(P<0.05),B组精子解冻后存活率和活力明显高于组A(P<0.05).冷冻后精子头部和尾部损伤比例均较冷冻前明显增加(P<0.05),而B组精子解冻后头部和尾部损伤比例均较A组明显下降(P<0.05).精子解冻后顶体完整性较冷冻前明显下降(P<0.05),B组精子解冻后精子顶体完整性较A组明显提高(P<0.05).结论 冷冻过程对人精子产生损伤,冷冻保护剂中添加7.5 mmol/L乙酰左旋肉碱可以提高精子解冻后顶体完整性,减轻冷冻损伤对于精子头尾超微结构的损伤,起到冷冻保护作用.     

海藻糖保护同种带瓣大动脉的最适浓度

背景：目前液氮低温保护组织和器官技术已经非常成熟,但其保护剂一直以来争论不一。 目的：观察不同冷冻保护剂对液氮深低温冻存同种带瓣大动脉组织细胞凋亡和代谢的影响,寻求最佳的冷冻保护剂及冷冻保护剂的最适浓度。 方法：取新西兰大白兔带瓣主动脉、肺动脉标本,将其在液氮中冻存12,15,18个月,冻存液分别使用0.1 mol/L二甲基亚砜,0.1 mol/L海藻糖,0.1 mol/L海藻糖+0.1 mol/L二甲基亚砜,0.2 mol/L海藻糖+0.1 mol/L二甲基亚砜,0.3 mol/L海藻      糖+0.1 mol/L二甲基亚砜。标本复温后,免疫组织化学方法检测标本的细胞凋亡情况,葡萄糖消耗量测定法检测组织细胞的代谢水平。 结果与结论：免疫组织化学和葡萄糖消耗量测定结果均显示经0.1 mol/L海藻糖+0.1 mol/L二甲基亚砜,0.2 mol/L海藻糖+  0.1 mol/L二甲基亚砜冻存液处理的标本冻存效果最好,其次是经0.3 mol/L海藻糖+0.1 mol/L二甲基亚砜处理和经0.1 mol/L海藻糖单独处理的标本,单独使用0.1 mol/L二甲基亚砜处理的标本冻存效果最差。说明海藻糖对液氮深低温保存同种带瓣大动脉活性有很好的保护作用,单独使用海藻糖优于单独使用二甲基亚砜,联合应用海藻糖和二甲基亚砜效果更好,且联合应用的海藻糖最佳浓度范围是0.10~0.20 mol/L。     

圆头精子的冷冻保存

目的:探讨圆头精子的冷冻保存效果及其精子功能改变。方法:6例圆头精子症患者各提供1-3份精液标本,应用不含卵黄冷冻保护剂和二步降温方法冷冻保存圆头精子症标本,检测冷冻精子活动率、头-尾膜完整率、存活时间和顶体蛋白酶活性。结果:圆头精子标本冷冻保存后,精子活动率显著减少,膜完整率显著下降,头膜损伤-尾膜完整的精子率显著升高(n=13,P＜0.01)。体外存活时间缩短。精子冷冻前后无顶体蛋白酶活性。不含卵黄冷冻保护剂与含卵黄冷冻保护剂,以及两步冷冻与多步冷冻的效果比较未见显著差异(n=7,P＞0.05)。结论:圆头精子可经历冷冻保存后存活,但冷冻复苏率低。冷冻-解冻过程显著降低精子功能。不含卵黄冷冻保护剂和两步降温可应用于冷冻保存圆头精子症标本。     

红细胞冻干保存中的渗透性损伤实验研究

红细胞长期保存中保护剂添加、洗涤过程会引入红细胞渗透性损伤.在冻干保存研究中,由于多种保护剂同时使用,保护剂的类型和功能一直是研究的重点,但很少有从渗透性损伤角度分析保存方案合理性的报道.目前相关文献、专利中所用的保护剂总渗透压差别很大,细胞保存后回收率差异也较大.文中用NaCl溶液实验模拟红细胞保存中保护剂添加、洗涤过程.结果表明,选择合适的添加、洗涤方法可以在一定程度上减小渗透损伤.就红细胞而言,1.5Osmol/kg左右是保护剂总渗透压的一个重要阈值,总渗透压低于该阈值时,渗透性损伤较小;高于该阈值时,渗透损伤随着渗透压的增大而迅速增大.所以选择保护剂时,首先应该根据总渗透压来排除渗透压过高的保存方案,否则红细胞在添加和洗涤保护剂时已经损伤很大.该研究对其它细胞长期保存中保护剂的选择也具有参考意义.     

不同低温保护剂对皮肤组织β1整合素表达影响的比较

目的比较两种不同低温保护剂对皮肤组织β1 integrin低温保存后表达的影响，为寻求皮肤组织低温保护剂的最佳配方提供试验依据。方法获取新鲜成人皮肤组织分为3组．新鲜对照组、海藻糖／二甲基亚砜（T／D）组、二甲基亚砜／丙二醇（D／P）作为低温保护剂保存组，-196℃液氮冻存7d、14d复温，免疫组织化学染色对各组间皮肤进行比较。并在此基础上，进一步采用RT-PCR方法对不同低温保护剂保存后皮肤的β1 integrin基因水平进行了深入的研究。结果通过光镜图象观察和基因水平分析结果说明，0．5M海藻糖／二甲基亚砜能够很好保护皮肤组织，β1 integrin的基因表达量与新鲜皮肤组相似。结论海藻糖与二甲基亚砜联合应用对皮肤组织β1 integrin的保护作用优于传统组。     

The effect of butylated hydroxytoluene (BHT) on bull spermatozoa frozen in two different extenders

This study evaluated the protective effect of butylated hydroxytoluene (BHT), a lipid-soluble antioxidant against cryopreservation damage on bull spermatozoa. Four BHT concentrations (0.5, 1, 2 and 4?mM) were evaluated. Sperm characteristics were evaluated when BHT was added to a post-thaw–freezing extender by measuring the degree of sperm lipid peroxidation (using malondialdehyde, MDA) and by measuring parameters such as motility and viability of spermatozoa. Production of MDA as an indicator of lipid peroxidation was obtained when BHT ranged from 0.5 to 1?mM in both extenders (P?<?0.0001). Sperm motility and viability evaluated immediately after thawing was higher in BHT-treated spermatozoa, this was significant (P?<?0.001) when the freezing egg yolk–citrate extender was supplemented with 0.5 and 1?mM BHT and when egg yolk–Tris extender was supplemented with 0.5?mM BHT. The addition of 0.5 and 1?mM BHT to semen egg yolk–citrate extender increased the viability of frozen semen after thawing (P?<?0.007). The addition of 0.5 and 1?mM BHT to both extenders resulted in a significant (P?<?0.0001) decrease in MDA production. In conclusion, the addition of BHT to freezing egg yolk–citrate extender improved the overall efficiency of thawed bull spermatozoa, but the addition of BHT to the freezing egg yolk–Tris extender did not.     

Stability of pseudorabies virus during freeze-drying and storage: effect of suspending media.

The effect of suspending media on the stability of pseudorabies virus upon freeze-drying and subsequent storage was studied. A variety of media was tested, including: sodium glutamate; sucrose; lactose; lactalbumin hydrolysate; peptone; a combination of sucrose, dextran, and glutamate; and various combinations of sucrose, glutamate, and potassium phosphates. Suspending media containing glutamate, either alone or in combination with sucrose and either dextran or phosphates, afforded the greatest degree of protection during the freeze-drying process and upon storage. Some possible functions of these additives in preventing injury to the virus during freezing and drying have been suggested.     

Studies on the Stability of Chicken IgY in Different Sugars, Complex Carbohydrates and Food Materials

The effect of non-reducing sugars (sucrose, lactose and trehalose), complex carbohydrates (cyclodextrin and dextran), infant formula or egg yolk on the stability of purified chicken IgY was evaluated under different conditions. Regardless of the protectant that was used, about 20% of the activity of IgY was lost during freeze-drying except in the presence of infant formula where the loss of IgY activity was approximately 75%. The lowest loss of activity (10%) was observed when no protectant was used. Trehalose was the best protectant followed by cyclodextrin and infant formula when IgY was stored for 6 or 14 weeks at different temperatures. Sucrose, lactose and dextran were not effective as protectants under these conditions. IgY activity was completely lost after pepsin treatment in the presence of sugars or complex carbohydrates while 34 and 40% of its activity was recovered when treated in the presence of infant formula and egg yolk, respectively. IgY was fairly stable after trypsin treatment with the recovery of residual activity being between 75-100% depending on the protectant. Finally, the effect of heat treatment on the stability of aqueous solutions of IgY was evaluated. IgY was stable at 50, 60 or 70°C while complete loss of IgY activity was observed at 80 and 90°C in the presence of all protectants except infant formula and egg yolk were about 5% residual activity was observed. These results demonstrate that the addition of different compounds to IgY during freeze-drying or storage at different temperatures or subjecting them to enzyme treatments provided varying and in most cases considerable protection against loss of biological activity.     

Preservation of the poliomyelitis virus when dried by the sublimation method (preliminary communication)

Summary The possibility of drying of the poliomyelitis virus is of great significance for obtaining not only the standard antigen for the diagnostic investigations, but also the stable live vaccine against poliomyelitis from nonpathogenic strains, one of which (type 11P-712[5]) was employed in the present work. The survival of the poliomyelitis virus during the process of drying of the culture fluid media was studied. Positive results were obtained. When drying the culture in the presence of egg yolk and saccharose the titer of the dry preparations was on the average lower than in the initial material by 1 point of ogarythim, viz., the viability reached 10%. A definite relationship between the survival of the virus and the concentration of the protective medium was revealed.Presented by Active Member AMN SSSR G. V.     

Preserving the structure of adsorbed protein films for time-of-flight secondary ion mass spectrometry analysis

The characterization of adsorbed protein films with ultrahigh vacuum (UHV) surface analysis techniques requires dehydration of the samples, which can cause significant alterations in protein structure. It is desirable to preserve the structure of adsorbed protein films during drying, so UHV analysis could be done in a state that is more representative of proteins' actual structure in the aqueous environment. In this study, two methods, trehalose protection and glutaraldehyde fixation, were explored for their feasibility in preserving adsorbed protein structure for a powerful UHV surface analysis technique, time-of-flight secondary ion mass spectrometry (ToF-SIMS). Trehalose protection had shown some promise for ToF-SIMS analysis in our previous study and was further examined with the model protein fibrinogen in this study. Using the combination of principal component analysis (PCA) and static ToF-SIMS analysis, we found that trehalose protection could reduce the conformation change of fibrinogen upon drying, and prevent it from unfolding and exposing hydrophobic domains. Moreover, when the adsorbed protein film became more densely packed, the drying-induced changes in protein structure were reduced. Thus, the protection afforded by trehalose coating was more significant at lower protein surface concentrations. The other method, glutaraldehyde fixation, was used in ToF-SIMS analysis for the first time. The epsilon-amino group of lysine was identified as the major reactive group in the protein structure toward glutaraldehyde fixation. Structural differences observed between fibrinogen films that were glutaraldehyde fixed before drying and after drying were similar to those observed between trehalose-protected and-unprotected dried fibrinogen films. Glutaraldehyde fixation was found to be a viable, alternative stabilizing method to trehalose protection for ToF-SIMS analysis.     

The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing

A firm biophysical basis for the cryopreservation of human spermatozoa is limited by a lack of knowledge regarding the water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPA). Cryomicroscopy cannot be used to measure dehydration during freezing in human spermatozoa because of their highly non-spherical shape and their small dimensions which are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of human sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPA. Using previously published data, the human sperm cell was modelled as a cylinder of length 40.2 micrometer and a radius of 0.42 micrometer with an osmotically inactive cell volume, V(b), of 0.23V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The 'combined best fit' membrane permeability parameters at 5 and 10 degrees C/min for human sperm cells in modified media are: L(pg) = 2. 4x10(-14) m(3)/Ns (0.14 micrometer/min-atm) and E(Lp) = 357.7 kJ/mol (85. 5 kcal/mol) (R(2) = 0.98), and in CPA media (with 6% glycerol and 10% egg yolk) are L(pg)[cpa] = 0.67x10(-14) m(3)/Ns (0.04 micrometer/min-atm) and E(Lp)[cpa] = 138.9 kJ/mol (33.2 kcal/mol) (R(2) = 0.98). These parameters are significantly different from previously published parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in human sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPA. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates (<100 degrees C/min) and the numerically predicted optimal cooling rates (>7000 degrees C/min) obtained using previously published suprazero human sperm permeability parameters which do not account for the presence of extracellular ice.