两种低温保护剂对皮肤储存后α-辅肌动蛋白表达及活力的影响

目的比较两种不同低温保护剂对皮肤组织α-辅肌动蛋白（α-actinin）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实验依据。方法新鲜成人皮肤组织分为3组,新鲜对照组和海藻糖-二甲基亚砜（T—D）组、二甲基亚砜-丙二醇（D-P）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。同时对各组皮片进行氧耗量测定。结果0．5mol／L海藻糖-二甲基亚砜能够很好保护皮肤组织,α-actinin的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α-actinin的保护作用优于传统组。     

低温保护剂载入关节软骨过程的传质建模

掌握加载过程中软骨组织内部低温保护剂浓度的时空分布特性,这对合理设计加载程序进而成功保存关节软骨至关重要,为此构建描述低温保护剂载入关节软骨过程的扩散传质模型。根据二元扩散热力学模型,将有效扩散系数与无限稀释扩散系数进行关联,活度系数采用UNIFAC模型估算,扩散系数的浓度依赖性采用Vignes公式表示,无限稀释扩散系数采用Siddiqi-Lucas公式计算。对于二甲亚砜(Me₂SO)、甘油(GLY)、乙二醇(EG)和丙二醇(PG)这4种典型的低温保护剂(CPA),由模型计算得到的软骨组织内CPA的平均浓度与文献报道的实验值之间的平均相对偏差(MRE)和决定系数(R²)分别为1.90%～36.29%和0.959～0.998(Me₂SO),13.56%～19.19%和0.990～0.995(GLY),8.89%～22.09%和0.969～0.988(EG),5.35%～23.76%和0.971～0.992(PG)。结果表明,该模型能较好地适用于这4种CPA,可用于直接预测加载过程中软骨组织内部CPA浓度的时空分布,从而指导加载程序的设计与优化。     

Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His-Phe-Arg-Trp of α-Melanotropin by NMR

Solution conformation of cyclo(Gly¹-His²-Phe³-Arg⁴-Trp⁵-Gly⁶) and its d -Phe analog corresponding to the message sequence [Gly-α-MSH_5-10] of α-MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)-d₆ solution and in a DMSO-d₆/H₂O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only -NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross-peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the d -analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low-energy conformers with two γ-turns, a γ-turn and a β-turn, two β-turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a γ-bend at Gly⁶, two γ-bends at Phe³ and Gly⁶ and a conformer with a single β-turn and a γ-bend for the l -Phe analog. On the other hand, a conformation with two fused β-turns around the two tetrads His²-d -Phe³-Arg⁴-Trp⁵ and Trp⁵-Gly⁶-Gly¹-His² dominates the equilibrium mixture for the d -Phe analog. For the d -Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.     

三种冷冻保护剂对人精子活力及生育潜力影响的研究

目的 通过比较甘油、甘油－卵黄－柠檬酸钠（GYG）和甘油－卵黄－柠檬酸钠－L－谷氨酰胺（GYCG）三种冷冻保护剂（CPM）的冷冻保护效果,旨在寻求简单、安全、有效、经济的精子冻贮方法。方法 应用甘油、GYG和GYCG三种冷冻保护剂（CPM）,通过冻贮前后精液常规分析,精尾低渗肿胀率（HOSR）、精子穿卵率（SPR）等精子功能的检测指标评价三种CPM的冷冻保护效果。结果 三种CPM对精子功能的冷冻保护作用各不相同,但GYCG较其他两种CPM具有更显著的保护人精子功能的作用。结论 在三种CPM中,GYCG的冷冻保护效果明显优于GYC和单一甘油,预示应用GYCG作为CPM可使人精子保持较高的生育潜力,具有安全、价廉、简单等优点,适合在我国推广应用。     

肝细胞的低温保存及应用研究进展

随着生物人工肝和肝细胞移植研究的开展 ,对肝细胞的需求不断增加 ,迫切需要有效的肝细胞长期保存方法以促进其在临床上的应用。低温保存是目前保存肝细胞的重要方法 ,其影响因素较多 ,主要有低温保存肝细胞的形式冻存液的组成、降温、复温速度 ,复苏肝细胞的功能检测及应用范围等。本文对国内外该方面的最新进展进行综述     

Vitrification and storage of oral mucosa epithelial cell sheets

Shipping time and shipping delays might affect the quality of the stem cells based engineered “organs.” In our laboratory, we have developed a limbal stem cell deficient (LSCD) rabbit model. To reverse the LSCD, we cultured oral mucosal epithelial cells for 2–3 weeks and engineered cultured autologous oral mucosa epithelial cell sheets (CAOMECS), which were grafted on the LSCD cornea. The purpose of this study was to vitrify CAOMECS and to store it until the CAOMECS can be grafted onto patients. CAOMECS were vitrified in LN₂ for up to 204 days. We tested two different methods of vitrification with different solutions; however, CAOMECS were only viable when they were not stored in a vitrification solution; results were only reported from this CAOMECS. On the basis of hematoxylin and eosin staining, we showed that the CAOMECS morphology was well preserved after long‐term storage in LN₂. Most of the preservation solutions maintained the CAOMECS phenotype (Ki67, proliferating cell nuclear antigen (PCNA), Beta‐Catenin, ZO‐1, E‐Cadherin, CK3, CK4, CK13). The exception was the solution composed with ethylene glycol and Dimethyl sulfoxide (DMSO): this resulted in loss of DeltaN‐p63 expression. DeltaN‐p63 is an important marker for cell proliferation. The expression of proteins involved in cell–cell connection and the differentiation markers were maintained. Apoptosis was not detected in the thawed CAOMECS. We demonstrated that CAOMECS can be stored long‐term in LN₂ without affecting their morphology and phenotype.     

Influence of seminal plasma on cryopreservation of human spermatozoa in a biological material-free medium: study of normal and low-quality semen

The objective was to evaluate the efficiency of a biological material-free medium and the role of seminal plasma (SP) in the cryopreservation of human spermatozoa. Normal semen samples and low-quality semen samples were used for this study. After centrifugation of 300 microL fractions of whole semen, pellets were resuspended either in autologous SP or in a chemically defined medium (BM) supplemented or not with 3% bovine serum albumin (BSA); after 15 min at 37 degrees C, the samples were diluted (V/V) with cryoprotective medium (30 mM NaCl; 22 mM sodium citrate, 19.4 mM fructose; 80 mM glutamine; 14%, V/V, glycerol) and maintained for 15 min at room temperature before freezing. Assessment of viability and motility was performed using fresh semen (T0), after centrifugation and resuspension prior to adding the cryoprotectant (T15), after adding the cryoprotectant (T30) and after freezing and thawing (Tpost). In all three resuspending media used, sperm viability and motility (forward and total) decreased (p < 0.05) during both the equilibration period especially before addition of the cryoprotective medium (between T0 and T15) and during the freeze-thaw process comparison between T30 and Tpost. The recovery of viable and motile spermatozoa (post-thaw values/values of fresh samples) was higher (p < 0.05) in normal semen than in low-quality semen. In both groups, the recovery was slightly, but significantly, higher with SP than with BM and the presence of BSA has no beneficial effect. To conclude, these data suggest that SP may reduce the deleterious effects of cryopreservation. Nevertheless cryopreservation of spermatozoa in a medium containing neither SP nor biological substances could offer an acceptable cryoprotection of spermatozoa to be used in assisted fertilization procedures, especially for intracytoplasmic sperm injection.     

The fate of cryopreserved nerve isografts and allografts in normal and immunosuppressed rats

Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1–1.5°C/minute down to –40°C at which point the vials were plunged into liquid nitrogen at –196°C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival. Published in 1993 by Wiley-Liss, Inc.     

乳猪肝细胞-196 ℃保存的初步研究

目的探索适合于乳猪肝细胞-196 ℃冷冻保存的条件和方法.方法体外分离新生乳猪肝细胞,以含不同浓度二甲亚砜(DMSO)的冻存液及不同的细胞浓度在液氮中保存.2个月后复苏接种培养,对其存活率、贴壁率、尿素合成能力、猪白蛋白mRNA的表达等进行检测,并观察其形态结构变化.结果不同DMSO浓度保存猪肝细胞复苏后的活力及形态均有所不同, 其中含5% DMSO冻存液组保存效果最差;10%组次之;15%组最佳.15% DMSO冻存液组复苏后肝细胞的存活率为(83±4)%,贴壁率是(81±5)%,细胞形态与未冻存组相似,均保持较强的尿素合成能力及猪白蛋白mRNA的表达,较5%与10% DMSO冻存液组有显著性差别(P<0.05).冻存时肝细胞浓度为(5～10)×106个/ml组复苏后细胞存活率明显优于(1～2.5)×106个/ml组.结论 15% DMSO浓度适合于乳猪肝细胞的长期保存.高浓度组猪肝细胞的冻存效果优于低浓度组.     

丙二醇-氯化钠-水三元溶液相图及其在血小板低温保存中的应用

为了获得血小板冰冻保护液（丙二醇/氯化钠/水，PG/NaCl/H2O）冰冻降温的数学模型，本研究使用差示扫描量热仪分别测定出不同浓度和不同比例条件下丙二醇/氯化钠/水三元溶液的凝固点（Tf），然后将已有的公式和实验所得的数据通过计算机进行拟合。结果获得了一个数学模型，该数学模型描述的是在PG/NaCl/H2O溶液中Tf变化与溶质浓度和PG/NaCl(质量比)两个参数变化之间的函数关系。结论：把该方程与血小板的渗透性特性的参数相结合，可以描述在平衡冷冻状态下，不同PG浓度时血小板的体积变化，指导血小板低温保存。