不同方法深低温保存兔肢体再植后血管组织的病理学变化

背景:虽然对于单一组织的冷冻保存获得了空前的成果,并且已逐渐应用于临床,但是对于复合组织的冷冻保存及应用还鲜有研究。目的:通过实验研究深低温冷冻不同复温方法下兔肢体再植后血管形态学变化,找出一种对复合组织中血管损伤最小的复温方法,从而为深低温处理后断肢再植可行性提供理论依据。方法:30只健康新西兰大白兔随机数字表法均分为对照组、慢速冷冻-慢速复温组、慢速冷冻-快速复温组,均给予大白兔右后肢自膝上1 cm处离断。慢速冷冻两组复温后均行断肢再植,再植肢体成活6 h后给予再次离断右后下肢。3组兔断肢均取股血管组织采用染色光镜及电镜进行形态学观察及大体观察,光镜病理计分结果用显著性分析。结果与结论:慢速冷冻-慢速复温组、慢速冷冻-快速复温组兔肢体再植6 h后的病理变化(大体标本、光镜、电镜)均较对照组差,但慢速冷冻-慢速复温组与慢速冷冻-快速复温组相比血管内皮细胞完整性较好,细胞器破坏较少。证实,经过深低温冷冻-复温处理后兔断肢再植6 h,离断肢体血管组织能保持一定的结构完整性,再植后6 h兔肢体获得成活,且慢速冷冻-慢速复温组更为适合离断肢体的保存,为深低温处理后离断肢体行断肢再植远期成活可行性提供了依据。     

冷冻预处理温度对胎兔许旺细胞活性的影响

背景：许旺细胞作为周围神经损伤修复的重要桥接材料,其活性对临床修复周围神经损伤成功与否具有重要影响。 目的：观察不同维持温度冷冻预处理超深低温保存对胎兔许旺细胞活性的影响。 方法：设计胎兔许旺细胞冷冻维持温度为-30~-70 ℃,每间隔-5 ℃为一组,以10%二甲基亚砜为低温保护剂,对胎兔许旺细胞进行上述温度冷冻预处理,液氮中保存48 h,快速复温后继续培养48 h,透射电镜观察细胞超微结构改变,MTT法和³H-TdR掺入法检测细胞活性。 结果与结论：透射电镜下见-45 ℃处理的细胞超微结构良好,其余各温度组细胞均出现细胞器肿胀、坏死等轻重不同的冷冻损伤表现。MTT法和³H-TdR检测结果均显示-45 ℃冷冻预处理对许旺细胞的生长抑制率最低,分别为(3.83±0.56)%、(4.41±0.71)%,和其余各温度组之间相比,差异有显著性意义(P < 0.05)。提示-45 ℃是胎兔许旺细胞冷冻预处理的最佳维持温度值,对细胞活性影响最小,可较好保持其生物学活性。     

快速冷冻与慢速冷冻条件下复合组织血管内皮的生物活性

背景：恢复良好血供对于复合组织保存及再植至关重要,但不同冷冻方法对血管活性影响不同。 目的：比较快速冷冻与慢速冷冻方法对复合组织血管内皮活性的影响。 方法：新西兰大白兔后肢分别进行快速冷冻与慢速冷冻,快速冷冻的兔后肢直接放入液氮中,慢速冷冻组经4 ℃,-20 ℃,-80 ℃冷冻后再投入到液氮中,各组依次保存12 h,3 d,7 d后快速复温,并设对照组。所有新西兰大白兔后肢均经甲醛溶液固定后行苏木精-伊红染色及免疫组织化学染色检测各组血管内皮的病理变化。 结果与结论：快速冷冻组和慢速冷冻组冷冻12 h,3 d,7 d时兔血管组织形态评分均低于对照组(P < 0.05)。快速冷冻组冷冻12 h,3,7 d时血管内皮组织血管内皮生长因子评分低于慢速冷冻组(P < 0.05)。证实,慢速冷冻法能更好的维持复合组织中的血管内皮细胞的生物学活性。关键词：深低温冷冻;复合组织;冷冻方法;内皮细胞活性;组织构建 doi:10.3969/j.issn.1673-8225.2012.20.011     

卵母细胞深低温保存的策略分析

背景：卵母细胞具有特殊而复杂的低温生物学性质,致使卵母细胞在深低温保存中易受到多因素的损伤而影响复温后的存活率、受精率和受精后发育潜能。 目的：综述卵母细胞深低温保存技术的研究进展,阐明存在的技术缺陷以及解决问题的思路与研究路线。 方法：以“卵母细胞,深低温保存/慢速冷冻,玻璃化”为中文捡索词,以“oocyte, cryopreservation/slow freezing, vitrification”为英文检索词,在中国知网(CNKI)期刊全文数据库和PubMed数据库检索2004 年1月至2014年10月有关卵母细胞深低温保存文献,排除陈旧性、重复性研究,最终纳入41篇文献进行综述。 结果与结论：慢速冷冻、玻璃化方法深低温保存卵母细胞得到较好的临床应用,但复温存活率、受精能力、发育能力损伤等仍困扰医生们。卵母细胞深低温保存技术的进一步完善或突破在于对深低温保存技术环节的细化、卵母细胞低温生物学特性的研究以及相关技术的创新,如深低温保护剂选择、冷冻载体的改进、卵母细胞时相选择、卵母细胞体外成熟技术、卵巢组织保存与移植等。     

不同保存方法对后交叉韧带保存后组织形态学影响*

目的观察不同的低温保存方法对异体骨-后交义韧带-骨复合组织形态学的影响。方法新西兰白兔膝关节96个随机分成4组:新鲜对照组、二步冷冻保存组、-80℃冷冻保存组、-196℃冷冻保存组,后三组经保存二周后于37℃水浴快速复温,随即与新鲜对照组一起进行光镜、电镜观察组织形态学方面的变化。结果HE染色光镜观察:新鲜对照组在胶原纤维形态、排列整齐性及分布致密度均优于其它各组,二步冷冻保存组较新鲜对照组稍差,但明显优于其他二组,-196℃冷冻保存组织学表现最差。电镜观察:新鲜对照组在胶原微纤维排列整齐性及分布致密度方面优于其它各组,二步冷冻组较新鲜对照组差,但优于其它二组,-196℃冷保存组最差。结论二步冷冻保存能够较好地维持后交叉韧带的组织形态学特性。     

猪肢体体外循中神经组织损伤和固有免疫的实验研究

目的:观察体外循环灌注保存猪肢体12 h后神经组织病理改变和固有免疫系统的改变, 探讨固有免疫系统在体外循环灌注中对猪肢体神经组织的影响.方法:实验分3组, 实验组: 随机取10头实验用landrace猪为实验对象, 取每头猪的1条前肢体进行实验, 经32℃体外循环灌注保存12 h;对照组: 实验组猪的另一前肢放置于4℃冰箱低温保存;再灌注组: 将2例对照组肢体体外循环灌注1 h.收集3组肢体神经组织、冰冻神经组织样品, 进行病理及补体C3b/c、 C4c和固有免疫抗体IgG和IgM抗体的免疫荧光强度的检测, 同时观测猪肢体跟腱反射.结果:实验组猪的神经反射正常, 而对照组猪未引出神经反射, 再灌注组神经反射很弱.病理结果显示实验组神经组织有轻微的水肿, 免疫荧光染色补体C3b/c、 C4c和固有免疫抗体IgG和IgM抗体免疫荧光强度增强, 而对照组神经组织轻微水肿, 免疫荧光染色补体C3b/c、 C4c和固有免疫抗体IgG和IgM抗体免疫荧光强度没有改变, 再灌注组神经损伤明显, 可见水肿和无髓鞘现象.结论:体外循环对肢体神经组织可能有保护作用.尽管体外循环也激活固有免疫, 但对神经组织仍有保护作用.     

深低温保存兔颈总动脉体外灌注后的结构和力学性能研究

 目的 探讨深低温保存对动脉血管组织学和力学性能的影响。方法 取 20 只新西兰兔的颈总动脉，用含 1.5 mol/L 1, 2-丙二醇的低温保护剂深低温（－196℃）保存 10 ~ 15 d，并在预冷的冰袋内缓慢复温；以新鲜血管作为对照。对新鲜和冷冻-复温血管的平滑肌细胞（SMC）进行体外培养，观察和分析新鲜、冷冻-复温和冷冻复温后体外灌注 6、12 和 24 h血管的内皮细胞、SMC、血管壁组织构和力学性能（轴向、周向弹性模量和断裂应力）的变化。结果 冷冻-复温后血管的 SMC 在体外培养中开始生长时间与新鲜血管基本相同（24 ~ 36 h），生长速度接近。深低温保存后，血管内皮细胞数量减少，电镜观察SMC数量和形态无明显变化，但血管的轴向弹性模量（5.82 ± 0.55）、轴向断裂应力（58.78 ± 6.96）和周向弹性模量（1.44 ± 0.22）、周向断裂应力（3.45 ± 0.40）均明显低于新鲜血管（分别为6.45 ± 0.80、146.96 ± 23.60 和 1.83 ± 0.17、5.33 ± 0.50， P < 0.05或 P < 0.01）。体外灌注后，冷冻-复温血管的内皮细胞脱落，SMC 变性、坏死，内弹力纤维和胶原出现不同程度崩裂，力学性能指标中除了轴向弹性模量外，均进一步降低（P < 0.05 或 P < 0.01）。结论 兔颈总动脉 SMC 深低温保存效果满意，但体外灌注对经深低温保存血管的内皮细胞、SMC 和血管壁结构具有明显损伤作用，导致血管力学性能显著降低。     

蟾蜍胃组织冻融碱性磷酸酶活性组化研究

本文用光镜组织化学分析方法分析了不同情况下冻融胃组织碱性磷酸酶活性。选择处于冬眠状态下蟾蜍，体温１０℃～１５℃，体重５０～７０ｇ，性别不限，随机分为４组，每组５只，将蟾蜍分为整体冷冻组，开胸冷冻组，冷冻复温组，正常对照组。在蟾蜍心跳呼吸停止，肢体僵直状态下，将冷冻蟾蜍放于５００ｍＬ溶液实验杯内。３０℃复温。复温溶液为４％任氏溶液，１０％甘油，５％葡萄糖。冷冻蟾蜍２～１０ｍｉｎ复活。蟾蜍心跳呼吸胃肠蠕动正常，速将胃取出。用冷丙酮固定２４ｈ。经组织化学常规处理，光镜组化：冷冻３组，胃组织结构清楚可见…     

透析法去除红细胞渗透性低温保护剂的实验研究

要实现人类红细胞深低温保存,必须在冷冻前添加、复温后去除低温保护剂.本研究提出了低温保护剂的新型的透析去除法,与常规的离心洗涤法比较而言,该方法能够快速、有效、安全地去除红细胞内的低温保护剂(甘油).使用该方法冰冻.复温.洗涤后的红细胞计数回收率为(89.17±2.46)%,血红蛋白回收率为(84.93±4.64)%,上清游离血红蛋白的含量为(0.66 ±0.13)g/L,所得冰冻-复温-洗涤后的红细胞悬液的渗透压(残余甘油的含量)为(340.33±20.56)mOsm,均达到了国家对冰冻洗涤红细胞的质量标准要求.     

组织工程化肌腱种子细胞深低温保存的实验研究

对肌腱种子细胞进行深低温保存，研究保存过程中多个环节的影响因索对细胞存活率的影响及深低温保存对种子细胞生物学特性、胶原分泌功能的影响。实验结果表明二甲基亚砜是肌腱种子细胞深低温保存中比较好的抗冻保护剂；冻存后使用与培养时不同的营养血清处理对细胞有损害，可降低细胞存活率；在冷冻保存过程中，细胞存活率与细胞浓度有一定关系，浓度太低可能降低细胞存活率；细胞在冷冻保存时，降温速度对细胞存活率有影响，慢速的分步降温组细胞存活率明显高于直接人液氮的快速降温组；采用10％二甲基亚砜加15%小牛血清加75% DMEM配方保存肌腱种子细胞，对其分泌胶原功能无明显影响，对其生长曲线、细胞周期及染色体众数无明显改变，适于肌腱种子细胞的保存。     

Optimal freezing and thawing for the survival of periphheral nerves in severed rabbit limbs

This study aimed to investigate the optimal freezing and thawing procedures for the survival of peripheral nerves in severed rabbit limbs. Twenty New Zealand White rabbits were randomized into four groups: normal control, slow-freezing fast-thawing, slow-freezing slow-thawing, fast-freezing fast-thawing, with five animals in each group. The hind limbs of the rabbits were severed at 1 cm above the knee joint. The severed limbs were cryopreserved with various freezing and thawing procedures. The sciatic nerves were harvested and trypsinized into single nerve fibers for morphological evaluation. The cell viability of the nerve fibers was examined by staining with Calcein-AM and propidium iodide. The fluorescent intensity of the nerve fibers was measured with a laser scanning confocal microscope. The morphology of the nerve fibers in the slow-freezing fast-thawing group was very similar with that of the normal control group, with only mild demyelination. The slow-freezing fast-thawing group and slow-freezing slow-thawing group showed severely damaged nerve fibers. The fluorescent intensities of the nerve fibers was significantly different among the four groups, with a decreasing order of normal control, slow-freezing fast-thawing, slow-freezing slow-thawing, and fast-freezing fast-thawing (P < 0.05). Of the various cryopreservative procedures, slow-freezing fast thawing has the minimal effects on the survival of nerve fibers in severed rabbit limbs.     

Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates

We investigated whether further in-vitro culture of human multicellular embryos that survive cryopreservation can select the viable embryos for transfer. Embryos for cryopreservation were supernumerary multicellular embryos obtained after in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatments, with <20% of their volume filled with anucleate fragments. These had been cryopreserved using a slow-freezing and slow-thawing protocol with 1.5 M dimethylsulphoxide as the cryoprotectant. From the start of our cryopreservation programme until September 12, 1994, the thawing strategy was to thaw frozen embryos up to the exact number needed for transfer. Embryos for transfer were selected on the basis of their morphological appearance and embryo transfer to the patient was done on the day of thawing. From September 12, 1994 onwards we used a more selective thawing strategy where a cohort of up to a maximum of 12 frozen embryos per patient is thawed from which embryos of the best morphological quality, and which are furthest advanced in terms of cleavage after a 24 h in-vitro culture period in Menezo B2 medium, are selected. We took delivery rates, embryo implantation rates and birth rates into account to see if there is any difference between the following three types of transfers used: 187 transfers exclusively of embryos having continued to cleave after thawing, 107 mixed transfers of embryos with and without further cleavage and 53 transfers exclusively of embryos with no further cleavage. The overall outcome in terms of delivery rate and embryo implantation and birth rates were not different between the new and the earlier thawing policies (6.6, 5.2 and 3.6% versus 6.0, 4.1 and 2.7% respectively). Only when a distinction was made between transfers on the basis of the presence of embryos with further cleavage, did the advantage of selection on the basis of cleavage capacity become evident. Significantly higher delivery and embryo implantation and birth rates (11.2, 7.7 and 6.5% respectively) were recorded with transfers exclusively of embryos with further cleavage versus mixed transfers of embryos with and without further cleavage (1.9, 2.9 and 0.6% respectively). Fifty-three transfers exclusively of embryos with no further cleavage did not lead to any delivery. Our results demonstrate that selection of human multicellular embryos which survive cryopreservation and continue to cleave in vitro can significantly improve the delivery rate per transfer and the implantation rate per transferred embryo.      

干式融化箱与水浴锅复苏细胞因子诱导的杀伤细胞的比较

目的对比干式融化箱与水浴锅复苏冻存细胞因子诱导的杀伤细胞（CIK细胞）的效果。方法提取10份健康志愿者外周血单个核细胞分别诱导培养CIK细胞并冻存于一80℃冰箱。1个月后从每份CIK细胞中取出相同的两个冻存管,分别采用干式融化箱和水浴锅进行复苏。记录复苏时间,检测复苏后细胞存活率及对K562细胞的杀伤作用。结果干式融化箱较水浴锅复苏时间较长[（4．33±0．19）min、（2．47±0．30）rain],差异有统计学意义（P〈0．05）。但两种方法复苏的CIK细胞存活率[（89．55＋5．36）％、（89．86±4．63）％]和对K562细胞的杀伤活性[（44．76±9．53）％、（46．97±10．17）％]差异无统计学意义（P〉0．05）。结论干式融化箱可以代替水浴锅进行冻存CIK细胞复苏。     

Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures

BACKGROUND: The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. METHODS: Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy system (Polscope). RESULTS: The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37 degrees C in culture medium. CONCLUSIONS: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.     

枸杞多糖对胎儿卵巢组织冷冻保存的影响

背景：抗氧化剂的应用对于提高卵巢组织冻存后的活力起着很重要的作用。 目的：通过观察冷冻保存的胎儿卵巢组织异种移植到裸鼠肾被膜下的发育情况,探索枸杞多糖在卵巢组织冷冻保存中的作用。 方法：实验分为4组。①新鲜移植组：取材后的新鲜胎儿卵巢组织直接进行移植。②冻存对照组：冷冻保护液为基液。③β-巯基乙醇组：冷冻保护液为基液添加100 μmol/L β-巯基乙醇。④枸杞多糖组：冷冻保护液为基液添加 400 mg/L枸杞多糖。对冷冻复温后的胎儿卵巢皮质块进行祼鼠的肾被膜下移植,并于移植后12周取材。 结果与结论：各组在移植物的存活率上差异无显著性意义(P > 0.05)。在卵泡计数上冷冻对照组最低(P < 0.05)。在卵泡的存活率上,各组间差异均有显著性意义,其中以冷冻对照组最低,枸杞多糖组最高。β-巯基乙醇组和枸杞多糖组卵巢超微结构较冷冻对照组保存的好。提示400 mg/L的枸杞多糖和100 μmol/L的β-巯基乙醇有利于卵巢组织的冷冻保存,并可显著提高冷冻卵巢组织移植后的存活率。     

Comparison of the survival of human biopsied embryos after cryopreservation with four different methods using non-transferable embryos

BACKGROUND: The standard embryo cryopreservation method is still less than optimal for biopsied embryos. The aim of this study was to compare the survival of biopsied embryos cryopreserved with four different methods using non-transferable embryos. METHODS: Abnormal embryos from one or three pronuclei and spare embryos of grade 3 and 4 were used for this study. Non-biopsied embryos were cryopreserved using the standard method as control. Biopsied embryos were cryopreserved using four methods as follows: standard method, modified freezing method, modified thawing method and vitrification. Blastomere survival and blastulation of frozen-thawed embryos were compared between the different methods. RESULTS: The proportion of embryos with > or = 50% blastomere survival and total blastomere survival rate of biopsied embryos were significantly higher with vitrification than the other three methods. Both the modified freezing and modified thawing methods had significantly higher embryo survival and total blastomere survival rates than standard methods. However, there was no significant difference in blastulation of surviving embryos in all the five groups. CONCLUSIONS: Non-transferable embryos derived from clinical IVF/ICSI are useful for evaluation of the optimal freezing procedures for biopsied embryos. Vitrification increases the survival rate of human biopsied embryos above standard and modified cryopreservation methods.     

种属和发育期及不同实验条件对卵裂期胚胎冷冻复苏结局的影响

背景：人卵裂期胚胎冷冻复苏的研究中,不同的实验条件及实验动物模型是否与人类胚胎具有相同的敏感性从而反映出实验方案的优劣值得探讨。 目的：观察胚胎种属和发育期及不同冷冻条件对卵裂期胚胎冷冻复苏结局的影响。 方法：将人卵裂期胚胎作为对照组,将KM小鼠胚分为2细胞,4细胞,8细胞组。各组胚胎随机采用以下实验方案：①冷冻操作环境温度18~20 ℃、24~26 ℃和37 ℃。②慢速程序化方案、自制straw叶片玻璃化方案和CPS玻璃化方案。③与玻璃化液接触时间< 40 s、40~60 s和60~90 s。各组胚胎比较不同实验条件下胚胎复苏率和培养24 h发育率。 结果与结论：①对照组24~26 ℃冷冻操作环境温度复苏率高于37 ℃冷冻操作环境(P < 0.05)。②对照组和4细胞鼠胚组采用自制straw叶片玻璃化方案复苏率高于慢速程序化方案(P < 0.05),培养24 h胚胎发育率差异无显著性意义(P > 0.05)。③各组胚胎与冷冻保护剂接触不同时间胚胎复苏率差异无显著性意义(P > 0.05)。表明卵裂期胚胎玻璃化冷冻复苏效果优于慢速程序化,24~26 ℃操作环境、减少冷冻保护剂剂量和缩短接触时间可改善玻璃化冷冻复苏结局;相同冷冻条件下,胚胎种属和发育阶段对冷冻复苏结局有影响,4细胞鼠胚更适合作为研究人类卵裂期胚胎冷冻复苏的实验模型。     

神经营养因子3纳米微球载体基因工程转染许旺细胞

背景：纳米微球基因转染技术携带或增强种植细胞可持续稳定延长其释放,保持其活性和作用时间。 目的：观察纳米微球载体转染神经营养因子3基因修饰的许旺细胞对坐骨神经缺损的促进和修复作用。 方法：采用纳米微球载体将神经营养因子3基因转染入体外培养的新生Wistar大鼠许旺细胞。取Wistar成年大鼠80只制作坐骨神经缺损模型,将4种不同的移植物注入细胞外基质凝胶-聚乳酸聚羟基乙酸共聚物管桥接管内：空白对照组未注入其他任何物质,细胞组注入许旺细胞,基因组注入神经营养因子3基因,实验组注入神经营养因子3基因修饰的许旺细胞。 结果与结论：术后坐骨神经及肌纤维横截面积染色比较,实验组优于细胞组、基因组(P < 0.01),细胞组、基因组均优于空白对照组(P < 0.01),细胞组、基因组组间比较差异无显著性意义(P > 0.05)。表明神经营养因子3基因转染许旺细胞移植,可相互促进,再造神经再生的微环境,促进并修复缺损坐骨神经缺损。     

Cryopreservation of suckling pig hepatocytes.

To determine the best and simplest method for cryopreservation of pig hepatocytes, we compared immediate cryopreservation with cryopreservation after short-term culture. Suckling pig hepatocytes were isolated by a modified 2-step in situ collagenase perfusion method, suspended in serum-free medium, and preserved for 10 da by two cryopreservation methods. Serial measurements were made of cell viability, LDH release, synthesis of protein, urea and glucose, glucose-6-phosphatase (G-6-Pase) activity, and diazepam transformation after thawing. These measurements were performed on both groups of cultured hepatocytes, and on freshly isolated hepatocytes, which served as a control. High viability (>95%)of thawed hepatocytes was obtained and maintained in both cryopreservation groups. There were no significant differences in cell viability, protein synthesis, glucose synthesis, G-6-Pase activity, or diazepam transformation between the two cryopreservation groups. In the immediate cryopreservation group, urea synthesis was less than in the group with cryopreservation after short-term culture. Protein synthesis, glucose synthesis, and diazepam transformation were lower in both cryopreserved groups than in the controls. The results showed that a protocol of immediate cryopreservation of hepatocytes in RPMI-1640 medium containing 10% DMSO, hormones, growth factors, and 10% newborn bovine serum, together with rate-controlled freezing and rapid thawing, provides indices of cell viability and function during subsequent serum-free culture that are comparable to hepatocytes cryopreserved after short-term culture, except for lower urea production. This simple procedure can be used in studies of bioartificial liver and hepatocyte transplantation.