深低温冷冻肌腱细胞活性的研究

目的研究深低温冷冻方法对肌腱细胞活性的影响,比较程序性降温和普通深低温冷冻法对腱细胞活性的影响.方法纯种SD大鼠24只（出生21 d）,随机分为3组,取双侧跟腱.新鲜肌腱对照组（A）,常规深低温冷冻组（B）,程序性降温深低温冷冻组（C）.采用相同的方法对3组肌腱细胞进行细胞培养.相差显微镜观察原代和传代后细胞的生长,绘制细胞的生长曲线,考察细胞的活性;对细胞进行成纤维细胞染色、胶原染色和对细胞进行形态观察（扫描电镜）;水解法定量分析细胞培养基中羟脯氨酸浓度的变化,检测细胞合成胶原的能力.结果原代细胞培养时A组细胞的生长速度快于B组和C组（P＜0.01）,C组细胞的生长速度快于B组（P＜0.01）,这种生长速度的差异在细胞传代后消失.细胞的形态学和组织学符合成纤维细胞形态.3组细胞培养基中羟脯氨酸浓度变化的差异无统计意义（P＞0.05）.结论经深低温冷冻处理的肌腱中仍存在具有活性的腱细胞,但数量显著少于新鲜肌腱中活细胞的数量.应用计算机控制程序性慢速降温方法处理的肌腱其活细胞的数量有所提高,但仍低于新鲜肌腱中活细胞的数量.     

The application of vitrification in human blastocyst cryopreservation: Analysis of clinical outcome of 117 cycles

Objective: To summarize the clinical outcomes of 117 human vitrified blastocyst transfer cycles and to determine the impact factors.Methods: In IVF-ET cycles, supernumerary embryos were cultured to 5-Day(D5) or 6-Day(D6), blastocysts of various stages were cryopreserved by vitrification using cryoloops. Survival rate and clinical pregnancy rate were observed.Results: A total of 312 blastocysts were thawed in 117 frozen embryo transfer cycles, the survival rate was 90.7% (283/312) after thawing. After the transfer of 230 blastocysts in 115 cycles, 69.6% (80/115) of the women got clinically pregnant, and 17.5% (14/80) of them suffered from miscarriage, 39 healthy babies were born in 28 deliveries, and the other 38 pregnancies are ongoing. The implantation rate was 47.4% (109/230). In 107 transfer cycles with 2 hatched blastocysts transferred in each cycle, 72.9% (78/107) got clinically pregnant, while in 8 cycles with 1 or no hatched blastocysts in the two transferred blastocysts, the clinical pregnancy rate is 25%(2/8). The clinical pregnancy rates were not statistically different between natural (77.4%, 24/31) or artificial endometrium preparation (66.7%, 56/84) cycles. Conclusions: These findings suggest that blastocyst vitrification is effective in terms of implantation rate and pregnancy outcome. Transferring of two hatched blastocyst can achieve a higher pregnancy rate.     

冷冻维持温度对大鼠血管组织超微结构的影响

目的：探讨不同维持温度冷冻对血管组织超微结构的影响。方法：以15％ DMSO为低温保护剂，采用两步冷冻保存步骤，设计维持温度为-45℃、-50℃、-55℃、-60℃、-65℃和-70℃，对SD大鼠血管组织进行冷冻预处理后，-196℃液氮保存1周。复温后光镜和透射电镜观察。结果：用维持温度-55℃、-60℃冷冻预处理的血管，经液氮保存、复温，血管组织超微结构保持良好，优于其它温度值的实验组。结论：-55℃～-60℃是适宜血管组织冷冻的维持温度值。     

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。     

东方田鼠室内繁殖与生长发育观察

近年来,逐渐增多的小鼠突变系资源已对现有的动物设施和品种（系）的保存方法提出严峻的挑战,小鼠胚胎和配合子的低温保存是目前保存小鼠种群资源中应用最为广泛的方法,通过低温保存不仅使原有的或新开发的小鼠品系得以安全的,长期的保存下来,同时也使这些小 模型的规模化生产,推广普及变得更为简单化,无疑,小鼠模型的保存将为21世纪哺乳类遗传学的飞速发展和最终攻克人类目前的疑难疾病创造了有利条件。     

How to improve donor skin availability: Pragmatic procedures to minimize the discard rate of cryopreserved allografts in skin banking

BackgroundMicrobial contamination of human skin allografts is a frequent cause of allograft discard. Our purpose was to evaluate the discard rate of skin bank contaminated allografts and specific procedures used to reduce allograft contamination without affecting safety.MethodsWe conducted at the Lille Tissue Bank a retrospective study of all deceased donors (n = 104) harvested from January 2018 to December 2018. Skin procurement was split into 3 zones: the back of the body and the two legs that were processed separately. It represented 433 cryopreserved skin allograft pouches of approximatively 500 cm² each. Donors were almost equally split between brain-dead (53%, 55/104) and cadaveric (47%, 49/104) donors.ResultsOut of all donors, 42 (40.5%) had at least one sampling zone with a positive microbiological test resulting in 106 (24%) contaminated skin pouches. The contamination rate did not vary according to the harvested zone or type of donor. Traumatic deaths showed significantly less contamination rates than other death types (p < 0.05). Contamination rate decreased with time spent in the antibiotic solution. The risk of having contaminated allografts was five-fold higher when the skin spent less than 96 h in the antibiotic cocktail (p < 0.05). According to our validation protocol, most donors (32/42, 76%) had skin allografts contaminated with bacteria (mainly Staphylococcus spp) compatible with clinical use. No recipient infection was recorded as a result of skin graft contaminated with saprophytic or non-pathogenic germs. By harvesting 3 separate zones per donor, the total surface area for clinical use increased by 53% for contaminated donors. Overall, the proportion of contamination-related discarded allografts was 3.2% (14/433 of pouches).ConclusionFew simple pragmatic measures (including skin incubation in the antibiotic bath for at least 96 h at 4 °C, splitting the skin harvesting areas to minimize the risk of cross-infection and clinical use of allografts contaminated with saprophytic and non-pathogenic germs) can reduce the discard rate of contaminated allografts without affecting clinical safety.     

人类胚胎冻存技术的初步研究

研究了不同冷冻与复苏方法以及不同移植周期法对人类胚胎冻存结果的影响。共复苏了１４９ 个胚胎,８９ 个（５９．７％ ）得以存活。在２０ 个移植周期中,５ 例（２５．０％ ）获得临床妊娠,２ 例已经分娩健康的婴儿。结果表明,采用缓慢降温至－ １２０℃再浸入液氮中冷冻及快速升温、逐步置换冷冻保护剂的方法复苏,胚胎的存活率（７２．７％ ）较高,但不同移植周期对冻胚移植的临床妊娠情况影响不大     

小鼠扩张囊胚颗粒法玻璃化冷冻保存技术研究

在２５℃室温下,用ＥＦＳ３０玻璃化溶液,对小鼠扩张囊胚进行颗粒法玻璃化冷冻保存。结果,胚胎在ＥＦＳ３０溶液中平衡１ｍｉｎ后滴入液氮中,解冻后的胚胎发育率达１００％,囊胚孵化率达７６％。而在１０％乙二醇溶液中将胚胎预处理５ｍｉｎ,再移入ＥＦＳ３０中平衡１ｍｉｎ后冷冻保存的胚胎,解冻后的发育率和囊胚孵化率分别为９８％和６３％。上述２组的胚胎发育率与对照组无明显差异,而囊胚发育率均明显低于对照组（Ｐ＜０．０１）。上述２组胚胎解冻后植入假孕小鼠子宫后的受体妊娠率分别为９０％和８１％,产出率分别为７１％和５８％,与对照组（６１％）相比均无显著性差异（Ｐ＞０．０５）。     

程序降温冷冻保存自体造血干细胞移植治疗恶性血液病

目的　探讨程序降温冷冻保存对造血干细胞活性的影响。方法　临床观察比较 4℃保存和深低温保存两组自体造血干细胞移植患者的造血恢复时间 ,同时采用台盼蓝拒染法与流式细胞仪Annexin V PI标记法检测两组造血干细胞的活性。结果　深低温保存移植组移植后血小板上升时间较 4℃保存移植组缓慢 (但P >0 0 5 ) ,4℃保存对造血干细胞活性影响不大 ,深低温保存可引起造血干细胞发生大量凋亡和死亡 ,导致存活率明显降低。采用流式细胞仪Annexin V PI标记凋亡测定法检测造血干细胞活性的敏感性明显优于传统的台盼蓝拒染法。结论　目前临床上应用的造血干细胞冷冻保护剂仍有较大缺陷 ,需开发具有抗凋亡作用的新型冷冻保护剂。造血干细胞活性检测选用流式细胞仪凋亡测定法较为可靠。     

深低温保存异体角膜缘干细胞羊膜移植治疗重度眼表疾病

目的 应用以羊膜为载体的深低温保存的角膜缘干细胞同种异体移植术治疗重度眼表疾病。方法 19例19眼重度眼表疾病患者采用羊膜移植联合深低温保存的角膜缘干细胞同种异体移植，2例术后半年行穿透性角膜移植术，随访观察术后疗效。结果 14例平均随访超过一年，眼表结构恢复正常，整个角膜上皮未见结膜杯状细胞，保存了眼球，视力均有不同程度的提高；1例植片脱落；4例发生角膜自溶的免疫排斥反应。结论 羊膜移植联合深低温保存的角膜缘干细胞同种异体移植术对于重度眼表疾病患者能快速、稳定的促进角膜表面重新上皮化，是一项可采纳的技术。