改良皮片4℃低温保存方法在创面二次植皮治疗中的应用研究

目的:探讨改良皮片4℃低温保存方法在创面二次植皮治疗中的应用效果。方法:①动物实验:健康成年豚鼠3只,处死后,取背部皮肤做成32个1 cm×1 cm小皮样,随机分为新鲜皮组、庆大盐水保存组、RPMI保存组,改良RPMI保存组进行4℃保存;1周后测定皮肤活力;②临床实验:观察自2018年10月至2018年12月,二期植皮患者33例,应用改良RPMI 4℃低温保存皮肤二期回植的患者16例,与重新取皮植皮患者17例比较其皮片成活率。结果:动物实验证实:改良RPMI保存组4℃保存组在皮肤储存1周时皮肤平均活力较庆大盐水保存组、RPMI保存组高(P0.05);临床实验证实:改良RPMI保存组与重新植皮的皮片平均成活率没有显著性差别(P0.05)。结论:改良4℃断层皮片低温保存方法可短期内保存皮肤较高的活力,是皮片再利用的一种有效方法。     

小腿挤压伤伴撕脱伤23例的临床治疗效果分析

目的:探讨小腿挤压伤伴撕脱伤患者的整体治疗方法,并分析其临床应用价值。方法:回顾性分析我院近5年来收治的23例小腿挤压伤伴撕脱伤患者的临床资料,分别采用行自体皮肤反削回植、异种皮覆盖或封闭负压吸引治疗+二期植皮、知名血管皮瓣转移、单纯清创缝合。结果:23例中,18例Ⅰ期愈合;5例局部皮肤坏死,经换药后Ⅱ期愈合2例,残余创面行植皮后Ⅱ期愈合1例;骨外露者经皮瓣转移修复后Ⅱ期愈合2例。随访3-16月,临床效果满意。结论:对于小腿挤压伤伴撕脱伤,依具体情况采用自体皮肤反削回植、异种皮覆盖或封闭负压吸引治疗+二期植皮、知名血管皮瓣转移、单纯清创缝合等方法修复创面对患者肢体功能恢复有较大的作用,临床效果较好,利于患者康复,具有一定的推广应用价值。     

VSD负压引流技术结合游离植皮治疗大面积皮肤缺损的 临床疗效研究

目的：研究VSD负压引流技术结合游离植皮治疗大面积皮肤缺损临床疗效。方法：选择2012年4月至2013年10月入我院接受治疗的大面积皮肤缺损患者65例,将其随机分为观察组（35例）与对照组（30例）。观察组患者术前先采用VSD治疗,游离植皮术后继续给予VSD治疗;对照组患者采取打压植皮或传统打包加压治疗。观察和比较两组患者的皮片成活时间、皮片成活率、术后住院治疗时间、换药次数、疼痛评分及家属满意度。结果：观察组患者的皮片成活时间短于对照组、皮片成活率高于对照组、住院时间短于对照组,换药次数少于对照组,两组之间的差异具有统计学意义（P〈0．05）。此外,观察组患者的疼痛视觉模拟评分明显低于对照组,患者家属的满意度评分明显高于对照组,差异具有统计学意义（P〈0．05）。结论：相对于传统的打压植皮或传统打包加压的技术而言,VSD负压引流技术结合游离植皮在治疗大面积皮肤缺损的临床疗效更好,值得在临床上推广使用。     

新生小鼠卵巢组织的玻璃化冻存、移植及分离卵泡的体外成熟培养初步研究

目的:系统地探索新生小鼠卵巢组织的玻璃化冻存建立卵巢库,移植和分离卵泡以及体外成熟培养的实验方法。方法:对1日龄小鼠卵巢组织进行冻存,解冻复苏和同种肾包膜下移植,从卵巢移植物种中进行卵泡分离和体外成熟培养。结果:①采用平衡液(ES)处理25min,玻璃化液(VS)处理3min方案冻存的卵巢组织具有更高比例的形态完整卵泡,其完整原始卵泡率均值达96.6%,显著性高于实验中其它四组方案(P0.05);②在分别移植2周和4周后回收卵巢移植物,发现二者的卵巢回收率无显著差异(P0.05),但移植4周组的卵泡回收数目要明显高于2周组(P0.05);③在培养基中添加适量的自体血清(10%,V/V)能显著提高卵子的体外成熟率,培养12h后对照组中生发泡破裂(GVBD)发生率为(34.74±4.26)%,添加血清后提高至(54.60±3.37)%,成熟的MⅡ期卵子获得率从(43.17±1.31)%升高至(57.75±5.31)%,有显著性差异(P0.05)。结论:通过该实验较好地建立了卵巢组织的玻璃化冻存、移植和卵泡分离以及体外成熟培养的实验方法。     

负压创面治疗技术对皮肤移植成活的临床观察及实验研究

目的:采用负压固定移植皮片方法,观察负压创面治疗技术(negative-pressure wound therapy,NPWT)对游离皮片成活的影响,初步探讨微血管形成与皮肤成活之间的关系。方法:采用回顾性研究的方法,对65例皮肤缺损的患者,根据皮肤移植术后皮片固定方法的不同,分为两组,其中I组为NPWT治疗组,有35例患者,刃厚游离皮片移植术后行创面负压吸引治疗;II组为常规治疗组,有30例患者,刃厚游离皮片移植术后用打包或加压包扎的方式固定。Balb/c小鼠20只,按皮片移植后不同固定加压方式,分为实验组:负压创面治疗技术使用组(10只),对照组:打包加压组(10只),于皮片移植术后第5天,大体观察移植皮片颜色、有无水疱、有无皮下积液及质地,计算并比较皮片成活率,以免疫组化染色标记毛细血管内皮,检测皮片中微血管情况。结果:临床观察表明:I组术后皮片成活时间平均较II组缩短,有统计学差异(P<0.01),I组术后住院治疗时间平均较II组缩短5天,有统计学差异(P<0.01),I组术后抗生素费用、换药次数及换药费用较II组减少,有统计学差异(P<0.01)。动物实验结果表明:术后第5天,实验组小鼠移植皮片中微血管增生较对照组明显增多(P<0.05)。结论:与常规打包或加压包扎固定皮片的方式相比,负压创面治疗技术的应用可以缩短皮片成活时间,缩短患者住院治疗时间,减少抗生素的使用及换药次数,促进移植皮片中毛细血管增生,提高皮片成活率。     

江西铅山红芽芋胚性愈伤组织的玻璃化法超低温保存及植株再生

以江西铅山红芽芋胚性愈伤组织为材料,研究各种因素对其玻璃化法超低温保存的影响。结果表明:江西铅山红芽芋胚性愈伤组织玻璃化法超低温保存较佳的预培养条件为0.3mol·L-1蔗糖预培养3d,较佳的60%PVS2装载时间为20min,较佳的100%PVS2脱水条件为25℃脱水30min,较佳的化冻温度为40℃,较佳的洗涤液蔗糖浓度为1.2mol·L-1,较佳的冻后培养条件为暗培养7d再转到光周期中培养。红芽芋胚性愈伤组织包埋玻璃化超低温保存后的平均成活率约为70%。红芽芋胚性愈伤组织冻后再生苗没有发生形态学、生理学和细胞学的变异。     

负压创面治疗技术对皮肤移植成活的临床观察及实验研究

目的：采用负压固定移植皮片方法,观察负压创面治疗技术（negative-pressure wound therapy,NPWT）对游离皮片成活的影响,初步探讨微血管形成与皮肤成活之间的关系。方法：采用回顾性研究的方法,对65例皮肤缺损的患者,根据皮肤移植术后皮片固定方法的不同,分为两组,其中Ⅰ组为NPWT治疗组,有35例患者,刃厚游离皮片移植术后行创面负压吸引治疗;Ⅱ组为常规治疗组。有30例患者,刃厚游离皮片移植术后用打包或加压包扎的方式固定。Balb／c小鼠20只,按皮片移植后不同固定加压方式,分为实验组：负压创面治疗技术使用组（10只）,对照组：打包加压组（10只）,于皮片移植术后第5天,大体观察移植皮片颜色、有无水疱、有无皮下积液及质地,计算并比较皮片成活率,以免疫组化染色标记毛细血管内皮,检测皮片中微血管情况。结果：临床观察表明：Ⅰ组术后皮片成活时间平均较Ⅱ组缩短,有统计学差异（P〈0．01）,Ⅰ组术后住院治疗时间平均较Ⅱ组缩短5天,有统计学差异（P〈0．01）,Ⅰ组术后抗生素费用、换药次数及换药费用较Ⅱ组减少,有统计学差异（P〈0．01）。动物实验结果表明：术后第5天,实验组小鼠移植皮片中微血管增生较对照组明显增多（P〈0．05）。结论：与常规打包或加压包扎固定皮片的方式相比,负压创面治疗技术的应用可以缩短皮片成活时间,缩短患者住院治疗时间,减少抗生素的使用及换药次数,促进移植皮片中毛细血管增生,提高皮片成活率。     

不同植皮方式修复大面积烧伤创面的临床效果分析

目的:比较邮票植皮、meek植皮以及自体微粒皮移植用于大面积烧伤患者创面修复的临床效果和可行性。方法:回顾性分析我院烧伤科收治的120例大面积烧伤患者的临床资料,根据不同的手术植皮方法分为微粒皮植皮组、Meek植皮组和邮票皮组,每组40例。三组患者入院后均给予常规基础治疗,再根据选择植皮方式的不同实施微粒皮植皮、Meek植皮和邮票皮植皮。对比三组患者的植皮成活率、创面一期愈合率、愈合时间、死亡率、1%烧伤面积(1%TBSA)治疗费用(元)以及康复效果(康复优良率)。结果:邮票皮组植皮成活率明显高于Meek植皮组(P0.05),Meek植皮组植皮成活率明显高于微粒皮植皮组(P0.05)。Meek植皮组和邮票皮组创面愈合时间均明显短于微粒皮植皮组(P0.05)。邮票皮组1%TBSA治疗费用明显低于Meek植皮组(P0.05),Meek植皮组1%TBSA治疗费用明显低于微粒皮植皮组(P0.05)。Meek植皮组和邮票皮组的创面一期愈合率均明显高于微粒皮植皮组(P0.05);Meek植皮组和邮票皮组的死亡率均明显低于微粒皮植皮组(P0.05);meek植皮组的瘢痕最轻,关节功能恢复最好;微粒皮植皮组瘢痕最重,关节功能恢复最差。结论:不同植皮方式用于大面积烧伤创面修复的临床效果各不相同。邮票皮成活率高、抗感染能力强,但扩张比例低,创面愈合效果差;meek皮扩张比例高、康复效果好,但抗感染能力差;微粒皮扩张比例最高,但成活率低,治疗成本高。临床上,应该根据患者的自身情况选择不同的植皮方式。     

游离植皮联合负压封闭引流治疗骨科创面的临床研究

目的：观察游离植皮联合负压封闭引流（vacuumsealingdrainage,后文简称VSD）对骨科创面的疗效,并与植皮后传统加压包扎相比较,为临床实践提供更好的治疗方法。方法：对广州中医药大学第一附属医院创伤骨科2008年3月至2010年2月收治的65例感染创面病例采取手术清创后予VSD引流,合理应用抗生素,创面感染得到控制后,创面干净,肉芽生成良好,外露的肌腱、骨膜表面有新鲜的肉芽组织覆盖,达到植皮的要求后,随机分成两组,其中30例（实验组）采用游离植皮联合VSD法闭合创面,35例（对照组）采用游离植皮加压包扎植皮区,对两组术后的平均换药次数、创面平均愈合时间、植皮成活率情况、平均住院时间（植皮后）、平均抗生素应用次数（植皮后）进行统计学分析,采用t检验和卡方检验,对此两种方法进行评价。结果：植皮联合VSD组与植皮加压包扎组,在平均换药次数、创面平均愈合时间、植皮成活率、平均住院时间（植皮后）、平均抗生素应用次数（植皮后）的对比,有显著性差异（P〈0.05）。结论：创面达到游离植皮条件后,游离植皮联合VSD负压引流可以促使皮片黏附,保持创面洁净,避免皮下渗液积聚,有利于皮片的存活,与植皮加压包扎组相比,减少了平均换药次数,缩短创面平均愈合时间及平均住院时间（植皮后）,减少抗生素平均应用次数,提高了植皮成活率,说明游离植皮联合VSD组优于游离植皮加压包扎组,游离植皮联合VSD法治疗骨科创面有显著疗效。该手术方法操作简单,术后护理方便,是一种较理想的植皮后的固定方法,有利于创面的愈合,值得临床推广应用。     

游离植皮联合负压封闭引流治疗骨科创面的临床研究

目的:观察游离植皮联合负压封闭引流(vacuumsealingdrainage,后文简称VSD)对骨科创面的疗效,并与植皮后传统加压包扎相比较,为临床实践提供更好的治疗方法。方法:对广州中医药大学第一附属医院创伤骨科2008年3月至2010年2月收治的65例感染创面病例采取手术清创后予VSD引流,合理应用抗生素,创面感染得到控制后,创面干净,肉芽生成良好,外露的肌腱、骨膜表面有新鲜的肉芽组织覆盖,达到植皮的要求后,随机分成两组,其中30例(实验组)采用游离植皮联合VSD法闭合创面,35例(对照组)采用游离植皮加压包扎植皮区,对两组术后的平均换药次数、创面平均愈合时间、植皮成活率情况、平均住院时间(植皮后)、平均抗生素应用次数(植皮后)进行统计学分析,采用t检验和卡方检验,对此两种方法进行评价。结果:植皮联合VSD组与植皮加压包扎组,在平均换药次数、创面平均愈合时间、植皮成活率、平均住院时间(植皮后)、平均抗生素应用次数(植皮后)的对比,有显著性差异(P<0.05)。结论:创面达到游离植皮条件后,游离植皮联合VSD负压引流可以促使皮片黏附,保持创面洁净,避免皮下渗液积聚,有利于皮片的存活,与植皮加压包扎组相比,减少了平均换药次数,缩短创面平均愈合时间及平均住院时间(植皮后),减少抗生素平均应用次数,提高了植皮成活率,说明游离植皮联合VSD组优于游离植皮加压包扎组,游离植皮联合VSD法治疗骨科创面有显著疗效。该手术方法操作简单,术后护理方便,是一种较理想的植皮后的固定方法,有利于创面的愈合,值得临床推广应用。     

山萘酚通过增强Treg细胞免疫抑制功能延长移植物生存时间

目的:探讨应用山萘酚增强Treg细胞免疫抑制功能,从而抑制大鼠移植物排斥反应并改善移植物生存的作用和机制。方法:以Wister大鼠和SD大鼠分别为供、受体,建立同种异体皮肤移植排斥反应动物模型。观察受体老鼠皮肤移植物的情况,记录移植物失功时间(移植物皮片80%面积发生排斥)。RT-PCR检测移植7天后脾细胞、淋巴细胞FOXP3、CTLA-4和IL-10的mRNA水平,用HE染色组织病理学观察术后7天移植皮片的淋巴细胞浸润程度。体外实验T细胞增殖抑制试验加入山萘酚作为对照,观察Treg功能情况。结果:1.山萘酚能增强移植后同种异体移植物的生存时间(DMSO组6.3±0.3天,山萘酚组13.7±0.39天,P<0.01);2.RT-PCR显示山萘酚可增强细胞CTLA-4(对照组9.24±0.17,山萘酚组12.48±0.145,P<0.05)、FOXP3(对照组0.96±0.07,山萘酚组1.41±0.07,P<0.01)和IL-10(对照组0.95±0.12,山萘酚组1.50±0.16,P<0.05)的mRNA水平;3.体外T细胞增殖抑制实验中,山萘酚可增强Treg细胞的免疫抑制功能。结论:在大鼠皮肤移植模型中,山萘酚可延长皮肤移植物的生存时间,提高Treg细胞相关IL-10、FOXP3和CTLA-4的mRNA水平;体外实验中,能抑制效应T细胞的增殖,表明山萘酚在提高移植物生存方面存在一定的价值。     

The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification

This report highlights the first successful cryopreservation protocol for shoot tips of Garcinia mangostana L. achieved by using vitrification technique. We investigated the effects of different temperatures and exposure periods to a plant vitrification solution 2 (PVS2), sucrose concentrations and preculture periods, and unloading treatments in steps of the vitrification protocol on the survival of G. mangostana shoot tips after cryopreservation. Exposure to PVS2 for 25 min gave beneficial effects with 10.4 ± 1.8 % survival at 0 °C with average water content of 1.1 ± 0.3 g g^?1 dry mass. Survival was 13.7 ± 5.5 % when using preculture medium with full-strength Murashige and Skoog (MS) medium supplemented with 0.6 M sucrose for 2 days. A significant difference was observed in survival of shoot tips when treated with various sucrose concentrations in preculture which strengthens their importance towards enhancing survival of shoot tips after cryopreservation. MS with 0.4 M sucrose and 2 M glycerol applied as an unloading solution increased the survival of shoot tips to 44.1 ± 6.5 %. Experiments on the effect of ascorbic acid were also conducted for each step of vitrification. Our results showed higher survival of 45.8 ± 3.8 % but there were no significant effects compared with the control (without ascorbic acid). Further study on the recovery dark/light period was conducted. Survival of shoot tips significantly increased to 50.0 ± 16.7 % when subjected to 7 days in the dark before transferring to 16 h/8 h light/dark photoperiod. These studies strengthen suggestions that cryopreservation through vitrification is possible for ex situ conservation of germplasm of this tropical recalcitrant species.     

丹参酮IIA磺酸钠对烧伤植皮创面愈合及瘢痕形成的影响

目的:探讨丹参酮IIA磺酸钠注射液对烧伤患者植皮创面愈合及瘢痕形成情况的影响。方法:选取2014年3月至2014年12月我院收治的烧伤植皮患者62例,根据临床用药分为试验组(使用丹参酮IIA磺酸钠注射组)与对照组(未使用丹参酮IIA磺酸钠注射液)。比较两组创面愈合情况,术后植皮成活率及愈合后瘢痕形成情况。结果:1经治疗,两组创面均愈合,试验组患者植皮成活率为(97.12±1.89)%,高于对照组(89.96±1.86)%,差异具有统计学意义(P0.01);试验组愈合时间较对照组短,试验组创面愈合时间为10.1±1.9天,对照组为14.3±2.3天,两组比较差异具有统计学意义(P0.001);瘢痕形成评价试验组均优于对照组,差异具有统计学意义,其中血肿面积(1.50±0.03 vs.3.04±0.08,P0.01)、畸形率[2(6.45)vs.8(25.81),P0.05]、感染率[2(6.45)vs.9(29.03),P0.05]。结论:丹参酮IIA磺酸钠注射液对于烧伤植皮创面的患者,能够提高植皮成活率,促进创面愈合,减轻瘢痕形成,改善创面愈合质量。     

Closed versus open vitrification for human blastocyst cryopreservation: A meta-analysis

Closed vitrification can minimize the risk of microbiological transmission through liquid nitrogen during the cooling, storage, and warming procedures. As cooling rates may reduce when closed vitrification is applied, clinical outcomes should be compared between closed and open vitrification in order to justify the use of closed vitrification. This study was conducted to investigate the differences in survival, implantation, clinical pregnancy, and live birth rates between closed and open vitrification for human blastocyst cryopreservation. This systematic review and meta-analysis included 7 studies that reported survival, implantation, clinical pregnancy, or live birth rates following closed or open vitrification. There were no statistically significant differences in survival rates (risk ratio [RR]: 1.00, 95% confidence interval [CI]: 0.98–1.02), implantation rates (RR: 1.02, 95% CI: 0.93–1.11), clinical pregnancy rates (RR: 0.99, 95% CI: 0.89–1.10), and live birth rates (RR: 0.77, 95% CI: 0.58–1.03) between closed and open vitrification. Although there was no statistical significance, the tendency of lower live birth rates with closed vitrification than with open vitrification could be clearly identified. Therefore, it is not yet possible to conclude that closed vitrification clearly provides an aseptic alternative to open vitrification in human blastocyst cryopreservation.     

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows?. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.     

西红花酸治疗DHT诱导的PCOS小鼠的机制研究

目的:观察西红花酸对双氢睾酮(Dihydrotestosterone,DHT)诱导的多囊卵巢综合症(Polycystic ovarian syndrome,PCOS)小鼠的疗效并探讨其作用机制。方法:妊娠15-18天给予孕鼠皮下注射DHT诱导子代雌鼠PCOS模型。待子鼠8周龄后,随机选择一半数量的PCOS小鼠连续4周西红花酸灌胃,作为西红花酸给药组(n=18)。给药期间检测体重和动情周期,待小鼠16周龄左右,通过眼球取血后处死,取出下丘脑、卵巢。采用HE染色观察卵巢组织的病理改变;ELISA试剂盒检测血清中雌二醇(Estradiol,E2)、睾酮(Testosterone,T)、孕酮(Progesterone,P4)、促黄体生成素(Luteinizing hormone,LH)、卵泡刺激素(Follicle-stimulating hormone,FSH);采用免疫组化、Western blot和实时荧光定量PCR法检测下丘脑的前腹侧视旁核(Anteroventral periventricular,AVPV)、弓状核(Arcuate,ARC)的kisspeptin以及视前区(Preoptic area,POA)里Gn RH的表达水平。结果:与对照组相比,PCOS小鼠卵巢与体重的比值上升了22.56%±6.77%,动情周期延长,卵巢内出现大的空泡,闭锁卵泡数量增加了138.74%±33.22%,窦状卵泡、成熟窦状卵泡和黄体数量分别减少了38.80%±4.69%、56.35%±7.32%和63.77%±7.25%,血清中E2、P4和FSH水平分别降低了40.99%±2.69%、56.91%±5.25%、和38.80%±4.69%,而T、LH水平分别升高了43.23%±4.70%和148.46%±28.16%,下丘脑中AVPV中kisspeptin神经元表达减少,ARC中kisspeptin神经元表达增多,POA中Gn RH神经元减少,而以上情况能够被西红花酸改善。结论:西红花酸分别通过促进和抑制下丘脑AVPV核和ARC核团的kisspeptin表达改善PCOS的病理变化。     

秦岭大熊猫精液的细管冷冻保存

2008年3月1日至4月27日和2009年3月3日至5月1日,在陕西省珍稀野生动物抢救饲养研究中心对处于繁殖期内的4只雄性秦岭大熊猫(Ailuropoda melanoleuca qinlingensis)精液进行了细管冻精实验。比较组成不同的4种稀释液:葡萄糖-果糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液1)、葡萄糖-蔗糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液2)、葡萄糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液3)和美国进口的TEST(加入3.5%甘油),以及直接降温平衡法(方法 1)与逐级降温平衡法(方法 2)2种冷冻保存操作方法,对秦岭大熊猫精液进行细管冷冻保存后精子活力和顶体完整率的影响。结果表明:稀释液1的精子活力为46.25%±11.67%,顶体完整率为80.75%±7.89%,TEST的精子活力为48.75%±8.54%,顶体完整率为84.50%±7.59%,两者的精子活力和顶体完整率均无明显差异(P0.05),但是都明显高于稀释液2(P﹤0.01)和稀释液3(P﹤0.01);采用方法 1冷冻保存秦岭大熊猫精液,解冻后精子的活力和顶体完整率分别为45.67%±10.54%和81.37%±8.42%,都显著高于方法 2(P﹤0.01);方法 1解冻后畸形率为23.50%±3.51%,明显低于方法 2(P﹤0.01)。经比较确定,方法 1(用稀释液1)是一种较好的细管冷冻保存秦岭大熊猫精液的方法。     

Vitrification of goat, sheep, and cattle skin samples from whole ear extirpated after death and maintained at different storage times and temperatures

Proper tissue preservation from a wide range of animals of different species is of paramount importance, as these tissue samples could be used to reintroduce lost genes back into the breeding pool by somatic cloning. We aim to study the temporal and thermal post-mortem limits, tested in rabbits and pigs, within which there will be guarantees of obtaining living skin cells in goat, sheep, and cattle. We also intend to study the effect of vitrification on the ability of ear skin cells, stored at different times and temperatures, to attach to the substratum and grow in vitro after warming. Ears were stored either at 4 degrees C for 12, 252, and 348 h post-mortem (hpm), or at room temperature (22-25 degrees C) for 60 and 96 hpm. In all cases, skin samples from these ears were sorted into two groups: one group was in vitro cultured immediately after storage, and the other group was vitrified after storage and further in vitro cultured. In goat and sheep, no differences in attachment (100%: goat; 90-100%: sheep) or subconfluence (75-100%: goat; 70-100%: sheep) rates were observed between experimental groups. However, in days of culture to reach subconfluence, significant differences between non-vitrified and vitrified groups were observed when ears were stored at 4 degrees C for 12 and 252 hpm. In cattle, with respect to attachment rate, vitrified samples from ears stored at 22-25 degrees C for 60 hpm were different from non-vitrified control group (60 vs. 100%, respectively; P < 0.05). Also, days of culture to reach subconfluence were analysed by a non-parametric Cox Survival Analysis. In general, results from ANOVA and Survival Analysis were similar, because the proportion of censored data was quite low (9%), so the bias when using ANOVA is not too high. In spite of all the above, the lowest survival rates (75%: goat; 70%: sheep; and 40%: cattle) were sufficiently high to enable collection of skin samples from the majority of dead animals and their cryopreservation.     

Droplet-vitrification methods for apical bud cryopreservation of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Rob.]

This study aimed to develop a cryopreservation protocol for the long-term preservation of yacon [Smallanthus sonchifolius (Poepp. and Endl.)], an Andean crop with high fructooligosaccharide content in its tuberous roots. Initially, the cryopreservation protocol was developed using a yacon clone originated from Ecuador classified as ECU 41. Osmotic dehydration of apical buds (2–3 mm long) was carried out by assessing two plant vitrification solutions, PVS2 (15, 30, and 60 min) at 0 °C and PVS3 (30, 45, 60, and 75 min) at 22 °C. After cryopreservation, the apical buds were thawed and placed on MS medium?±?0.1 mg l^?1 N⁶-benzyladenine (BA). The survival rates ranged from 37 to 90% within all treatments, with those subjected to PVS2 and PVS3 for 60 min showing the highest survival rates on MS medium without BA (87 and 90%, respectively). At 12 weeks post cryopreservation, these treatments also provided the highest regrowth rates, both reaching 73% of normally growing (shooting, rooting) plantlets. Survival rates on MS?+?0.1 mg l^?1 BA regrowth medium reached up to 90%; however, regrowth into normally rooted plantlets did not exceed 67% post cryopreservation. The optimized protocols were then applied to 4 additional yacon clones originated from Bolivia and Peru, classified as BOL 22, BOL 23, PER 12, and PER 14. This resulted in survival and regeneration rates ranging between 79.7–94.1% and 66.3–75.4% respectively. Our study shows that optimal cryopreservation protocols for the long-term conservation of yacon can be based on both PVS2 and PVS3 vitrification solutions.

     

Cryopreservation of mouse embryos by vitrification: A meta-analysis

A meta-analysis of cryopreservation studies vitrifying mouse embryos was undertaken to determine the treatment effect of vitrification. Treatment by vitrification decreased embryo viability compared with controls: the odds ratio was 9.02 (CI: 3.73-21.78; P < 0.001), a 24.90% (CI: 14.88-34.91; P < 0.001) reduction in risk was associated with embryos in the control group, and for every 4.00 (CI: 3.91-4.09) embryos treated by vitrification, one does not survive. A multiple regression analysis evaluated covariates of embryo survival. For each hour increase post-hCG treatment when embryos were cryopreserved, there was a decrease of 0.36% (SEM ± 0.01) in survival (P < 0.001). The number of embryos surviving vitrification decreased 0.25% (SEM ± 0.02) per day increase in age of the female mouse (P < 0.001), whereas there was no significant difference for control group embryos. For each 1 h increase post-hCG treatment after cryopreservation when blastocysts were assessed for viability, there was a decrease of 0.13% (SEM ± 0.01) in survival. The later interval post-hCG treatment when blastocysts were assessed, the less viable they were compared with earlier blastocysts, independent of the vitrification protocol. This effect was not observed for control embryos. A high percentage of variability in the treatment effect for vitrification was likely due to underlying heterogeneity among studies. A portion of the risk associated with vitrification could be attributed to the general effects of cryopreservation. Future research should identify effects in a cryopreservation protocol specific to vitrification that affect viability of mouse embryos.