囊胚培养液对小鼠胚胎的毒性:体外生殖辅助医疗器械安全性评价

背景:囊胚培养液是可以支持胚胎从受精卵发育到囊胚阶段的一种重要培养液,其安全性直接影响囊胚的质量。目的:观察囊胚培养液对小鼠胚胎发育的影响。方法:选用B6D2F1品系小鼠,将雌性小鼠进行超数排卵处理,与同品系雄鼠合笼过夜,次日收集1细胞期受精卵,在M16培养液中培养至4细胞期后,检查胚胎发育情况并转移到受试囊胚培养液中继续培养(实验组),5 d后检查囊胚发育情况并计算囊胚形成率。同时,用含有内毒素的囊胚培养液M16培养小鼠受精卵,作为阳性对照组;用已被证实没有胚胎毒性的囊胚培养液M16培养液培养1细胞期受精卵,作为阴性对照组。结果与结论:阳性对照组囊胚形成率为0,阴性对照组囊胚形成率为87.1%,实验组囊胚形成率为87.3%,实验组囊胚形成率大于80%,说明受试囊胚培养液可以支持小鼠受精卵正常发育到囊胚阶段,对胚胎没有毒性效应。

液相质谱联用法同时检测体外生殖培养液中的18种氨基酸

背景:目前,对于体外生殖培养液的质量控制文件或技术标准中,对成分含量指标及检测方法未做明确规定,为保证该类产品的安全有效性,应及早建立质量标准。目的:采用液相质谱联用法检测体外生殖培养液中18种氨基酸指标成分,分析不同用途培养液中18种氨基酸的含量。方法:采用超高效液相色谱三重四级杆质谱联用法,检测体外生殖培养液中18种氨基酸(甘氨酸、亮氨酸、蛋氨酸、酪氨酸、组氨酸、苏氨酸、丙氨酸、异亮氨酸、色氨酸、胱氨酸、赖氨酸、天门冬氨酸、缬氨酸、苯丙氨酸、脯氨酸、丝氨酸、谷氨酸、精氨酸)指标成分,色谱柱为SUPELCO Discovery HS F5-3(15 cm×2.1 mm,3μm),流动相为0.1%甲酸水溶液-0.1%甲酸乙腈溶液,梯度洗脱,流速0.35 m L/min,柱温40℃;质谱采用电喷雾离子源,负离子模式扫面,扫描方式为多反应监测。结果与结论:18种氨基酸在0.25-12.5 mmol/L浓度范围内线性关系练好;18种氨基酸的平均加样回收率在86.3%-125.3%之间,精密度实验与重复性实验的相对标准偏差均小于4.7%,说明采用液相质谱联用法检测体外生殖培养液灵敏、快速、准确、专属性强,可为体外生殖培养液的质量标准研究提供依据。

液质谱联用法同时检测体外生殖培养液中的3种能量成分

背景:目前,对于体外生殖培养液的质量控制文件或技术标准中,对成分含量的指标及检测方法未做明确规定,为保证该类产品的安全有效性,应及早建立质量标准。目的:建立采用液相质谱联用法检测体外生殖培养液中葡萄糖、乳酸钠、丙酮酸钠3种指标成分的方法,分析不同用途培养液中3种成分的含量。方法:采用超高效液相色谱三重四级杆质谱联用法,使用SUPELCO Discovery HS F5-3色谱柱(15 cm×2.1 mm,3μm),流动相为0.1%甲酸水溶液-0.1%甲酸乙腈溶液,梯度洗脱,流速0.35 m L·min-1,柱温40℃;质谱采用电喷雾离子源,负离子模式扫面,扫描方式为多反应监测。结果与讨论:葡萄糖、丙酮酸钠、乳酸钠分别在0.1-10 mg/L(r=0.999 8)、0.05-5 mg/L(r=0.999 4)、0.1-10 mg/L(r=0.999 4)范围线性关系练好;平均加样回收率在96.4%-98.1%之间,相对标准偏差均小于2.8%。说明采用液相质谱联用法检测体外生殖培养液灵敏、快速、准确、专属性强,可为体外生殖培养液的质量标准研究提供依据。

体外辅助生殖用耗材产品环氧乙烷残留限量的风险探讨

体外辅助生殖用耗材产品的作用对象是配子、合子及不同细胞阶段的胚胎。因此在评价环氧乙烷灭菌残留限量时,不仅要考虑环氧乙烷的细胞毒性,还应考虑其胚胎毒性和发育毒性。本文对环氧乙烷的毒理作用机制、生殖细胞毒性及基因毒性进行阐述,探讨体外辅助生殖用耗材产品环氧乙烷残留的风险及控制措施。

液相色谱-三重四级杆质谱联用法检测人类辅助生殖技术用液中N(2)-L-丙酰胺-L-谷氨酰胺含量

目的:建立采用液相质谱联用法检测体外生殖培养液中N(2)-L-丙酰胺-L-谷氨酰胺指标成分的方法,分析不同用途培养液中N(2)-L-丙酰胺-L-谷氨酰胺的含量。方法:采用超高效液相色谱-三重四级杆质谱联用法,使用SUPELCO Discovery HS F5-3色谱柱(150 mm×2.1 mm,3μm),流动相为0.1%甲酸水溶液-0.1%甲酸乙腈溶液,梯度洗脱,流速0.35 mL·min^(-1),柱温40℃;质谱采用电喷雾离子源,负离子模式扫描,扫描方式为多反应监测。结果与结论:N(2)-L-丙酰胺-L-谷氨酰胺在1.276~12.76μg·mL^(-1)浓度范围内线性关系良好;平均加样回收率在100.5%~103.0%,相对标准偏差小于1.98%。说明采用液相质谱联用法检测体外生殖培养液灵敏、快速、准确、专属性强,可为体外生殖培养液的质量标准研究提供依据。

In Vitro Picloram-lnduced Somatic Embryogenesis from Leaflets of Cherry （Prunus incisa Thunb,）

Cherry regeneration via somatic embryogenesis is a powerful tool to breeding. In this way, the embryogenic capacity of Prunus incisa specie has been tested from leaves under different interactions of picloram concentrations and darkness exposures. Induction culture was achieved on MS medium supplemented with picloram concentrations at 0.5, 1 and 1.5 mg.L1 and submitted to 10, 20, 30 and 40 days of darkness. The best rate of embryogenic leaves was obtained with the interaction of 30 days darkness exposure＊ 1 mg.L^-1 picloram. According to their age, leaves were differently reacted to somatic embryogenesis; indeed, the 2nd expanded leaf from the apex was the most embryogenic one. Concerning the effect of additional auxin to picloram （1 mg·L^-1）, IAA at 0.1 mg·L^-1 and IBA at 0.1 mg·L^-1 gave significantly higher induction rates than all other concentrations, but regenerating somatic embryos showed some teratological abnormalities probably due to seconda;y embryogenesis. At the opposite, NAA at 0.5 mg·L^-1 didn＇t improve embryogenic rate but affected positively embryo development. Furthermore, embryogenesis preferentially took place on the basal part of leaf. Satisfactory rates of somatic embryogenesis are obtained but further improvement remains possible.

Anti-angiogenesis effect of Demethyl bryoanthrathiophene(DBT) in vitro

Objective:The aim of the study was to investigate the effect of Demethyl bryoanthrathiophene(DBT) on proliferations of human umbilical vein endothelial cells(HUVECs) and human lung adenocarcinoma cell line A549,and antiangiogenic effect of DBT on HUVECs in vitro.Methods:MTT assay was used to observe the effect of DBT on proliferations of HUVECs and A549 cells,flat plate scarification assay and tube formation in vitro test were used to observe the impact of DBT on migration and vaso-formed ability of HUVECs.The effects of DBT on apoptosis and cell cycle of HUVECs were calculated by flow cytometry.Results:MTT assay showed that treatment with DBT resulted in strong inhibition to the growth of HUVECs and A549 cells.The inhibition effects of DBT on HUVECs and A549 cells were related to dosage and times of dependency.In different doses of DBT(0.16,0.32 and 0.48 μmol/L) of flat plate scarification for 24 h,inhibition rates of DBT to migration of HUVECs were 14.70%,38.23% and 58.82%,respectively.In dose of DBT from 0.04,0.20 to 0.40 μmol/L for 24 h in tube formation,there were significance differences(P < 0.01) in the decreasing number of angiogenesis and incomplete blood vessel compared with control groups.All results showed that DBT promoted the apoptosis rate of HUVECs,and the increase of concentration of DBT accompanied the acceleration of apoptosis rate.Conclusion:DBT could inhibit the proliferations of HUVECs and A549 cells,and effectively suppress angiogenesis in vitro.

Effect of three suture lines on the proliferation and cell cycle of lung adenocarcinoma cell A549 in vitro

Objective： The interaction of cell and medical biomaterial is one of the significant factors to affect clinical application of medical biomaterial. This research is to investigate three of suture lines how to affect the proliferation and cell cycle of lung adenocarcinoma cell A549 in vitro. Methods： Three of suture lines were respectively cultivated with lung adenocarcinoma cell A549, after of 72 hours, we detected absorptions of each group by MTT method in order to reflect the proliferation of lung adenocarcinoma cell A549, and also examined percentage of G1 period cells and S period cells of each group by flow cytometry. Results： Different of suture lines had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 （P 〈 0.05）. The effect of absorbent suture line was the strongest on the proliferation and cell cycle of lung arlenecarcinoma cell A549, the effect of chorda serica chirurgicalis was medium, and the effect of slide wire was poor. Different length of each suture line had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 （P 〈 0.05）, Conclusion： Three of suture line materials have different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549, with dose-effect relationship.

The off-host survival and viability of a native and non-native fish louse （Argulus, Crustacea： Branchiura）

Fish ectoparasites are introduced to water bodies or aquaculture systems along with their hosts. Argulid fish lice can survive off-host for a given time, and in spite of quarantine measures might also be introduced via the transfer of water. However, they must cope with varying abiotic conditions without access to food. We investigated the effect of temperature （5 to 28℃） on the off-host survival time of Argulus japonicus, an invasive species which is apparently increasing its distribution, and compared this with the European A. foliaceus. A clear effect of temperature on the off-host survival time of all stages of both species was demonstrated. Larval and juvenile A. japonicus survived up to 9 days at 22℃ and in the case of adults up to 13 days at 15℃. By comparison, larval A. foliaceus survived up to 5 days at 15℃, adults up to 14 days at 9℃ and juveniles up to 7 days at 9℃ and 15℃. Thus, A. japonicus is more resistant to starvation at higher temperatures under controlled off-host conditions and A. foli- aceus is more resistant to starvation at lower temperatures. Infectivity of A. japonicus decreased linearly with the time spent off-host after 2 days for larvae and 4 days for adults. Temperature only had a significant effect on the infectivity of both develop- mental stages after 24 hours off-host between 13-23℃ for larvae and 13-18℃ for adults. In conclusion both species can potentially survive off-host pelods in water transports for up to 13-14 days as adults. Their survival is shorter as larvae. This study demonstrates that transportation of water has the potential to introduce Argulus species

Functional spermatid-like cells derived from the ground-state embryonic stem cells in vitro

Embryonic stem cells（ESCs）are pluripotent and can self-assemble to form cell clusters or embryoid bodies（EBs）,which can then differentiate into all cell types of the three germ layers,as among which one is the primordial germ cell（PGCs）（Daley,2007）.In vivo PGCs are the gamete founder cells（the ooctyes and sperms）,which transmit genetic information from one generation to the next generation to maintain mammalian life cycles.About 15%of couples in China are infertile;