密度梯度离心联合上游法的精液处理在供精体外受精胚胎移植中的应用

目的:观察供精体外受精-胚胎移植术(D-IVF)中采用密度梯度离心法联合上游法处理精液后的胚胎情况。方法:回顾本院2013年1月-2014年7月D-IVF 135个周期,观察冷冻精液复苏后经采用密度梯度离心法联合上游法处理后精子总活动率、前向运动精子活动率等精液情况,以及受精以后的受精率、卵裂率、临床妊娠率,对照组为同期46个因丈夫梗阻性无精行ICSI的周期。结果:处理后精子总活动率、前向运动精子活动率均大大提高,但是精子密度却大大下降,处理前后比较差异均有统计学意义(P0.05);受精以后的实验室资料和对照组比较,各项参数差异无统计学意义(P0.05)。结论:密度梯度离心法联合上游法的精液处理方法在D-IVF中是一种稳定可行的方法。     

SHR大鼠前额叶、纹状体突触体的提取方法

目的 介绍SHR大鼠前额叶、纹状体突触体的提取方法。方法 采用Percoll密度梯度离心法制备突触体,透射电镜观察其形态和结构完整性。结果 本实验所获取的突触体形态上呈连续膜封闭的椭圆结构,周围有完整的膜包围;突触前成分可见一个或多个线粒体和大量突触小囊泡;突触间隙清晰可见,且突触后部分特征性的致密部结构完整,形态清晰,密度较高。突触体突触前膜、突触间隙、突触后膜保存完好,突触体分布密度较高,具有典型的突触体形态结构特征。结论 本实验提供了一种耗时少,产出率高,结构完整、形态典型且可运用于精细化研究的突触体制备新技术,补充了传统技术的不足,为神经系统疾病的研究提供技术支持。     

Leukocyte Esterase From Synovial Fluid Aspirate : A Technical Note

Accurate and efficient diagnosis of periprosthetic joint infection remains one of the most challenging tasks for orthopedic surgeons. Currently, no widely used diagnostic test allows for quick and efficient performance, low cost, and high sensitivity and specificity. Aspiration of synovial fluid from a patient's joint can be done in the clinic both quickly and easily; oftentimes, the aspirate obtained is bloody, thus rendering the use of colorimetric strip testing impractical. We describe a simple, inexpensive, and effective protocol using centrifugation to allow for leukocyte esterase (LE) testing after bloody joint aspirations. In all cases, both septic and aseptic, there was a 100% concordance in LE enzyme test results. Although further validation may be necessary, these initial results demonstrate that accuracy of LE testing is not affected by centrifugation.     

Giant panda (Ailuropoda melanoleuca) sperm morphometry and function after repeated freezing and thawing

This work examines the effects of subsequent cycles of freezing–thawing on giant panda (Ailuropoda melanoleuca) sperm morphometry and function, and assesses whether density‐gradient centrifugation (DGC) can increase the number of freezing–thawing cycles this sperm can withstand. A sperm sample was collected by electroejaculation from a mature giant panda and subjected to five freezing–thawing cycles. Although repeated freezing–thawing negatively affected (P < 0.05) sperm motility and membrane integrity, in both nonselected and DCG‐selected sperm samples, >60% of the sperm cells in both treatments showed acrosome integrity even after the fifth freezing cycle. In fresh semen, the sperm head length was 4.7 μm, the head width 3.6 μm, area 14.3 μm² and perimeter length 14.1 μm. The present results suggest that giant panda sperm trends to be resistant to repeated freezing–thawing, even without DGC selection.     

葡聚糖凝胶微柱结合高效液相色谱法测定奥沙利铂脂质体包封率

目的：对奥沙利铂脂质体进行质量评价,建立奥沙利铂脂质体包封率的测定方法。方法：采用凝胶微柱离心法分离游离药物与脂质体,以HPLC法测定奥沙利铂的药物含量,计算脂质体的包封率。结果：奥沙利铂浓度在0.02~1mg.mL-1范围内线性关系良好（r=0.9999）,结果3批次奥沙利铂脂质体的包封率分别为56.6%,57.5%,60.0%。结论：该方法简便、迅速,可准确地测定出奥沙利铂脂质体的包封率。     

An Advanced Protocol for the Quantification of Marine Sediment Viruses via Flow Cytometry

Viruses are highly abundant, diverse, and active components of marine environments. Flow cytometry has helped to increase the understanding of their impact on shaping microbial communities and biogeochemical cycles in the pelagic zone. However, to date, flow cytometric quantification of sediment viruses is still hindered by interference from the sediment matrix. Here, we developed a protocol for the enumeration of marine sediment viruses by flow cytometry based on separation of viruses from sediment particles using a Nycodenz density gradient. Results indicated that there was sufficient removal of background interference to allow for flow cytometric quantification. Applying this new protocol to deep-sea and tidal-flat samples, viral abundances enumerated by flow cytometry correlated well (R² = 0.899) with counts assessed by epifluorescence microscopy over several orders of magnitude from marine sediments of various compositions. Further optimization may be needed for sediments with low biomass or high organic content. Overall, the new protocol enables fast and accurate quantification of marine sediment viruses, and opens up the options for virus sorting, targeted viromics, and single-virus sequencing.     

密度梯度离心结合Keihuaer抗酸染色法富集孕妇外周血中FNRBC的研究

目的 探索建立一种简便、快速、较低成本的用于孕妇外周血胎儿有核红细胞(FNRBC)富集的实验方法.方法 采集18名孕周在8～16周的孕妇外周血10 ml,经Percoll不连续密度梯度离心初步分离后,运用Keihuaer抗酸染色法对玻片上的细胞进行染色标记,显微镜下观察、辨别FNRBC的特殊染色及形态,对阳性涂片者进一步行胎儿血红蛋白(HBF)特异性抗体标记以证实其胎源性.结果 经Keihuaer抗酸染色处理,阳性FNRBC的胞浆染色呈深红色,核蓝色,而母亲的有核红细胞胞浆不着色.18名孕妇外周血中有14名可检出FNRBC,平均1～6个阳性FNRBC/病例,阳性检出率为77.8%.14例FNRBC阳性玻片经HBF抗体标记亦证实相同结果,而4例Keihuaer抗酸染色阴性病例亦表现HBF标记阴性,两者检出符合率100.0%.结论 密度梯度离心结合Keihuaer抗酸染色法用于富集FNRBC 具有较高的特异性,实验操作方法简便、快速,有望用于无创性产前基因诊断的推广应用.     

成年鼠被毛毛乳头细胞分离方法的建立与评价

目的 寻求一种简单高效的分离培养小鼠被毛毛乳头细胞的方法.方法 取5周龄C57小鼠背部皮肤,刮去毛发后予0.2％Ⅰ型胶原酶37℃消化2h,刮下毛球部,经过2次Ficoll密度梯度离心后,得到纯净的毛乳头进行培养.检测毛乳头贴壁率、毛乳头细胞特异性标记表达及其体内毛囊重建能力.结果 该分离方法能显著降低工作强度,减少污染机会,获得的毛乳头贴壁快、细胞迁出快.qRT-PCR、细胞免疫荧光实验结果均证实体外培养的小鼠被毛毛乳头细胞可表达与毛囊诱导能力相关的特异性标记物ALP、β-catenin及Versican,且与触须毛乳头细胞相比差异无统计学意义(P＞0.05).体外培养的小鼠被毛毛乳头细胞具有诱导毛囊再生的能力.结论 胶原酶消化结合Ficoll梯度离心是一种简单、有效地分离获取小鼠背部毛乳头的方法.     

Comparison of the in vitro fertilization rate by human sperm capacitated by multiple-tube swim-up and Percoll gradient centrifugation

Two sperm preparation methods, a multiple-tube swim-up and Percoll-gradient centrifugation, were employed in our human in vitro fertilization program. The fertilization rate of these two sperm preparation methods was compared when they were employed in semen samples of <60 million motile sperm/ml. The results described here suggest that both of these methods gave a similar fertilization rate in these semen samples, i.e., 72±8% for the Percoll-gradient centrifugation method and 66±8% for the multiple-tube swim-up method.