New and sensitive standard cell culture technique for the detection of cytomegalovirus in clinical specimens

Conventional tube cell culture has been recognised as the most sensitive technique available for human cytomegalovirus (HCMV) detection. Low-speed centrifugation of specimen inocula onto cell culture monolayers has been shown to increase the efficiency of infection with the AD 169 strain of HCMV. Therefore a centrifugal force of 900g for 1 hour at 37 degrees C was used to enhance the detection of HCMV cytopathic effect (CPE) in shell vials that contained circular coverslips with a monolayer of human embryonic lung (HEL) fibroblasts. Of 195 specimens, HCMV CPE was detected in 18 specimens (9.02%) on shell vial culture assay, whereas conventional tube cell culture was positive in only 13 specimens (6.6%). The shell vial culture assay was significantly more sensitive (P less than 0.05). Furthermore the development of the cytopathic effect on shell vial culture assay was significantly earlier (P less than 0.01) and more extensive. Urine samples were sonicated and the results obtained with immunofluorescence using human immune serum demonstrated that sonication increased both the intensity of fluorescence and number of fluorescent foci of HCMV-infected cells and also decreased the non-specific fluorescence of the background.     

离心作用测定细胞与材料间的粘附力及其初步应用

目的：介绍一种简单易行的检测细胞与材料间黏附力的方法。方法：本方法的建立基于离心力在转速一定时与半径成正比的原理。医用石膏制备培养皿固定装置，于培养皿底制备材料薄膜（包括有PLGA，PLGA COLI，PLGA COL I FN），待大鼠骨骼肌卫星细胞悬液与其作用一定时间后，一定转速开动离心机。测定未脱落细胞斑的半径R,取均值。结果：上述三种材料表面未脱落大鼠骨骼肌卫星细胞圆斑半径（R）分别为7.96、15.11、26.10mm，三组间有显著性差异。结论：（1）R的变化可反映细胞与材料间粘附力的改变。（2）利用离心作用检测细胞与材料间的粘附力是可行的，尤其适用于同一种细胞与不同材料间粘附力的比较。（3）COL I和FN的使用可以增加大鼠骨骼肌卫星细胞与PLGA间的粘附。     

人睾丸组织中成熟精子的分离方法初探

探索简单、适用的体外分离睾丸组织中成熟精子的方法,以用于男性无精子症病人行卵母细胞内单精子注射受精-胚胎移植为目的。用含不同浓度透明质酸酶的10 %人血白蛋白人类输卵管液培养人睾丸曲细精管组织匀浆2 4 h,再用Percoll梯度离心法洗涤后计算所获各级精子的总数,并设置前后对照和空白对照进行比较。用不同浓度透明质酸酶处理后的4个实验组分别与2个对照组比较,实验组所获得的各级精子的总数高于对照组,有显著差异(P<0 .0 1)。实验组中透明质酸酶浓度分别为5 0、10 0、15 0 u/ ml的10 %人血白蛋白人类输卵管液处理组获得的各级精子总数组间比较,处理前对照和处理后空白对照之间比较,无显著差异(P>0 .0 5 )。而用浓度为2 0 0 u/ ml的10 %人血白蛋白人类输卵管液处理组获得的各级精子的总数与其它浓度处理组之间比较存在统计学差异(P<0 .0 1) ,其所获得的各级精子总数相似,但活动能力较其它浓度处理组低。透明质酸酶结合Percoll梯度离心可从人睾丸曲细精管组织匀浆中分离出较多更纯的成熟精子,但过高浓度的透明质酸酶可能会影响分离出的精子的活动能力。     

快速大量分离血浆低密度脂蛋白亚组分的方法

目的 :研究快速大量分离低密度脂蛋白亚组分的方法。方法 :通过2步超速离心法分离LDL亚组分 ,并用电泳法及电镜观察鉴定2种亚组分。结果 :仅用10h就将LDL亚组分分开 ,并证实经分离的2种亚组分为纯品。结论 :本方法是1种分离LDL亚组分较好方法     

基于免疫磁珠法的不同筛选方案对胎儿有核红细胞富集效果的比较

目的：将密度梯度离心(density gradient centrifugation,DGC)与免疫磁珠法(magnetic-activated cell sorting,MACS)相结合,应用4种不同的筛选方案,对脐血中的胎儿有核红细胞(fetal nucleated red blood cells,FNRBC)进行分离,评估不同策略富集FNRBC的效果。方法：对10例脐血标本(12 mL/例)按4种不同方案进行筛选：DGC;DGC⁺MACS阴性筛选(CD45^-);DGC⁺MACS阳性筛选(CD71⁺);DGC⁺MACS阴性/阳性筛选(CD45-/CD71⁺)。通过细胞计数板计数,K-B染色后显微镜下人工FNRBC计数,从获得总细胞量、FNRBC量和FNRBC比例经t检验后评估不同方案富集FNRBC的效果。结果：(1)与其他3组相比,DGC方案可获得的总细胞量(0.95×10⁷/mL)最多,FNRBC数量(39.87/mL)最多,F...     

PEG6000沉淀结合差速离心方法浓缩水中脊髓灰质炎病毒

建立一种简便,有效的从细菌培养物中纯化ＰＶ１病毒的方法用于水病毒消毒实验。方法ＰＶ１Ｈｅｎａｎ株在Ｈｅｐ－２细胞中增殖后用聚乙二醇６０００沉淀结合差速离心的方法纯化病毒,ＲＴ－ＰＣＲ和透射电镜鉴定提纯病毒的形态学和分子生物学特性。结论ＰＥＧ６０００结合差速离心的方法较之蔗糖密度梯度区带离方法易于操作且比单独ＰＥＧ沉淀有更高的纯化系数,是一种简便,有效的方法。     

梯度离心法在肿瘤药敏试验中的应用及临床相关性研究

目的　研究梯度离心法在肿瘤药敏试验中的应用及肿瘤药敏试验与临床个体化化疗的相关性。方法　取 4 6例肿瘤患者新鲜癌细胞 ,梯度离心后用MTT法检测癌细胞对 8种常用抗癌药的敏感性 ;根据药敏结果对患者进行化疗 ,无敏感药物者按经验方案化疗 ,观察实验与临床疗效的相关性。结果　改良法癌细胞获得百分率为 94 6 5± 2 8 4 7,与传统方法比较差异有显著性 (P <0 0 1) ;药敏试验阳性者 2 5例 ,阴性者2 1例。实验提示敏感药物化疗者的有效率为 76 % (19/ 2 5 ) ;耐药者按经验方案化疗 ,有效率为 2 8 6 % (6 /2 1)。P <0 0 1,差异有显著性 ;实验与临床符合率达 73 9% (34/ 4 6 )。结论　梯度离心MTT法体外癌细胞药敏试验能更好的指导临床个体化治疗 ,具有预见癌症患者临床个体化化疗疗效的重要价值。     

体外诱导小鼠骨髓单个核细胞向内皮分化的实验研究

目的:为组织工程研究做准备,分离培养小鼠骨髓单个核细胞并诱导向血管内皮分化。方法:成年Balb/c小鼠,应用淋巴细胞分离液(密度1.077g/m l),将长骨骨髓经F icoll非连续密度梯度离心,收集中层的单个核细胞,加入诱导培养基并置于纤维连接素包被的培养板上进行诱导分化,观察细胞生长状况,以透射电镜及Ⅷ因子、CD31、Lectin免疫组化以及流式细胞仪方法对培养细胞进行鉴定。结果:细胞圆形、纺锤形单层融合贴壁生长;Ⅷ因子、CD31、Lectin免疫组化染色阳性;透射电镜显示细胞具有内皮特征性的W-P小体。结论:采用F icoll密度梯度离心可获得较高纯度的骨髓单个核细胞,经体外培养并诱导分化,具有血管内皮细胞的特征。     

Long-term clinical outcomes of fat grafting by low-pressure aspiration and slow centrifugation (Lopasce technique) for different indications

Abstract

Autologous fat grafts have been used successfully for structural fat grafting in facial, lip, and hand rejuvenation, body contour improvement, and traumatic defect restoration. The purposes of this study were to define a new fat graft harvesting and processing technique, which is named the “Lopasce technique” (low-pressure aspiration and slow centrifugation technique), and to evaluate the late clinical outcomes of fat grafting by this technique for different indications. A retrospective study was performed using the medical records of 21 patients (17 women and four men). The mean injected fat volume was 33.2 ± 34 cc (range 6–125 cc). The mean follow-up period was 13.2 ± 5.6 months (range 6–26 months). Postoperative results were evaluated by subjective and objective methods. In the subjective evaluation, 19 patients stated that there had been little resorption and that it was not necessary to repeat the fat grafting, one patient reported that the fat was resorbed in part, and one patient reported that the fat was resorbed completely. In the objective evaluation, the amount of fat graft taken in the recipient sites was between 60%–80% (average 70%) when compared with preoperative and late postoperative photographs of the patients at the 6- and 26-month follow-ups. Fat grafting is a simple, effective, and reproducible technique with a high satisfaction rate and few disadvantages or complications. We consider that structural fat grafting with the lopasce technique is an easy, effective, and long-lasting treatment for correction of congenital or acquired defects associated with various medical conditions.