小鼠睾丸支持细胞体外分离培养的研究

为了研究一种能快速、高效地分离、纯化小鼠睾丸支持细胞的方法,试验采用颈部脱臼的方法处死3周龄的小白鼠,取其睾丸并去除附属组织(血管、白膜脂肪等)后剪碎,用Ⅳ型胶原酶和胰蛋白酶分步消化制备细胞悬液进行培养,台盼蓝染色以鉴定细胞的活率;再对细胞悬液进行低渗溶液处理并利用支持细胞贴壁而其他细胞不贴壁的特性对其进行分离、纯化;最后对支持细胞采用H.E.和油红O染色的方法进行鉴定,观察其形态、结构和生长增殖情况。结果表明:该方法能够有效地分离、纯化以及培养小白鼠睾丸支持细胞。     

VEGF对绵羊卵泡颗粒细胞FSHR、E2R表达的影响

血管内皮生长因子(VEGF)可以促进颗粒细胞的增殖,但是否影响同样分布于颗粒细胞上的FSHR、E2R表达效果尚属未知.因此,通过Western blotting和荧光定量PCR分别对添加0、5、10、15、20、25 μg/L VEGF卵泡颗粒细胞中FSHR、E2R表达量及FSHR mRNA、E2R mRNA表达量进行检测.Western blotting蛋白检测结果表明,空白对照组和各个不同质量浓度VEGF处理组的颗粒细胞上均有FSHR和E2R蛋白表达,FSHR相对分子质量为75 000,E2R相对分子质量为56 000；荧光定量PCR检测结果表明,添加VEGF的各个处理组中FSHR mR-NA、E2R mRNA表达量均显著高于对照组(P＜0.05),各处理组间的FSHR mRNA之间差异不显著(P＞0.05),而VEGF添加量20、25 μg/L组的E2R mRNA表达量显著高于其他3个处理组(P＜0.05),并且这2个组间则差异不显著(P＞0.05),其余3个处理组间亦差异不显著(P＞0.05).     

山羊乳腺上皮细胞基因转染条件的优化

为了优化外源基因转入乳腺上皮细胞的条件,本研究采用组织块培养法,从山羊乳腺组织中分离并获得了纯化的乳腺上皮细胞,通过染色角蛋白和测定生长曲线对其进行了鉴定和检测。然后利用电穿孔法将PEGFPC1载体导入乳腺上皮细胞,分别比较了在不同电压(120、140、160、180、200V)、不同电击时长(5、10、15、20ms)以及2株细胞(GMEC1、GMEC2)的不同代数(第1代、第3代、第5代、第9代)条件下的转染效率,并利用优化条件对山羊乳腺上皮细胞进行人β-防御素3基因转染,经过G418筛选,最终获得单克隆细胞,同时对获得的单克隆阳性细胞进行体外诱导表达,并对诱导产物进行Western blotting鉴定。结果表明,不同遗传背景的细胞对转染效率的影响较小,而第1代培养的细胞转染效率要显著高于传代细胞,乳腺上皮细胞在电压180V、电击时长15ms、电击1次的条件下转染效率最高,优化条件下转染、筛选和扩增后获得稳定表达人β-防御素3蛋白的单克隆乳腺上皮细胞。研究工作为乳蛋白基因表达调控机制的研究及乳腺特异性表达载体的检测提供参考资料。     

Platycodin D protects oral epithelial cells against infection of Candida albicans

AIM: To investigate the effect of platycodin D on Candida albicans infection in oral epithelial cells. METHODS: The viability of the oral squamous carcinoma KB cells was detected by MTT assay after treated with different concentrations of platycodin D. The KB cells were infected with Candida albicans, and then were incubated with platycodin D at different concentrations. Adherent numbers of the Candida albicans were counted by Gram staining, and the bacterial activity and conversion were measured by Trypan blue staining. Furthermore, the protein levels of IL-18 and human β-defensin 2 (HBD-2) were analyzed by ELISA, and the expression of HBD-2 at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTS: The viability of the KB cells was not affected by platycodin D at the concentrations used. The adherent numbers, bacterial activity and conversion were decreased by treatment with platycodin D in a dose-dependent manner. In addition, the protein level of IL-18 in the culture supernatant and the mRNA expression of HBD-2 in the KB cells were also reduced after platycodin D treatment.CONCLUSION: Platycodin D has a bacteriostasis effect and prevents oral epithelial cells from Candida albicans infection.     

Culture of human cardiac stem cells with improved cardiosphere-growing medium in vitro

AIM: To prepare an improved medium for culturing human cardiac stem cells. METHODS: The heart samples of the right auricle obtained from the patients after cardiac surgery were minced into pieces (about 1 mm×1 mm×1 mm), digested and cultured. The primary cells obtained were cultured with improved cardiosphere-growing medium (CGM) for proliferation, and the cells were identified by flow cytometry. Finally, purer c-kit+ cells were obtained by the method of magnetic bead sorting. RESULTS: After about 2 weeks of culture, small, round and phase-bright cells migrated from the well-adherent explants over a layer of fibroblast-like cells. These cells were collected by a brief digestion with Accutase, washed and cultured with improved CGM. No significant difference of the proliferative capacity between using traditional CGM and improved CGM was observed. After subculture and proliferation, the identification result by flow cytometry showed that the positive rate of c-Kit surface marker on these cells was (6.8±2.1)%. By the method of anti-c-Kit magnetic bead sorting, purer c-Kit+ cardiac stem cells were obtained and differentiated into cardiomyocytes. CONCLUSION: Purer c-Kit+ cardiac stem cells are isolated with the improved CGM culture.     

病毒受体及其研究方法概况

病毒受体在介导病毒与靶细胞的特异性结合过程中起着非常重要的作用。病毒受体的特性与病毒的宿主范围和组织特异性密切相关,是病毒致病性的关键因素之一。研究病毒受体的方法很多,笔者综述了病毒受体特征及其几种主要研究方法,以期为有效控制、治疗病毒感染提供理论参考。     

Effects of extracellular potassium on expression of HERG gene nonsense mutant L539fs/47

AIM: To study the effects of extracellular potassium on the protein expression of wild- type HERG and its mutant L539fs/47. METHODS: Wild-type HERG (WT) or its mutant HERG-L539fs/47 (MT) were transfected into HEK293 cells for 36 h. The cells were incubated in different media containing 0.8, 4.3 or 10 mmol/L potassium. After 6 h of incubation, the protein expression of HERG was detected by flow cytometry.After 12 h of incubation, the localization and quantity of the proteins were detected by laser confocal imaging and Western blot. RESULTS: Different from the retention of mutant protein in cytoplasm, wild-type HERG protein was mainly distributed in the cell membrane. The 2 proteins both increased with the changes of extracellular potassium. Flow cytometry showed that the fluorescence in the 2 groups both increased with the changes of extracellular potassium (P < 0.01). The fluorescence in WT group was significantly higher than that in MT group (P < 0.01). Western blot showed that mutant HERG protein included only one 60 kD band, different from the 135 kD and 155 kD bands in wild-type HERG, which were affected by the changes of extracellular potassium (P < 0.05). CONCLUSION: The retention of HERG mutant L539fs/47 protein in the cytoplasm is more than wild-type HERG. Chronic high extracellular potassium keeps the stability of wild-type and mutant HERG proteins on the cell membrane. Chronic low potassium reduces the expression of HERG channel proteins in a time-dependent manner.     

武安国家森林公园蕨类植物叶表皮细胞的显微观察

利用光学显微镜对武安国家森林公园14种观赏蕨类植物叶表皮细胞的显微结构进行了观察.结果表明:几种蕨类植物不同种属间具有一定的共性,表现为气孔器全部分布在叶的下表皮,即全部为下生气孔.但在叶表皮细胞的大小、形态、垂周壁式样及气孔器的类型、保卫细胞的长宽、气孔指数等特征上存在一定的差异,基于14个分类群和35项表型性状特征相似性的聚类分析结果,表明蕨类植物各个种之间的亲缘关系和传统分类方法基本相同.