翅果油树休眠芽和种子的试管萌发

目的：为了扩大珍稀濒危植物翅果油树组织培养的取材范围和研究其种子休眠的原因。方法：以翅果油树一年生枝条的休眠芽（分别于10,11,12,1,2,3,月份取材）和当年生种子为材料,采用MS为基本培养基,附加6－BA,NAA,GA3等外源激素进行试管苗萌发研究。结果：1．休眠芽的萌发率表现为从秋季到冬季逐渐下降和从冬季到春季逐渐提高的规律性变化。其中以3月份取材最佳,休眠芽可萌发为小苗。2．对种子进行试管内和试管外萌发研究,表明翅果油树种子休眠的原因有：中果皮坚硬,种皮革质化,透水透气性差,中果皮和种皮内有抑制物质存在;种仁营养丰富,易引起土壤,空气中微生物的繁殖而烂种;此外,裸露的种仁在试管中的萌发率高达70％,可发育为无菌苗。结论：3月份取材的休眠芽和种仁无菌萌发的实生苗可为组织培养提供新的材料来源。     

New method of <Emphasis Type="Italic">in vitro</Emphasis> culturing of pigment retinal epithelium in the structure of the posterior eye sector of adult rat

We propose a new method of organotypic roller 3D-culturing of the posterior sector of the eye. The method allows maintaining tissue viability in vitro for 14 days (which considerably surpasses the capacities of stationary culturing) and studying of the behavior of pigment retinal epithelial cells and choriocapillary membrane. Using this method we demonstrated phenotypic transformation, migration, and proliferation of pigment retinal epithelial cells under conditions of roller organotypic culture. In the absence of the retina, these cells exhibit properties of scavenger cells (phagocytes) both within and outside the layer. Under conditions of roller culturing in vitro, cells of the pigment retinal epithelium undergo changes similar to those observed in various retinal pathologies in vivo, including age-associated changes. Here we discuss the possibility of using the proposed method for evaluation of the effect of various factors added to the culture medium on the pigment epithelium, for modeling of processes developing in damaged pigment epithelium or under conditions of various pathologies, and for the study of regeneration responses in cells of pigment retinal epithelium in adult vertebrates. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 4, pp. 207–215, October, 2007     

小鼠耳蜗毛细胞体外培养研究方法

目的建立耳蜗Corti器体外培养模型.方法取刚出生2～3天小鼠耳蜗,采用鼠尾胶表面平铺培养并结合荧光染色方法观察耳蜗毛细胞的生长情况.结果耳蜗基底膜经1～5天培养,内、外毛细胞和支持细胞生长良好,排列有序,无任何衰亡和缺损.结论耳蜗Corti器可以在体外培养环境存活一定时间并健康生长.     

Optimal sampling time after preparation of platelet concentrates for detection of bacterial contamination by quantitative real-time polymerase chain reaction

BACKGROUND AND OBJECTIVES: A universal quantitative real-time polymerase chain reaction (PCR), based on bacterial 16S rDNA, to detect bacterial contamination of platelet concentrates (PCs), was developed previously and compared with automated culturing. In the present study, this real-time PCR method was evaluated to determine the optimal sampling time for screening of bacterial contamination in PCs. MATERIALS AND METHODS: Routinely prepared PCs were spiked with suspensions of Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Propionibacterium acnes to 1, 10 and 100 colony-forming units (CFU)/ml and stored at room temperature for 7 days. The presence of bacteria in these PCs was monitored by quantitative real-time PCR. As a reference method (additional control), BacT/Alert automated culturing was used. For PCR, 1-ml aliquots were drawn from all (spiked) PCs on days 0, 1, 2, 3, 6 and 7 of storage. As a control, triplicate samples (10 ml) were inoculated into aerobic and anaerobic BacT/Alert culture bottles immediately after spiking (day 0) and after storage for 1, 2, 3, 6 or 7 days. RESULTS: With quantitative real-time PCR, all bacterial species tested were reproducibly detected on day 1 after spiking at original concentrations of 10 and 100 CFU/ml. Bacteria were also detected on day 1 from PCs spiked with an initial concentration of 1 CFU/ml, except for E. coli, which was detected in only one of the three samples and P. aeruginosa, for which analysis was not performed on day 1. With the reference method, bacteria were detected in culture bottles (inoculated on day 0) within a mean time of 20.1 h, with the exception of P. acnes which was detected at a mean time of 102.3 and 49.3 h (for original spiking concentrations of 10 and 100 CFU/ml respectively). CONCLUSIONS: PCR enables the rapid detection of low initial numbers of bacteria in PCs. For reliable detection, our results support that sampling of PCs for real-time PCR screening should not be carried out earlier than 1 day after preparation (48 h after blood collection). Importantly, the real-time PCR approach has the potential to be used before the release of PCs from the blood centre or shortly before they are transfused in the hospital.     

药用植物商陆毛状根培养系统的建立

目的:建立药用植物商陆(Phytolacca acinosa Roxb.)的毛状根离体培养系统.方法:分别用发根农杆菌A4、R1600、C58C1 3个菌株感染商陆的子叶外植体,获得毛状根,并筛选了优质株系;考察了影响毛状根生长的因素;测定了毛状根的生长曲线;利用PCR和高压纸电泳法对毛状根进行tDNA转化的检测.结果:利用发根农杆菌A4、R1600、C58C1 3种菌株成功地从商陆中诱导出毛状根;以水解酪蛋白为氮源、以麦芽糖为碳源最有利于商陆毛状根的生长;毛状根的生长曲线呈"S"形,24 d时质量达到最大(增长18倍);PCR及高压纸电泳检测结果表明Ri质粒的tDNA已整合进毛状根中.结论:成功建立了商陆毛状根离体培养系统,为进一步进行药用成分的工业化生产和引入外源基因改良性状奠定了基础.     

In vitro and in vivo genotoxicity evaluation of hormonal drugs. I. Hydrocortisone

Genotoxic evaluation of a widely used glucocorticoid, hydrocortisone, was undertaken using a battery of in vitro and in vivo test systems. Human lymphocyte cultures and mouse bone marrow studies (micronuclei and sister chromatid exchange analyses) showed the drug to be very potent clastogen. However, the Ames/Salmonella assay both with and without S9 did not show an increase in the His+ revertants.     

诱导多潜能干细胞的研究进展

诱导多潜能干细胞(IPSC)自2006年问世,就备受全球各大实验室广泛关注,成为生命科学领域新的里程碑。本篇综述旨在回顾多潜能性的研究历史,重点介绍IPSC培养技术,探讨IPSC培养技术在细胞治疗方面的应用前景。通过大量查阅国内外相关领域的综述及论著,本文归纳总结近年来IPSC领域的最新研究成果,以期为该领域后续的探索性研究提供参考。     

单纯Ⅱ型胶原酶消化法分离、培养人退变椎间盘髓核细胞的形态学观察

目的:探讨单纯Ⅱ型胶原酶消化法体外培养扩增人退变椎间盘髓核细胞的可行性。方法:收集20例人退变椎间盘髓核,单纯Ⅱ型胶原酶消化分离出髓核细胞并连续培养传代,倒置相差显微镜和HE染色观察细胞形态学变化,甲苯胺蓝染色检测髓核细胞内聚集蛋白聚糖的表达,免疫细胞化学法行Ⅱ型胶原染色,观察髓核细胞的类软骨表型表达情况。结果:单纯Ⅱ型胶原酶消化法可较好的分离培养人退变椎间盘髓核细胞,20例人退变椎间盘髓核,培养成功16例;原代髓核细胞平均7d贴壁,呈类圆形或多角形,P1代髓核细胞平均12h贴壁,呈大梭形或多角形,两代细胞融合95%所需时间分别为30d和7d,差异有统计学意义(P0.01);聚集蛋白聚糖和Ⅱ型胶原主要表达于原代和P1代髓核细胞浆内,被甲苯胺蓝染成天蓝色,免疫细胞化学染色主要表现为黄褐色沉淀,两代间聚集蛋白聚糖和Ⅱ型胶原的表达无统计学差异(P0.05)。结论:单纯Ⅱ型胶原酶消化法可简化髓核细胞分离步骤,提高培养效率;传一代后髓核细胞增殖速率提高,但仍维持类软骨表型,表达聚集蛋白聚糖和Ⅱ型胶原。