T载体介导的基于attL核心区LR反应的简化快速Gateway克隆系统

Gateway克隆技术已得到广泛的应用。该技术先通过BP反应将目标片段连到带有完整attL特异识别位点的入门载体,然后与终载体通过LR反应得到表达载体。Gateway克隆方法与传统的酶切连接方法相比有快速简单等优点。但是,BP和LR酶都非常昂贵。本研究首先对3个常用Gateway载体的atts特异位点序列比对发现,attL序列核心交换位点“core attL”的21~22 bp长的碱基是保守和必要的。由此,设计含有core-attL序列的引物,通过PCR克隆得到DNA片段并连入pMD18-T载体,然后进行LR反应,可成功得到目标表达载体,并在保守的位点上正确重组。本研究还对其中一个带有绿色荧光蛋白基因的表达载体转化至烟草,能够正常表达该蛋白质。结果表明,通过将含有attL核心位点基因片段连接到pMD18-T载体上,可以省略BP反应而将目标片段连接到终载体上,节约了反应时间和成本。     

Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat

The success of stem cell application in regenerative medicine, usually require a stable source of stem or progenitor cells. Fat tissue represents a good source of stem cells because it is rich in stem cells and there are fewer ethical issues related to the use of such stem cells, unlike embryonic stem cells. Therefore, there has been increased interest in adipose-derived stem cells (ADSCs) for tissue engineering applications. Here, we aim to provide an easy processing method for isolating adult stem cells from human adipose tissue harvested from the subcutaneous fat of the abdominal wall during gynecologic surgery. We used a homogenizer to mince fat and compared the results with those obtained from the traditional cut method involving a sterile scalpel and forceps. Our results showed that our method provides another stable and quality source of stem cells that could be used in cases with a large quantity of fat. Furthermore, we found that pregnancy adipose-derived stem cells (P-ADSCs) could be maintained in vitro for extended periods with a stable population doubling and low senescence levels. P-ADSCs could also differentiate in vitro into adipogenic, osteogenic, chondrogenic, and insulin-producing cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirates, adipose tissues obtained from pregnant women contain multipotent cells with better proliferation and showed great promise for use in both stem cell banking studies as well as in stem cell therapy.     

Antheridiogen production and response in Polypodiaceae species

Antheridiogen chemicals secreted by living fern gametophytes have been shown to influence production of male gametangia and thus mating systems in a large number of terrestrial fern species. Antheridiogens have not previously been thought to be prevalent in the Polypodiaceae, a large family composed mostly of tropical epiphytes. This study presents bioassay methods more sensitive than previously used to detect antheridiogen and demonstrates that antheridiogens are also operative in the Polypodiaceae and in epiphytic species. Seven species in six genera (Campyloneurum angustifolium, C. phyllitidis, Lepisorus thunbergianus, Microgramma heterophylla, Phlebodium aureum, Phymatosorus scolopendria, and Polypodium pellucidum) were tested for the presence of an antheridiogen system. All species tested except P. aureum were induced to produce antheridia precociously by their own antheridiogen and by that of Pteridium aquilinum (APt). Phlebodium aureum responded to APt and promoted antheridium formation in Onoclea sensibilis but did not respond to its own antheridiogen. Spores of all species except P. aureum were induced to germinate in darkness by antheridiogen of the same species and by APt and to form antheridia in the dark, further enhancing the possibility of intergametophytic mating.     

极低密度脂蛋白对巨噬细胞载脂蛋白E基因表达的调节

巨噬细胞（M）是动脉粥样硬化（AS）斑块中泡沫细胞的主要来源之一,它可通过其表面的多种脂蛋白受体介导摄取合APOB或APOE的脂蛋白,导致M的脂质增加．此外,M有较强的分泌ApOE的能力[1],这一现象虽已发现多年,但意义尚不清楚VLDL是一种含APOE的脂蛋白,它能通过M膜上识别APOE的受体进入M,从而促进Mg内脂质堆积,并与AS密切相关[2,3]．流行病学调查发现在我国的高脂血症中2／3为VLDL升高为主的高甘油三酯（HTG）血症．另外,Evans等报道HTG血症患者的血浆中以ApoE－poorVLDL升高为主,它们在血浆中清除的速率较Apo…     

Utilizing the macroporous media for insect cell/baculovirus expression

Due to their bio-compatibility and high porosity, cellulose foam and polyurethane foam (PUF) were selected as the media to immobilize insect cells for recombinant baculovirus expression. In packed beds, entrapment efficiency and axial cell distribution are two prime important parameters associated with reactor performance. These two parameters were found to be affected by initial cell loading density and liquid circulation rate. Based on the entrapment kinetic model, the entrapment parameters as K ₀, σ _m could be evaluated. The establishment of the correlation between K ₀, σ _m and Reynolds number, N _Re, described in this study provided convenient tools to achieve an efficient cell entrapment in the packed bed. The appropriated circulation velocity for uniform cell distribution was also determined.