接种密度对Vero细胞在微载体表面生长的影响

研究了接种密度对Ｖｅｒｏ细胞在微载体表面生长行为的影响,发现培养过程中所获得的细胞最高密度随接种密度的增加而有所提高,当细胞的表面接种密度高于１．０×１０＾５ｃｅｌｌｓ／ｍｇＭＣ时,因贴壁后细胞扩展受到限制而不利于进入对数生长期。就整个Ｖｅｒｏ细胞培养过程,适宜的表面接种密度为３×１０＾－４－７×１０＾４ｃｅｌｌｓ／ｍｇＭＣ,此时可以获得较高的比生长速率和细胞增殖倍数。     

外源性H2S通过调节β-位淀粉样前体蛋白裂解酶1表达对PC12细胞APP／Aβ代谢的影响

目的观察外源性硫化氢对嗜铬细胞瘤细胞β-位淀粉样前体蛋白裂解酶1（BACE1）的调节作用,进而探讨其对淀粉样前体蛋白／β-位淀粉样蛋白代谢途径的影响。方法用硫氢化钠作外源性H2s供体,实验设空白对照组、NaHs50μmol／L组、NaHS100μmol／L组和NariS200μmol／L组,按分组浓度处理PC12细胞24h后,RT-PCR和Western blot法检测细胞内BACE1 mRNA及蛋白表达,并用Western blot法继而检测APP代谢过程中关键蛋白APP、C99、C83表达变化,EusA法检测细胞培养液中AB40和AB42水平。结果NaHS在实验浓度范围内从基因与蛋白两个水平上呈剂量依赖性下调BACE1表达,并下调C99、Ap40和Ap42蛋白表达,上调C83蛋白,各NaHS组分别与对照组比较,差别均有统计学意义（P〈0．05）,而对APP蛋白表达没有影响,各组间比较差别无显著性（P〉0．05）。结论外源性H2s具有通过调节PC12细胞BACE1表达下调APP／Aβ代谢的作用。     

Human stem cells as a model for cardiac differentiation and disease

Studies on identification, derivation and characterization of human stem cells in the last decade have led to high expectations in the field of regenerative medicine. Although it is clear that for successful stem cell-based therapy several obstacles have to be overcome, other opportunities lay ahead for the use of human stem cells. A more immediate application would be the development of human models for cell-type specific differentiation and disease in vitro. Cardiomyocytes can be generated from stem cells, which have been shown to follow similar molecular events of cardiac development in vivo. Furthermore, several monogenic cardiovascular diseases have been described, for which in vitro models in stem cells could be generated. Here, we will discuss the potential of human embryonic stem cells, cardiac stem cells and the recently described induced pluripotent stem cells as models for cardiac differentiation and disease. Received 07 August 2008; received after revision 26 September 2008; accepted 03 October 2008     

The protease-activated receptor-3 (PAR-3) can signal autonomously to induce interleukin-8 release

Protease-activated receptors (PARs) play a clear role in the burst of inflammatory reactions and immune responses. However, for PAR-3, the most elusive member of the PAR family, the functional role is still largely unclear. It has been claimed that PAR-3 does not signal autonomously, although the wide expression of human PAR-3 indicates its important physiological roles. We demonstrate that in HEK-293 cells, stably transfected with human PAR-3, thrombin induced calcium signaling, IL-8 gene expression and IL-8 release. We confirmed this finding using human lung epithelial and human astrocytoma cells that express endogenous PAR-3. Moreover, thrombin exposure of HEK-293 cells resulted in ERK1/2 activation coinciding with IL-8 release. The effects of thrombin were not dependent on PAR-1 activation, as confirmed by PAR-1 gene silencing. Thus, we propose that PAR-3 is able to signal autonomously to induce IL-8 release mediated by ERK1/2 phosphorylation, which contributes actively to inflammatory responses. Received 9 December 2007; received after revision 16 January 2008; accepted 18 January 2008     

Olfactory ensheathing cells are attracted to, and can endocytose, bacteria

Olfactory ensheathing cells (OECs) have been shown previously to express Toll-like receptors and to respond to bacteria by translocating nuclear factor-kappaB from the cytoplasm to the nucleus. In this study, we show that OECs extended significantly more pseudopodia when they were exposed to Escherichia coli than in the absence of bacteria (p=0.019). Co-immunoprecipitation showed that E. coli binding to OECs was mediated by Toll-like receptor 4. Lyso-Tracker, a fluorescent probe that accumulates selectively in lysosomes, and staining for type 1 lysosome-associated membrane proteins demonstrated that endocytosed FITC-conjugated E. coli were translocated to lysosomes. They appeared to be subsequently broken down, as shown by transmission electron microscopy. No obvious adherence to the membrane and less phagocytosis was observed when OECs were incubated with inert fluorescent microspheres. The ability of OECs to endocytose bacteria supports the notion that OECs play an innate immune function by protecting olfactory tissues from bacterial infection.     

In vivo matrix-guided human mesenchymal stem cells

Microfracture of subchondral bone results in intrinsic repair of cartilage defects. Stem or progenitor cells from bone marrow have been proposed to be involved in this regenerative process. Here, we demonstrate for the first time that mesenchymal stem (MS) cells can in fact be recovered from matrix material saturated with cells from bone marrow after microfracture. This also introduces a new technique for MS cell isolation during arthroscopic treatment. MS cells were phenotyped using specific cell surface antibodies. Differentiation of the MS cells into the adipogenic, chondrogenic and osteogenic lineage could be demonstrated by cultivation of MS cells as a monolayer, as micromass bodies or mesenchymal microspheres. This study demonstrates that MS cells can be attracted to a cartilage defect by guidance of a collagenous matrix after perforating subchondral bone. Protocols for application of MS cells in restoration of cartilage tissue include an initial invasive biopsy to obtain the MS cells and time-wasting in vitro proliferation and possibly differentiation of the cells before implantation. The new technique already includes attraction of MS cells to sites of cartilage defects and therefore may overcome the necessity of in vitro proliferation and differentiation of MS cells prior to transplantation. Received 3 November 2005; received after revision 15 December 2005; accepted 4 January 2006     

盐酸戊乙奎醚对大鼠心肌微血管内皮细胞缺氧/复氧后的保护作用

目的：从细胞水平建立大鼠心肌微血管内皮细胞缺氧/复氧模型，观察了盐酸戊乙奎醚对心肌微血管内皮细胞的H/R损伤的保护作用。方法：实验采用酶消化法进行细胞的原代培养，1：2传代。采用荧光标记乙酰化Dil-Ac-LDL的方法鉴定。培养的细胞随机分为4组，①对照组，常氧环境下培养，不给予任何处理；②H/R组，改培养液为D-Hank,s液，于37℃、5%CO2、95%N2 密闭缺氧鑵内培养2h后，弃D-Hank,s液，改为完全培养液，放入常氧培养箱中继续培养4h;③PHC预处理组，在细胞缺氧2h前给予PHC（终浓度为0.1μm•L-1）预处理，④H/R +PHC组，在细胞H/R后，给予PHC（终浓度为0.1μm•L-1）孵育2h。MTT自动比色法测细胞活力，流式细胞法检测细胞凋亡。结果：①成功培养大鼠心肌微血管内皮细胞，用荧光标记乙酰化Dil-Ac-LDL的方法鉴定为微血管内皮细胞。②成功制备出微血管内皮细胞的H/R模型 将试验细胞培养液改为D-Hank,s 液，置于37℃、5%CO2、92%N2 密闭缺氧罐中环缺氧2h，后改为完全培养液放入常氧培养箱中继续培养4h可制备出微血管内皮细胞的I/R模型。③. H/R组细胞死亡率和凋亡率与对照组相比明显增高，差异有统计学意义（P<0.01），PHC+H/R组的细胞死亡率和凋亡率下降，差异有统计学意义（P<0.05）,H/R+PHC组与对照组相比，虽然细胞的死亡率和凋亡率有所下降，但差异没有统计学意义（P>0.05）。结论：PHC预处理对大鼠微血管内皮细胞的H/RI有保护作用。 关键词：盐酸戊乙奎醚；心肌微血管内皮细胞；缺氧/复氧     

一种有效的富氢中选择氧化CO催化剂Au-CuO/Al2O3

采用等体积浸渍法制备了1.0%Au-CuO/Al2O3催化剂,考察了不同Au与Cu的原子比对催化剂在富氢气氛下选择氧化CO性能的影响,并对该催化剂稳定性进行了初步测试.结果表明,该催化剂在30 ℃～85 ℃范围内可以将CO浓度由1.0%降至0.005%以下.催化剂在80 ℃,模拟气氛条件下10 h内失活很小.通过BET、XRD、UV-vis漫反射等表征发现:催化剂体系中Cu以CuO的形式存在,较小粒子的Au在该反应中表现出较好的活性与选择性.     

王瓜根抗癌糖蛋白的生理生化特征及其对肺腺癌细胞杀伤作用初步研究

经电泳分析,王瓜根活性蛋白可分为两种:其一是酸性蛋白,具细胞毒作用,等电点测定 P~I 为3.2,PAGE 电泳测定分子量为6.76×10~4道尔顿;其二是碱性蛋白,等电点为9.3,PAGE 电泳测定分子量为1.58×10~4道尔顿;微具细胞毒作用。王瓜根活性蛋白属糖蛋白,经纸层析和薄层硅胶 H 层析证实含半乳糖和木糖。扫描电镜观察和光镜连续观察,显示酸性蛋白对肺腺癌细胞的体外培齐有杀伤作用,当培养液含120μg/ml 时杀伤率达58%,对正常肺细胞作用弱;碱性蛋白对肺腺癌细胞杀伤作用较差,在同样剂量时只有47.6%杀伤率。新鲜王瓜根原汁有很强的杀伤肺腺癌细胞作用,剂量在26g/ml 时,杀伤率达60%;当提高到105g/ml 时,杀伤率达74%。试验结果提示:王瓜根活性抗癌物质,除指三萜—葫芦素之外,可能还存在另一种活性物质。这种活性抗癌物质对肺腺癌细胞表面微绒毛无作用,经处理24小时后,癌细胞开始变形,表现为不同程度内陷,32小时癌细胞膜开始溶解,48小时癌细胞溶成团块。