Comparison of the inducibility of CYP mRNA exposed to typical inducers in fresh and cryopreserved cynomolgus monkey hepatocytes

Herein, we evaluated CYPs and their nuclear receptor mRNA induction by exposure to typical inducers, omeprazole, rifampicin, and phenobarbital in cynomolgus monkey hepatocytes. Six freshly-isolated hepatocytes and 6 cryopreserved hepatocytes from cynomolgus monkey liver were prepared for a 14-day monolayer culture, 28-day co-culture with feeder cells, and 28-day 3D spheroid culture with feeder cells. Omeprazole and rifampicin respectively induced CYP1A1 and CYP3A8 mRNAs, while phenobarbital induced CYP2C43, CYP2C75, and CYP3A8, and slightly induced CYP2B6. The nuclear receptors AHR, PXR, and CAR mRNA levels, which were activated by omeprazole, rifampicin, and phenobarbital, respectively, tended to decrease via exposure to inducers despite the increase in CYP mRNA levels. These trends were similar for all three culture methods. No evident difference was observed in CYP mRNA induction between fresh and cryopreserved hepatocytes. Based on mRNA levels, the co-culture and 3D spheroid culture methods are more reasonable than monolayer culture for CYP evaluation, because the use of feeder cells can reduce the number of hepatocytes, improve the cell adhesion, and maintain the mRNA expression levels. In addition, co-culture method is more cost-effective, as common culture plates can be used.     

RU486对人绒毛膜滋养层、蜕膜细胞以及小鼠胚胎与子宫内膜“共培养”的影响

本文报道RU 486对离体人绒毛膜滋养层、蜕膜细胞的影响。并在小鼠胚胎与子宫内膜“共培养”模型上,观察不同浓度的RU 486对着床前、后胚胎及子宫内膜的作用。实验结果发现:当RU 486浓度为10～60μg/ml时,对绒毛滋养层细胞无直接影响;仅在浓度60μg/ml时,对蜕膜有轻度抑制作用。RU 486对小鼠胚胎及子宫内膜均有不同程度的影响,且随药物浓度的增加而作用增强。当浓度达10μg/ml时,几乎完全抑制胚胎的发育,并发生萎缩及退行性变化,同时子宫内膜也遭严重破坏。     

TNF-α诱导后的小鼠成骨细胞对破骨前体细胞增殖的影响

目的：通过将小鼠成骨细胞与破骨前体细胞间接共培养,研究经肿瘤坏死因子-α( tumour necrosis fac-tor-α,TNF-α)诱导后模拟炎症状态下的成骨细胞对破骨前体细胞增殖功能的影响。方法按照TNF-α10 ng/mL的浓度对鼠成骨细胞分别培养2、4、8、16 h,以诱导其进入炎症状态,并分别提取其上清液,与加入了核因子κB受体活化因子配体(receptor activator for nuclear factor-κ B ligand,RANKL)30μg/L 的鼠单核细胞共培养,利用激光共聚焦显微镜观察和细胞计数试剂盒(cell counting kit-8,CCK-8)法进行增殖活性检测。结果 TNF-α诱导2 h后的成骨细胞上清液与加入RANKL 的单核细胞共培养时,细胞增殖活性实验组与对照组之间差异无统计学意义(P>0.05),诱导4 h实验组破骨前体细胞的增殖活性高于对照组,8、16 h实验组破骨前体细胞的增殖活性低于对照组。结论短暂炎症刺激下,成骨细胞可能对破骨前体细胞增殖有促进作用;长时间炎症刺激下,成骨细胞对破骨前体细胞的增殖可能有抑制作用。     

Extent and features of liver steatosis in vitro pave the way to endothelial dysfunction without physical cell-to-cell contact

Background and aimsSeveral chronic multifactorial diseases originate from energy unbalance between food intake and body energy expenditure, including non-alcoholic fatty liver disease (NAFLD), diabetes, and cardiovascular disorders. Vascular endothelium plays a central role in body homeostasis, and NAFLD is often associated with endothelial dysfunction (ED), the first step in atherosclerosis. Both sugars and fatty acids (FAs) are fuel sources for energy production, but their excess leads to liver steatosis which may trigger ED through a network of mechanisms which need to be clarified. Here, we investigated the crosstalk pathways between in vitro cultured steatotic hepatocytes (FaO) and endothelial cells (HECV) being mediated by soluble factors.Methods and resultsWe employed the conditioned medium approach to test how different extent and features of hepatic steatosis distinctively affect endothelium leading to ED. The steatogenic media collected from steatotic hepatocytes were characterized by high triglyceride content and led to lipid accumulation and fat-dependent dysfunction in HECV cells. We found a parallelism between (i) extent of hepatocyte steatosis and level of lipid accumulation in HECV cells; (ii) type of hepatocyte steatosis (with macro- or microvesicular LDs) and extent of oxidative stress, lipid peroxidation, nitric oxide release and expression of ED markers in HECV cells.ConclusionsThe present findings seem to suggest that, in addition to triglycerides, other soluble mediators should be released by steatotic hepatocytes and may influence lipid accumulation and function of HECV cells. Further studies need to depict the exact profile of soluble factors involved in steatotic hepatocyte-endothelium crosstalk.     

大鼠原代神经细胞放射性损伤敏感性与ROS含量关系及依达拉奉的保护作用

目的 探讨大鼠原代神经细胞培养体系放射性损伤敏感性与ROS含量关系以及依达拉奉的保护作用。方法 X射线单次照射来源于大鼠海马的原代神经元,星形胶质细胞以及星形胶质细胞-神经元共培养体系,对比评价正常培养或依达拉奉干预与否条件下细胞死亡、凋亡以及活性氧（ROS）含量变化。结果 X射线照射引起原代神经元培养体系ROS含量及细胞死亡率明显升高。共培养体系细胞损伤较轻,星形胶质细胞培养体系则无明显损伤。依达拉奉通过清除ROS可以阻止细胞死亡。结论 放射损伤后原代神经元培养体系ROS含量明显增高,导致神经元凋亡失调。依达拉奉通过清除ROS逆转这一病理过程而产生神经元保护作用,值得临床推广。     

A biomimetic Schlemm's canal inner wall: A model to study outflow physiology,glaucoma pathology and high-throughput drug screening

Glaucoma is a disease that damages the optic nerve, frequently leading to blindness. Elevated intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, which is expected to affect 80 million people by 2020, causing bilateral blindness in over 10 million individuals. Because pathological changes to Schlemm's canal (SC) may account for significant resistance to outflow, there is considerable interest in characterizing and evaluating the Schlemm's canal as a target for glaucoma therapeutics. In conventional, two-dimensional culture, human Schlemm's canal (HSC) cells lose spatial, mechanical and biochemical cues, resulting in altered gene expression and cell signaling than observed in vivo, compromising the clinical relevance of data obtained from such systems. Here, we report, for the first time, that 3D culture of HSC cells on microfabricated scaffolds with defined physical and biochemical cues, rescued expression of key HSC markers, VE-cadherin and PECAM1, and mediated pore formation, crucial for the Schlemm's canal regulation of IOP. We demonstrated that following treatment with the glaucopathogenic agent, TGF-β2, HSC cells undergo an endothelial–mesenchymal transition, which together with the increase in extracellular matrix (ECM) proteins might account for the decrease in outflow facility observed in patients with high TGF-β2 levels in their aqueous humor. We also demonstrated that unlike 2D cultures, 3D cultures of HSC cells are amenable to gene transfer. Thus, our data imply that 3D culture of HSC cells may be used as a platform to advance our understanding of HSC physiology and pathology and as a model for high-throughput drug and gene screening.     

脐血源性神经球分化为类雪旺细胞的研究

目的 研究脐血间充质干细胞(CB-MSC)向雪旺细胞谱系分化的生物表型和特征.方法诱导CB-MSC为悬浮的神经球,然后用胶质生长因子诱导分化为类雪旺细胞.分化的CB-MSC表现出和雪旺细胞类似的形态学的改变.通过免疫化学染色和Western blotting鉴定这些细胞雪旺细胞标志的表达情况;通过分化的CB-MSC与背根神经节神经元共培养观察其促进其轴突生长的作用.结果 未分化的CB-MSC很少表达nestin、GFAP和S-100.在CB-MSC诱导成神经球以后,82.38%±3.70%的细胞表达nestin,而GFAP和S-100仍然阴性.接种于分化液36 h后细胞表达nestin、GFAP、S-100,阳性率分别为43.78%±3.21%、35.42%±1.82%和29.49%±2.54%.相比未分化CB-MSC,分化的CB-MSC中神经轴突的形成率和轴突的长度明显增加,差异有统计学意义(P＜0.05).结论CB-MSC可以通过诱导转变为神经球,神经球可以诱导成雪旺细胞的形态,表型和功能类似雪旺细胞,可做为一种替代骨髓间充质干细胞的细胞来源,对于治疗神经系统疾病具有潜在的应用价值.     

医科和理工科联合培养医学研究生的优势

本文对医科和理工科联合培养医学研究生的优势进行了探讨,认为这种培养方式可以达到医工结合、资源共享,可以培养医学研究生的良好思维方式和科研能力,可以增强医学研究生的创新意识.同时,对高校创新教育模式及其存在问题进行了探讨.     

Osteoblast: Osteoclast co-cultures on silk fibroin, chitosan and PLLA films

This study investigates the growth of a co-culture of osteoblasts and osteoclasts on four different types of degradable biomaterials with bone tissue engineering potential. Single or co-cultures of osteoblasts and osteoclasts (used at a ratio of 1:100 osteoblast:osteoclasts) were cultured on vapour stabilised silk fibroin, methanol stabilised silk fibroin, chitosan and poly (l lactic acid) (PLLA) films for 10 days. Osteoclast differentiation was determined by tartrate resistant acid phosphatase (TRAP) staining, total cell number by a picogreen DNA assay, cell morphology by scanning electron microscopy (SEM) and the material topography by atomic force microscopy (AFM). Samples were also monitored for degradation by differential scanning calorimetry (DSC) and fourier transform infrared (FTIR). Results demonstrated that vapour stabilised silk fibroin, methanol stabilised silk fibroin and chitosan all support the growth of osteoblasts and osteoclasts in both single and co-cultures. PLLA showed poor osteoclast differentiation in both single and co-cultures but supported osteoblast attachment and proliferation. Both silk fibroin materials showed sign of early degradation in the ten-day period, but very little change was seen in chitosan and PLLA samples. This study indicates that this novel co-culture approach for bone tissue engineering may be possible if scaffolds are created from silk fibroin or chitosan.     

Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing

The purpose of the present study is to develop a novel method for the fabrication of transferable micropatterned cell sheets for tissue engineering. To achieve this development, microcontact printing of fibronectin on commercially available temperature-responsive dishes was employed. Primary rat hepatocytes were seeded on the dish surfaces printed with fibronectin. Under serum-free conditions, hepatocytes were attached onto fibronectin domains selectively. Then, a second cell type of endothelial cells was seeded in the presence of serum. Double fluorescent staining revealed that endothelial cells successfully adhered to the intervals of hepatocyte domains. Finally, all the cells were harvested as a single contiguous micropatterned cell sheet upon temperature-reduction. With a cell sheet manipulator having a gelatin layer for the support of harvested cell sheets, harvested micropatterned cell sheets were transferred to new dish surfaces. This technique would be useful for the fabrication of thick tissue constructs having a complex microarchitecture.