蒙古绵羊输卵管上皮细胞培养体系的建立

为了建立蒙古绵羊输卵管上皮细胞培养体系作为良好的体外研究模型,本试验取蒙古绵羊输卵管,运用0.25%胰蛋白酶消化的方法分离上皮细胞进行体外培养。对培养细胞进行形态学、免疫组织化学方法鉴定。原代培养细胞用胰蛋白酶/EDTA方法成功传代,传三代后检测其染色体核型。结果显示,成功获得了原代和传代培养细胞,经鉴定为上皮细胞,传三代后经染色体核型分析显示细胞核型正常,培养的细胞健康状况良好,可以用做体外研究模型。

鼻咽活检组织的上皮细胞培养

鼻咽癌是与Epstein—Barr病毒(EBV)关系非常密切的人类恶性肿瘤。但EBV在鼻咽癌发病机理中的作用尚不清楚。目前还未证实EBV能直接转化正常鼻咽上皮细胞。其部分原因是不能长期和大量培养正常的或异常的鼻咽上皮细胞,难以作转化等实验。本实验应用低血清和无血清培养基。

淋巴细胞分层液提高上皮细胞培养效率

目的尝试应用淋巴细胞分离液收集纯度高、活性强的上皮细胞进行体外培养,探讨是否可以加快上皮细胞培养的速度。方法根据上皮细胞和成纤维细胞比重上的差异,将经过胰酶处理后的小白鼠皮肤,用镊子把上皮和真皮层撕开,收集两层之间的基底细胞,并把这些细胞悬液加入淋巴细胞分层液(LP)中,收集中间层的细胞作为实验组进行体外培养,以传统方法分离的细胞培养作为对照组。两组同时进行对细胞生长速度的三种测试:上皮细胞生长曲线测定、Lowry蛋白测定和H^3-胸腺嘧啶核甙测定。结果经统计学分析证明,实验组的细胞生长速度与对照组比较有明显加快的表现。结论淋巴细胞分层液能获得活力强、生长快的细胞,可缩短培养成上皮细胞皮片的时间,有一定的临床意义。

人乳头瘤病毒体内外培养模型研究进展

人乳头瘤病毒(HPV)复制增殖与上皮细胞分化有关,尚不能在常规细胞培养条件下复制其感染过程。近年来,裸鼠异种移植模型及三维上皮细胞培养模型的出现,使部分型别HPV在人体外复制和增殖成为可能。该文综述HPV体内外培养模型研究进展。

Primary Culture of Bovine Mammary Epithelial Cells

[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.

A Study of Rabbit Lens Epithelial Cells Survival and Growth on the Rabbit Capsular Bag in Vitro

Objective: To study the proliferation, migration and metaplasm of residual rabbit lens epithelial cells (LECs) after extracapsular cataract extraction(ECCE)based on the rabbit capsular bag model in vitro. Methods: Sham cataract surgery, including anterior capsulorhexis, nucleus hydroexpression and aspiration of lens fibers, was performed on 20 rabbit lens. The capsular bags were isolated and pinned to sterile non-toxic silicone rings on petri dishes. The capsular bags were incubated with Eagle's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) and monitored for 3 weeks by phase-contrast microscopy, after which light microscopy was performed on them.Results: After a latent period of 2-3 d, outgrowth was observed across the posterior capsule. Growth proceeded rapidly so that the posterior capsule was totally covered by a confluent monolayer of cell at 6-8 day. Capsular wrinkles became increasingly apparent as time progressed, causing a marked rise in light scatter. An increase in capsular tension also came.Conclusion: This model exhibits many of the in vito characteristics of the lens capsule after extracapsular surgery and may prove useful in further elucidating the cellular mechanisms of posterior capsule opacification and developing strategies for inhibiting cell growth with this system.

大面积深度烧伤治疗技术应用效果调查报告

目前,大面积深度烧伤治疗的主要方法包括皮肤原位再生复原技术(MEBT/MEBO),传统外科削切痂植皮(自体皮)技术以及外科削切痂+体外上皮细胞培养外供皮源技术等。其中,利用皮肤原位再生复原技术治疗大面积深度烧伤已经成为中国烧伤治疗的主体技术,而且也逐渐成为国外烧伤治疗的主流技术,它成功地解决了烧伤创面疼痛、创面感染、创面进行性坏死以及深Ⅱ度烧伤创面瘢痕愈合的四大国际性技术难题,在挽救患行生命的同时大幅度降低了致残率,使烧伤总面积超过90%TBSA的特重度烧伤患者的总治愈率从以往的5.7%提高至92%,致残率从100%下降至2.05%。其它两种技术因在大面积深度烧伤治疗中的高死亡率、高致残率以及患者痛苦大、创面愈合质量差、治疗费用高等原因逐渐被医患双方所放弃。下图是各种疗法治疗深Ⅱ度烧伤创面的疗效对比,详细病例资料请观看本期杂志所附光盘。

Probiotic modulation of dendritic cells co-cultured with intestinal epithelial cells

AIM： To investigate cytokine production and cell surface phenotypes of dendritic cells （DC） in the presence of epithelial cells stimulated by probiotics.METHODS： Mouse DC were cultured alone or together with mouse epithelial cell monolayers in normal or in- verted systems and were stimulated with heat-killed probiotic bacteria, Bifidobacterium lactis ADO 11 （BL）, Bifidobacterium bilfidum BGN4 （BB）, Lactobacillus casei IBS041 （LC）, and Lactobacillus acidophilus AD031 （LA）, for 12 h. Cytokine levels in the culture supernatants were determined by enzyme-linked immunosorbent as say and phenotypic analysis of DC was investigated by flow cytometry.RESULTS： BB and LC in singlecultured DC increased the expression of I-Ad, CD86 and CD40 （I-Ad, 18.51 vs 30.88, 46.11, CD86, 62.74 vs 92.7, 104.12; CD40, 0.67 vs 6.39, 3.37, P 〈 0.05）. All of the experimental probiot-ics increased the production of inflammatory cytokines, interleukin （IL）-6 and tumor necrosis factor （TNF）-α. However, in the normal coculture systems, LC and LA decreased the expression of I-A^α （39.46 vs 30.32, 33.26, P 〈 0.05）, and none of the experimental probiotics increased the levels of IL-6 or TNF-α. In the inverted coculture systems, LC decreased the expression of CD40 （1.36 vs -2.27, P 〈 0.05）, and all of the experimental probiotics decreased the levels of IL-6. In addition, BL increased the production of IL-10 （103.8 vs 166.0, P 〈 0.05） and LC and LA increased transforming growth factor-13 secretion （235.9 vs 618.9, 607.6, P 〈 0.05）.CONCLUSION： These results suggest that specific pro- biotic strains exert differential immune modulation mediated by the interaction of dendritic cells and epithelial cells in the homeostasis of gastrointestinal tract.

Interactions of primary neuroepithelial progenitor and brain endothelial cells: distinct effect on neural progenitor maintenance and differentiation by soluble factors and direct contact

Neurovascular interactions are crucial for the normal development of the central nervous system. To study such interactions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and endothelial cell fractions from embryonic mouse brains. Depending on the culture conditions endothelial cells were found to favor maintenance of the neuroprogenitor phenotype through the production of soluble factors, or to promote neuronal differentiation of neural progenitors through direct contact. These apparently opposing effects could reflect differential cellular interactions needed for the proper development of the brain.

Comparative genomic study of gastric epithelial cells co-cultured with Helicobacter pylori

AIM:To identify genes potentially involved in Helicobacter pylori(H.pylori)-induced gastric carcinogenesis.METHODS:GES-1 cells were co-cultured with H.pylori strains isolated from patients with gastric carcinoma(GC,n = 10) or chronic gastritis(CG,n = 10) for in vitro proliferation and apoptosis assays to identify the most and least virulent strains.These two strains were cagA-genotyped and used for further in vivo carcinogenic virulence assays by infecting Mongolian gerbils for 52 wk,respectively;a broth free of H.pylori was lavaged as control.Genomic profiles of GES-1 cells cocultured with the most and least virulent strains were determined by microarray analysis.The most differentially expressed genes were further verified using quantitative real-time polymerase chain reaction in GES-1cells infected with the most and least virulent strains,and by immunohistochemistry in H.pylori positive CG,precancerous diseases,and GC biopsy specimens in an independent experiment.RESULTS:GC-derived H.pylori strains induced a potent proliferative effect in GES-1 cells in co-culture,whereas CG-derived strains did not.The most(from a GC patient) and least(from a CG patient) virulent strains were cagA-positive and negative,respectively.At week 52,CG,atrophy,metaplasia,dysplasia,and GC were observed in 90.0%,80.0%,80.0%,90%,and 60.0%,respectively,of the animals lavaged with the most virulent strain.However,only mild CG was observed in 90% of the animals lavaged with the least virulent strain.On microarray analysis,800 differentially expressed genes(49 up-and 751 down-regulated),involving those associated with cell cycle regulation,cell apoptosis,cytoskeleton,immune response,and substance and energy metabolisms,were identified in cells co-cultured with the most virulent strain as compared with those co-cultured with the least virulent strain.The six most differentially expressed genes(with a betweenness centrality of 0.1-0.2) were identified among the significant differential gene profile network,including JUN,KRAS,BRCA1,SMAD2,TRAF1,and HDAC6.Quantitative real-time polymerase chain reaction analyses verified that HDAC6 and TRFA1 mRNA expressions were significantly more up-regulated in GES-1 cells cocultured with the most virulent strain than in those cocultured with the least virulent strain.Immunohistochemistry of gastric mucosal specimens from H.pyloripositive patients with CG,intestinal metaplasia(IM),dysplasia,and GC showed that moderately positive and strongly positive HDAC6 expression was detected in 21.7% of CG patients,30.0% of IM patients,54.5% of dysplasia patients,and 77.8% of GC patients(P < 0.001).The up-regulation of TRAF1 expressions was detected in 34.8%,53.3%,72.7%,and 88.9% specimens of CG,IM,dysplasia,and GC,respectively(P < 0.001).CONCLUSION:The overexpression of HDAC6 and TRAF1 in GES-1 cells co-cultured with the GC-derived strain and in H.pylori-positive dysplasia and GC suggests that HDAC6 and TRAF1 may be involved in H.pyloriinduced gastric carcinogenesis.

In vitro assessment of gastrointestinal viability of two photosynthetic bacteria,Rhodopseudomonas palustris and Rhodobacter sphaeroides

The objectives of this study were to assess the potential of two photosynthetic bacteria （PSB）, Rhodopseudomonas palustris HZ0301 and Rhodobacter sphaeroides HZ0302, as probiotics in aquaculture. The viability of HZ0301 and HZ0302 in simulated gastric transit conditions （pH 2.0, pH 3.0 and pH 4.0 gastric juices） and in simulated small intestinal transit conditions （pH 8.0, with or without 0.3% bile salts） was tested. The effects of HZ0301 and HZ0302 on the viability and permeability of intestinal epithelial cell in primary culture of tilapias, Oreochrornis nilotica, were also detected. All the treatments were deter- mined with three replicates. The simulated gastric transit tolerance of HZ0301 and HZ0302 strains was pH-dependent and correspondingly showed lower viability at pH 2.0 after 180 min compared with pH 3.0 and pH 4.0. Both HZ0301 and HZ0302 were tolerant to simulated small intestine transit with or without bile salts in our research. Moreover, there was no significant difference （P〉0.05） among three treatments including the control and the groups treated with HZ0301 or HZ0302 both in intestinal epithelial cell viability and membrane permeability, showing no cell damage. In summary, this study demonstrated that HZ0301 and HZ0302 had high capacity of upper gastrointestinal transit tolerance and were relatively safe for intestinal epithelial cells of tilapias.

Studies on Proliferationpromoting Effect of Supernatant from Human Thymic Epithelial Cell Culture on Mouse Thymocyte and Tcell Line

Supernatants from human primary thymic epithelial cell culture were collected. The proliferationpromoting effect of the supernatant on mouse thymocyte, as well as on Tcell line 85, Be 13, HDMar, Peer, Loucy, Molt4, Reh and Jurkat, were observed. The results demonstrated that the supernatants could increase spontaneous 3 HTdR intake of mouse thymocyte, promote ConAinduced thymocyte proliferation and stimulate the proliferation of 85 cell or the cells of HDMar, Loucy and Jurkat at stationary phase, but did not display any effect on Be13, Peer, Molt4 and Reh cells.

3D （Three-Dimensional） Caco-2 Spheroids： Optimized in vitro Protocols to Favor Their Differentiation Process and to Analyze Their Cell Growth Behavior

3D （Three-dimensional） Caco-2 spheroids closely recapitulating in vivo physiological organization of intestinal epithelial cells, provide an excellent in vitro model system to study their pathophysiology and their response to stressful stimuli. The objective of this technical note is to provide optimized in vitro experimental protocols for culturing 3D Caco-2 spheroids and for analyzing their cell growth features. An optimized 3D Caco-2 spheroid culturing technique based on a new configuration of the culture medium is provided A methodological approach to determine the distribution of the cell cycle phases in disaggregated Caco-2 spheroids by using cytofluorimetric analysis is also described. The optimized culturing protocol favors 3D Caco-2 spheroid differentiation process, as evaluated by the number of well-differentiated spheroids with a single hollow lumen. The cytofluorimetric analysis allows rapid collection of cell cycle phase data from high numbers of spheroid samples, thus, permitting to estimate their growth dynamics in a relatively short time. The optimized technical approaches described here can be applied in systematic manner to a variety of research activities utilizing 3D Caco-2 spheroids. Ease of use, time and economic saving advantages deriving from these protocols further highlight their potential.

Studies on Biological Characteristics of Rabbit Oral Mucosal Epithelial Cells

Objective:The biological characteristics of rabbit oral mucosal epithelial cells(OMECs) were studied and the suitable procedure for the culture of OMECs was explored.Methods:Cultured OMECs by different surface disinfection,by different Dispase Ⅱ preparation,in different medium,in different calciumion concentration of K-SFM medium.Results:Microbial contamination of cell culture could significantly reduce with 2% iodine solution used for sterilization of oral cavity before cell culture.The OMECs digested with Dispase Ⅱin K-SFM could attach to the culture flask quickly,the OMECs digested with Dispase Ⅱ in PBS rare attached to the culture flask.The proliferations of OMECs cultured in 0 or 0.09 mmol/L calcium concentrations of K-SFM medium showed not statistically significant.the OMECs grew fast with serum,but it was easy to differentiate into fibroblast-like cells.Conclusion:OMECs could grow well and had the typical oral mucosal epithelial cell morphology by the procedure that described in this experiments.