pEGFP-ORF2质粒在Vero细胞中的有效表达

目的：确立pEGFP—ORF2质粒在不同细胞密度,不同转染时间,及血清存在与否时在细胞中的有效表达,为进一步研究HSV-2LATORF2对细胞的作用,及阐明HSV-2潜伏复发机制打下基础。方法：采用脂质体法将pEGFP—ORF2与pEGFP—C2两个真核表达载体转染Vero细胞,观察荧光蛋白表达。结果：有无血清对转染效果无明显差异。在细胞密度为4．0×10^4cells／ml,转染后84hEGFP表达量较其它条件下高,且在相同条件下pEGFP—ORF2与pEGFP—C2的表达无明显差异。结论：血清对表达效果的影响不大,细胞的接种密度及转染时间对转染有明显作用,以荧光蛋白作为标签鉴定蛋白表达的方法切实可行。     

Vero细胞最佳培养体系筛选

为了筛选出适合Vero细胞生长的最佳条件,本研究分别对Vero细胞的培养液、传代比例和接种浓度进行了优化.首先将Vero细胞在不同组合培养液中进行连续3代传代培养,筛选出适宜Vero细胞连续稳定传代的最佳培养液组成.然后分别在不同的传代比例和接种浓度下进行连续3代培养,筛选出适宜Vero细胞连续稳定传代的最佳传代比例和接种浓度.最终结果显示,Vero细胞在1.296%MEM溶液与特级胎牛血清的组合优于其他组合,可满足Vero细胞连续稳定传代.并且在该培养基的培养下,1∶6的传代比例为最适宜Vero细胞的传代比例,3×10^4/cm^2的接种密度为最适宜Vero细胞的接种密度.本研究最终完成了Vero细胞最佳培养体系条件筛选,并证实该条件满足Vero细胞连续稳定传代.     

Vero细胞微载体培养的化学成分明确无血清培养基的设计

目的：设计适用于Vero细胞微载体培养的化学成分明确无血清培养基。方法：以商品化的DMEM／F12合成培养基为基础培养基,应用Plackett—Burman实验设计和响应面分析法设计支持Vero细胞微载体培养的化学成分明确无血清培养基。结果：以细胞密度为评价指标,在单因素实验的基础上采用Plackett-Burman实验设计考察10种培养基添加成分对Vero细胞生长的影响,确定了3种对Vero细胞生长起明显促进作用的培养基添加成分,为胰岛素、血清素和腐胺。继而利用响应面法分析了这3种添加成分的最佳水平范围,设计了一种支持Vero细胞贴附培养的无血清培养基（VERO—SFM—A）。在Bellco搅拌式培养瓶中采用VERO-SFM．A和Cytodex1微载体培养Vero细胞,细胞密度由接种时的4×10^5cells／ml增加到培养6d后的22．3×10^cells／ml,细胞活力保持在96％以上。结论：VERO—SFM—A能够有效地支持Vero细胞在微载体表面固定化生长并达到较高的细胞密度,具有实际应用于Vero细胞微载体规模化培养的应用潜力。     

Vero及Vero-dst细胞对犬瘟热病毒敏感性比较

目的为了更好地分离犬瘟热病毒（CDV）并确诊犬瘟热,本实验比较了Vero及Vero-dst细胞对此病毒的敏感性。方法将CDV标准毒株Snyder Hill株及临床犬瘟热阳性犬组织匀浆分别接种Vero及Vero-dst两种细胞,通过观察细胞病变、检测病毒滴度（TCID50）,并通过RT-PCR法进行比较,分析两种细胞对CDV的敏感性。结果接种病毒后Vero细胞盲传5代始终未见细胞病变,而Vero-dst细胞12 h出现了明显的合胞样细胞病变,且RT-PCR扩增出了CDV基因特异性片段。结论 Vero-dst细胞对CDV表现了良好的敏感性,是体外分离培养CDV的一个有效细胞系。而所本实验中使用的Vero细胞并不适于CDV的分离与培养。另外,本实验利用Vero-dst细胞从临床犬瘟热阳性病例中成功分离到了野毒株,并确定其毒力较标准毒株毒力强,可用于进一步的研究。     

Globotriaosyl ceramide receptor function - Where membrane structure and pathology intersect

The glycosphingolipid globotriaosyl ceramide, (Galα1-4Galß1-4 glucosyl ceramide-Gb₃) also known as CD77 and the P^k blood group antigen, is bound by both verotoxins and by the HIV adhesin, gp120. Gb₃ plays an important receptor role in VT induced hemolytic uremic syndrome (HUS) and HIV infection. The organization of glycolipids, including Gb₃, into lipid rafts is central to both pathologies. The fatty acid heterogeneity within the Gb₃ lipid moiety plays a central role in assembly within such ordered domains. Differential binding of verotoxins and gp120 to such Gb₃ isoforms in model and cell membranes indicates a significant role in the eventual pathogenic outcome. HUS may provide the first example whereby membrane Gb₃ organization provides a predictor for tissue selective in vivo pathology.     

A new material of cryopreserving cell samples

Cryopreservation was first studied in early twentieth century, and the cryopreservation of cell and tissue samples has become an inseparable process in biology research labs, helping scientists to store living materials and to accumulate specimens.Recently, a new and simplified cryopreservation product, BioFlash Drive™ SP developed by a US firm Fibulas, came to the attention of many researchers. It integrates the sample container with control-released cryoprotectant, and the container itself can achieve slow-freezing in deep freezers. This means no reagents mixing or extra steps for slow-freezing are needed. The design of this product aims to simplify the current cell-freezing procedures and reduce the lengthy and error prone processes.In this research, we compared the post-thaw cell viability using two of the most widely used protocols, with the one with BioFlash Drive™ SP (Fibulas). Cell lines tested in this research include Vero (ATCC® CCL-81™) and HEp-2 (ATCC® CCL-23™). Results show there is not statistically significant difference in cell viability between the conventional protocols and the new protocols. However, this new protocol is less manual and less time consuming. With this new method, it might be possible for researchers to archive research progress more often, because saving cell can be an easier but still reliable experience.     

Lipid rafts are involved in SARS-CoV entry into Vero E6 cells

Lipid rafts often serve as an entry site for certain viruses. Here, we report that lipid rafts in Vero E6 cells are involved in the entry of severe acute respiratory syndrome coronavirus (SARS-CoV). Infectivity assay showed the integrity of lipid rafts was required for productive infection of pseudotyped SARS-CoV. Depletion of plasma membrane cholesterol with MβCD relocalized raft-resident marker caveolin-1 as well as SARS-CoV receptor ACE2 to a nonraft environment, but did not significantly change the surface expression of ACE2. MβCD-treatment inhibited infectivity of pseudotyped SARS-CoV by 90%. Biochemical fractionation and confocal imaging confirmed that ACE2 colocalized with raft-resident markers. Furthermore, an ectodomain of SARS-CoV S protein (S1188HA) could associate with lipid rafts after binding to its receptor, and colocalize with raft-resident marker ganglioside GM1. The binding of S1188HA was not affected by depleting plasma membrane cholesterol. Taken together, our results support that lipid rafts serve as an entry port for SARS-CoV.     

狂犬病毒aG株在Vero细胞上传代适应研究

为研制有效、安全和稳定的Vero细胞狂犬病疫苗提供实验室基础资料。采用狂犬病固定毒4aG株在Vero细胞上进行传代适应,同时对该毒株在Vero细胞上的增殖条件,病毒液的回收方法进行研究。结果显示,狂犬病固定毒4aG株在Vero细胞上多次传代后获得一株Vero细胞适应株(4aG-V株),该毒株的毒力可达8.50 logLD50/ml,且具有很好的抗原性及免疫原性。结果表明,感染Vero细胞最适种毒比例为1∶103,病毒滴度随着时间的延长而增强到第12天后逐渐减弱,且采用低温冻融破碎法回收的病毒液滴度优于直接收液法。4aG-V株在Vero细胞上维持时间长,可连续收液,有望其生产高滴度的狂犬病毒液。     

Non‐hemolytic enterotoxin of Bacillus cereus induces apoptosis in Vero cells

Bacillus cereus is an opportunistic pathogen that often causes foodborne infectious diseases and food poisoning. Non‐hemolytic enterotoxin (Nhe) is the major toxin found in almost all enteropathogenic B. cereus and B. thuringiensis isolates. However, little is known about the cellular response after Nhe triggered pore formation on cell membrane. Here, we demonstrate that Nhe induced cell cycle arrest at G₀/G₁ phase and provoked apoptosis in Vero cells, most likely associated with mitogen‐activated protein kinase (MAPK) and death receptor pathways. The influx of extracellular calcium ions and increased level of reactive oxygen species in cytoplasm were sensed by apoptosis signal‐regulating kinase 1 (ASK1) and p38 MAPK. Extrinsic death receptor Fas could also promote the activation of p38 MAPK. Subsequently, ASK1 and p38 MAPK triggered downstream caspase‐8 and 3 to initiate apoptosis. Our results clearly demonstrate that ASK1, and Fas‐p38 MAPK‐mediated caspase‐8 dependent pathways are involved in apoptotic cell death provoked by the pore‐forming enterotoxin Nhe.     

转瓶培养与生物反应器微载体培养乙脑病毒的比较

分别用15L转瓶与15L生物反应器微载体(2.5g/L CytodexⅢ)系统培养Vero细胞并接种乙型脑炎病毒(简称乙脑病毒)。转瓶培养Vero细胞7~8d,细胞数最高能达到8×108;当单层细胞长至3.0~4.5×108时接种乙脑病毒,病毒滴度能达到6.5~6.98 lg PFU/ml,并能够连续收获4~5次;采用微载体系统培养Vero细胞,细胞密度最高能达到170×108;当单层细胞长至60~70×108时接种乙脑病毒,病毒滴度能达到7~7.5 lg PFU/ml,并能够连续收获13~15次。两种方式培养的乙脑病毒收获液分别经灭活、浓缩、柱层析纯化后制备Vero细胞乙脑纯化疫苗,各项检定指标均符合《中国药典》的相关要求。