融合细胞株Eahy926是人肺腺癌细胞株A549和人脐静脉内皮细胞杂交而成的永生化细胞株,具有血管内皮细胞的特性,已广泛用于内皮细胞相关研究.本研究应用蛋白质组学技术分析融合细胞株Eahy926与亲本人肺腺癌细胞株A549的蛋白质差异表达,探...融合细胞株Eahy926是人肺腺癌细胞株A549和人脐静脉内皮细胞杂交而成的永生化细胞株,具有血管内皮细胞的特性,已广泛用于内皮细胞相关研究.本研究应用蛋白质组学技术分析融合细胞株Eahy926与亲本人肺腺癌细胞株A549的蛋白质差异表达,探讨融合细胞生物学特性变化及其机制.对Eahy926和亲本A549的细胞总蛋白质进行双向凝胶电泳,在PDQuest软件辅助下找出差异表达蛋白质点,经肽质量指纹图谱(peptide mass fingerprinting,PMF)和串级质谱(tandem massspectrometry,TMS)分析,SWISS-PROT数据库检索,成功鉴定出28个差异蛋白,如CATB、CK8、CK18、annexin A2、GRP78、HSP90、HSP60、vimentin等一些与分子伴侣、氧化应激、能量代谢、信号转导等有关,并与肿瘤细胞分裂增殖、分化凋亡、侵袭转移、免疫逃逸以及肿瘤血管生长密切关联的蛋白质.研究发现,融合细胞株Eahy926和人肺腺癌细胞株A549的蛋白质组表达谱存在明显差异,这将有助于今后进一步探讨肿瘤细胞与内皮细胞的相互作用机制及其融合细胞的特性,筛选肿瘤增殖和转移相关蛋白质及分子标志物,亦可为肿瘤的抗血管生成治疗提供新思路.展开更多
目的观察胱硫醚γ裂解酶(CSE)siRNA转染人脐静脉内皮EAhy926细胞后CSE的表达及对H2S的影响。方法体外培养EAhy926细胞,经siRNA转染以沉默CSE基因表达,分为5组进行培养:正常对照组(A组):只加入转染试剂;阴性对照组(B组):转染Neg-siRNA;转...目的观察胱硫醚γ裂解酶(CSE)siRNA转染人脐静脉内皮EAhy926细胞后CSE的表达及对H2S的影响。方法体外培养EAhy926细胞,经siRNA转染以沉默CSE基因表达,分为5组进行培养:正常对照组(A组):只加入转染试剂;阴性对照组(B组):转染Neg-siRNA;转染CSEsiRNA1组(C组);转染CSEsiRNA2组(D组);转染CSEsiRNA3组(E组)。采用Lipofectamine 2000介导转染EAhy926细胞,在荧光显微镜下观察细胞以检测转染效率;Western blot法检测CSE蛋白表达;收集细胞检测H2S含量。结果观察转染效率为60%左右;A组CSE蛋白表达为(153.41±23.43),B组24 h CSE蛋白表达为(150.22±17.56),而D组24 h CSE蛋白表达降至(59.43±21.81),差异有统计学意义(P<0.01);并且A组H2S含量为(0.96±0.05),B组24 h H2S含量为(0.95±0.06),D组24 h H2S含量降至(0.54±0.08),差异有统计学意义(P<0.01)。结论 CSE-siRNA能够有效地抑制EAhy926中CSE的表达,并且CSE-siRNA2沉默作用最强。展开更多
Based on the physiological structure of the intestine, a Caco-2/EAhy926 tandem compound model was constructed in order to simulate the intestinal-vascular barrier. This model was applied in the study of transcytosis o...Based on the physiological structure of the intestine, a Caco-2/EAhy926 tandem compound model was constructed in order to simulate the intestinal-vascular barrier. This model was applied in the study of transcytosis of nanoparticles, and it was compared with the traditional intestinal cell model in the whole study. Briefly, Fe3O4 nanoparticles with a size about 30 nm were used as model nanoparticles, which remained steady during transcytosis. The nanoparticles hardly had cytotoxicity to Caco-2 cells and EAhy926 cells within the incubation concentrations. The cell tandem model was established by connecting upper Caco-2 monolayer and lower EAhy926 monolayer. Based on the FD4 permeability or TEER, all cell models remained integrity within certain period of culture time. The expression of Claudin-4 or VE Cadherin demonstrated the presence of tight junctions. The intact morphology of microfilament F-actin indicated the favorable intracellular connection. It was found that the two-layer cell tandem model created a bigger barrier for the transcytosis of FD4 than Caco-2 and EAhy926 monolayer models, and the translocation of Fe3O4 nanoparticles showed a similar pattern. Interestingly, we found that the main barrier of tandem model for nanoparticles was caused by the upper Caco-2 cell monolayer, while the lower layer of EAhy926 monolayer remained high permeability. Generally, the cell tandem compound model established here enabled us to evaluate the impact of both intestinal epithelial and endothelial layer on transcytosis, and it might provide a novel approach to study bio-nano interaction in the intestine.展开更多
本研究旨在制备和鉴定小鼠抗Ⅱ型登革病毒（DENV‐2）10种蛋白的抗体，为后续相关研究提供实验材料。利用真核表达载体pReceiver构建DENV‐210种蛋白的重组质粒，提取质粒 DNA ，肌内注射免疫小鼠，共免疫4次。末次免疫后2周取小鼠血清...本研究旨在制备和鉴定小鼠抗Ⅱ型登革病毒（DENV‐2）10种蛋白的抗体，为后续相关研究提供实验材料。利用真核表达载体pReceiver构建DENV‐210种蛋白的重组质粒，提取质粒 DNA ，肌内注射免疫小鼠，共免疫4次。末次免疫后2周取小鼠血清，利用DENV‐2感染的Vero细胞和DENV‐2各蛋白的稳定表达细胞，通过酶联免疫吸附试验（ELISA）、间接免疫荧光法（IFA）和蛋白免疫印迹法评价免疫效果，分析抗体的特点。DNA免疫小鼠后获得抗DENV‐210种蛋白的抗血清，抗体效价波动于1∶400～1∶16127之间，以抗E蛋白抗体效价最高，达1∶16127，而抗NS3、NS4b、NS5蛋白抗体效价较低，仅为1∶400。利用DENV‐2感染的Vero细胞和稳定表达病毒蛋白的EAhy926细胞进行IFA染色，抗DENV‐2各蛋白的抗血清均可特异性识别DENV‐2抗原。蛋白免疫印迹结果显示，抗E、NS1、NS4b和NS5蛋白抗体能识别热变性蛋白，其他抗血清未呈现阳性反应条带。本研究提示，DNA免疫小鼠所获得的抗DENV‐2各蛋白抗体能特异性识别自然感染或模拟自然感染状态下的DENV‐2蛋白，可为后续相关研究提供工具，也表明DNA免疫法可作为抗体制备的一种策略。展开更多
文摘融合细胞株Eahy926是人肺腺癌细胞株A549和人脐静脉内皮细胞杂交而成的永生化细胞株,具有血管内皮细胞的特性,已广泛用于内皮细胞相关研究.本研究应用蛋白质组学技术分析融合细胞株Eahy926与亲本人肺腺癌细胞株A549的蛋白质差异表达,探讨融合细胞生物学特性变化及其机制.对Eahy926和亲本A549的细胞总蛋白质进行双向凝胶电泳,在PDQuest软件辅助下找出差异表达蛋白质点,经肽质量指纹图谱(peptide mass fingerprinting,PMF)和串级质谱(tandem massspectrometry,TMS)分析,SWISS-PROT数据库检索,成功鉴定出28个差异蛋白,如CATB、CK8、CK18、annexin A2、GRP78、HSP90、HSP60、vimentin等一些与分子伴侣、氧化应激、能量代谢、信号转导等有关,并与肿瘤细胞分裂增殖、分化凋亡、侵袭转移、免疫逃逸以及肿瘤血管生长密切关联的蛋白质.研究发现,融合细胞株Eahy926和人肺腺癌细胞株A549的蛋白质组表达谱存在明显差异,这将有助于今后进一步探讨肿瘤细胞与内皮细胞的相互作用机制及其融合细胞的特性,筛选肿瘤增殖和转移相关蛋白质及分子标志物,亦可为肿瘤的抗血管生成治疗提供新思路.
文摘目的观察胱硫醚γ裂解酶(CSE)siRNA转染人脐静脉内皮EAhy926细胞后CSE的表达及对H2S的影响。方法体外培养EAhy926细胞,经siRNA转染以沉默CSE基因表达,分为5组进行培养:正常对照组(A组):只加入转染试剂;阴性对照组(B组):转染Neg-siRNA;转染CSEsiRNA1组(C组);转染CSEsiRNA2组(D组);转染CSEsiRNA3组(E组)。采用Lipofectamine 2000介导转染EAhy926细胞,在荧光显微镜下观察细胞以检测转染效率;Western blot法检测CSE蛋白表达;收集细胞检测H2S含量。结果观察转染效率为60%左右;A组CSE蛋白表达为(153.41±23.43),B组24 h CSE蛋白表达为(150.22±17.56),而D组24 h CSE蛋白表达降至(59.43±21.81),差异有统计学意义(P<0.01);并且A组H2S含量为(0.96±0.05),B组24 h H2S含量为(0.95±0.06),D组24 h H2S含量降至(0.54±0.08),差异有统计学意义(P<0.01)。结论 CSE-siRNA能够有效地抑制EAhy926中CSE的表达,并且CSE-siRNA2沉默作用最强。
基金The National Basic Research Program of China(973 program,Grant No.2015CB932100)the National Basic Research Program of China(Grant No.2015CB932100)+1 种基金National Natural Science Foundation of China(Grant No.81690264)the Innovation Team of the Ministry of Education(Grant No.BMU20110263)
文摘Based on the physiological structure of the intestine, a Caco-2/EAhy926 tandem compound model was constructed in order to simulate the intestinal-vascular barrier. This model was applied in the study of transcytosis of nanoparticles, and it was compared with the traditional intestinal cell model in the whole study. Briefly, Fe3O4 nanoparticles with a size about 30 nm were used as model nanoparticles, which remained steady during transcytosis. The nanoparticles hardly had cytotoxicity to Caco-2 cells and EAhy926 cells within the incubation concentrations. The cell tandem model was established by connecting upper Caco-2 monolayer and lower EAhy926 monolayer. Based on the FD4 permeability or TEER, all cell models remained integrity within certain period of culture time. The expression of Claudin-4 or VE Cadherin demonstrated the presence of tight junctions. The intact morphology of microfilament F-actin indicated the favorable intracellular connection. It was found that the two-layer cell tandem model created a bigger barrier for the transcytosis of FD4 than Caco-2 and EAhy926 monolayer models, and the translocation of Fe3O4 nanoparticles showed a similar pattern. Interestingly, we found that the main barrier of tandem model for nanoparticles was caused by the upper Caco-2 cell monolayer, while the lower layer of EAhy926 monolayer remained high permeability. Generally, the cell tandem compound model established here enabled us to evaluate the impact of both intestinal epithelial and endothelial layer on transcytosis, and it might provide a novel approach to study bio-nano interaction in the intestine.
文摘本研究旨在制备和鉴定小鼠抗Ⅱ型登革病毒（DENV‐2）10种蛋白的抗体，为后续相关研究提供实验材料。利用真核表达载体pReceiver构建DENV‐210种蛋白的重组质粒，提取质粒 DNA ，肌内注射免疫小鼠，共免疫4次。末次免疫后2周取小鼠血清，利用DENV‐2感染的Vero细胞和DENV‐2各蛋白的稳定表达细胞，通过酶联免疫吸附试验（ELISA）、间接免疫荧光法（IFA）和蛋白免疫印迹法评价免疫效果，分析抗体的特点。DNA免疫小鼠后获得抗DENV‐210种蛋白的抗血清，抗体效价波动于1∶400～1∶16127之间，以抗E蛋白抗体效价最高，达1∶16127，而抗NS3、NS4b、NS5蛋白抗体效价较低，仅为1∶400。利用DENV‐2感染的Vero细胞和稳定表达病毒蛋白的EAhy926细胞进行IFA染色，抗DENV‐2各蛋白的抗血清均可特异性识别DENV‐2抗原。蛋白免疫印迹结果显示，抗E、NS1、NS4b和NS5蛋白抗体能识别热变性蛋白，其他抗血清未呈现阳性反应条带。本研究提示，DNA免疫小鼠所获得的抗DENV‐2各蛋白抗体能特异性识别自然感染或模拟自然感染状态下的DENV‐2蛋白，可为后续相关研究提供工具，也表明DNA免疫法可作为抗体制备的一种策略。
