miRNA93和miRNA192在H3N2亚型SIV病毒复制及宿主抗病毒免疫中的作用

目的 探讨分离于广西猪流感病毒SW/Guangxi/NS2783/2010和SW/Guangxi/NS650/2012跨种属感染人肺腺癌A549细胞的miRNA93和miRNA192对病毒复制及宿主抗病毒免疫的影响。方法 通过测定不同稀释浓度的病毒感染细胞HA滴度值,确定病毒最佳稀释浓度。荧光定量PCR检测病毒感染细胞后miRNA93和miRNA192表达,Western blot检测病毒NP和HA蛋白表达水平;转染miRNA93和miRNA192抑制剂后,重新检测miRNA93、miRNA192、IFN-β及病毒NP、HA蛋白表达水平。结果 不同稀释度病毒感染人A549细胞后HA滴度的结果显示病毒SW2783的最佳稀释度为10^-3,而SW650的HA滴度无明显变化趋势,提示病毒SW2783对人A549细胞具有较好的适应性和感染能力。荧光定量PCR和Western blot结果提示两株病毒感染细胞后病毒NP和HA蛋白表达均先升高后降低,加入miRNA93抑制剂,两株病毒NP和HA蛋白的表达均上调;加入miRNA192抑制剂,病毒SW2783的HA蛋白表达下调（P=2.10×10^-4）,而SW650的HA蛋白表达上调（P=5.45×10^-5）,NP蛋白反而下调（P=0.034）;ELISA结果提示病毒SW2783和SW650感染细胞后炎症因子IFN-β表达水平随感染时间延长而升高。结论 研究表明miRNA93和miRNA192的表达与SIV病毒增殖及宿主抗病毒免疫有关,可作为干预猪流感病毒跨种属感染的新靶点。     

新城疫病毒D817对裸小鼠人结肠癌移植瘤的抑制作用

Objective To study the anti-tumor effects of Newcastle disease virus (NDV) strain D817 on human colon carcinoma model in nude mice. Methods The nude mouse model of human colon carcinoma was established by subcutaneous inoculation of human colon cancer LOVO cells. The tumor-bearing mice were given PBS, 5-Fu, high-dose NDV D817, moderate-dese NDV D817 or low-dose NDV D817 via caudal vein injection. The tumor size and weight of mice were measured. The liver damages were examined by histopathology. Apoptosis and necrosis of tumor ceils were detected by flow cytometry. The endotumoral content of TNF-α was detected using a mouse TNF-a ELISA kit. The live vires was detected by bemagglutination (HA) test. Results The moderate-dose NDV D817 inhibited the tumor growth more apparently than 5-Fu. The tumor growth inhibition rate reached to 48.1%. The liver damage and the weight change caused by NDV were less severe. NDV D817 made an increased apoptosis index and induced production of TNF-α. Live virus was not detected in important organs except in the tumor of nude mice by HA test. Conclusion In the anti-tumor process in nude mice bearing xenografts of human colon carcinoma, a suitable dose of NDV D817 is more safe and effective.     

新城疫病毒D817对裸小鼠人结肠癌移植瘤的抑制作用

Objective To study the anti-tumor effects of Newcastle disease virus (NDV) strain D817 on human colon carcinoma model in nude mice. Methods The nude mouse model of human colon carcinoma was established by subcutaneous inoculation of human colon cancer LOVO cells. The tumor-bearing mice were given PBS, 5-Fu, high-dose NDV D817, moderate-dese NDV D817 or low-dose NDV D817 via caudal vein injection. The tumor size and weight of mice were measured. The liver damages were examined by histopathology. Apoptosis and necrosis of tumor ceils were detected by flow cytometry. The endotumoral content of TNF-α was detected using a mouse TNF-a ELISA kit. The live vires was detected by bemagglutination (HA) test. Results The moderate-dose NDV D817 inhibited the tumor growth more apparently than 5-Fu. The tumor growth inhibition rate reached to 48.1%. The liver damage and the weight change caused by NDV were less severe. NDV D817 made an increased apoptosis index and induced production of TNF-α. Live virus was not detected in important organs except in the tumor of nude mice by HA test. Conclusion In the anti-tumor process in nude mice bearing xenografts of human colon carcinoma, a suitable dose of NDV D817 is more safe and effective.     

小鼠腹腔注射流感病毒后外周血IFN-γTNF-α和IL-2的变化及意义

目的 观察和比较小鼠腹腔注射人流感病毒H3N2和禽流感病毒H9N2后外周血干扰素γ(interferon-γ,IFN-γ)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和白细胞介素2(interleukin-2,IL-2)质量浓度的变化,探讨其变化规律.方法 采用腹腔注射方式给小鼠注射人流感病毒H3N2、禽流感病毒H9N2或无病毒尿囊液,分离血清,用ELISA法检测注射后第1、2、3、4和5天外周血IFN-γ、TNF-α和IL-2质量浓度.分别比较人流感病毒H3N2和禽流感病毒H9N2注射组与阴性对照组在不同时间的IFN-γ、TNF-α和IL-2质量浓度.用方差分析和LSD-t检验进行统计学分析.结果 在某些时间段,病毒注射组外周血IFN-γ和IL-2质量浓度与阴性对照组问的差别有统计学意义,而病毒注射组TNF-α质量浓度与阴性对照组间的差别则无统计学意义.结论 小鼠腹腔注射人流感病毒H3N2和禽流感病毒H9N2可促进IFN-γ和IL-2产生,而对TNF-α产生则无显著影响.     

新城疫病毒D817对裸小鼠人结肠癌移植瘤的抑制作用

目的 观察新城疫病毒DK/HK/817/1980(NDV D817)对裸小鼠人结肠癌移植瘤的抑制作用.方法 采用LoVo细胞株建立裸小鼠人结肠癌移植瘤模型,分别给予裸小鼠尾静脉注射PBS、氟尿嘧啶(5-Fu)以及NDV D817高、中、低3个剂量,观察不同剂量NDV D817对移植瘤的抑制作用,病理学观察NDV D817对移植瘤和肝细胞的损伤程度,流式细胞术检测肿瘤细胞的凋亡和坏死情况,酶联免疫吸附试验(ELISA)试剂盒检测荷瘤小鼠体内肿瘤坏死因子-α(TNF-α)的含量,血凝实验检测荷瘤小鼠体内活病毒量.结果 中剂量NDV D817可明显抑制移植瘤的生长,肿瘤抑制率达48.1%.NDV D817对荷瘤小鼠体重和肝脏的损伤较小,且具有诱导肿瘤细胞凋亡和诱导机体产生TNF-α的能力.荷瘤小鼠处死后只在瘤内检测到活病毒,而在血清和其他脏器中并没有检出活病毒.结论 在对裸小鼠人结肠癌移植瘤的抑制过程中,NDV D817有明显的抑瘤效果,适当剂量的NDVD817更安全有效.     

NDV7793促进人CD4~+T细胞表达TRAIL的实验研究

目的:研究新城疫病毒(Newcastle disease virus,NDV)弱毒株NDV7793能否促进人CD4+T细胞表达肿瘤坏死因子相关凋亡因子诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)。方法:首先用免疫磁珠分选法(magnetic activated cell sorting,MACS)分选外周血静止CD4+T细胞,然后加抗CD3抗体、抗CD28抗体和白细胞介素-2(interleukin-2,IL-2)使之成为能被NDV激活的CD4+T细胞。用流式细胞技术(flow cytometry,FCM)检测NDV刺激的CD4+T细胞的TRAIL的表达水平。结果:MACS法分选外周血得到的CD4+T细胞纯度达到(97.38±0.28)%;能被NDV激活的CD4+T细胞表面分子CD25和CD69双阳性表达率可达(29.30±1.08)%,与PBS阴性对照组(2.40±1.30)%相比,差异有统计学意义(P0.05);FCM检测结果显示,与对照组比较,NDV7793刺激的CD4+T细胞TRAIL表达水平均有显著升高,且在NDV7793效价为25 HU时达到最大值。结论:NDV7793可刺激CD3抗体、CD28抗体和IL-2预先活化的CD4+T细胞表达TRAIL。     

Changes and their significances of IFN-γ, TNF-α and IL-2 level in mouse peripheral blood following intraperitoneal injection with influenza viruses

Objective To observe changes of IFN-γ, TNF-α and IL-2 levels in mouse peripheral blood following intraperitoneal injection with influenza viruses. Methods Mice were divided into 3 groups randomly and injected with human H3N2 influenza virus, avian H9N2 influenza virus and sterilized virus-free allantoic fluid, respectively. Sera in different groups were collected at several time points after virus injection, and the levels of IFN-γ, TNF-α and IL-2 in peripheral blood were tested by sandwich ELISA. Statistical analysis was made using analysis of variance and LSD-t test. Results The levels of IFN-γ and IL-2 in peripheral blood in influenza viruses injection groups were significantly higher than those in negative control group, and no significant difference in TNF-α level was found between influenza viruses injection groups and negative control group. Conclusion Intraperitoneal injection with human or avian influenza viruses can promote the production of IFN-γ and IL-2 in mouse peripheral blood, but it has little effect on the production of TNF-α.     

新城疫病毒7793株对人结肠癌细胞的杀伤作用

目的:研究新城疫病毒NDV7793对人结肠癌细胞的体外杀伤作用,为结肠癌的生物疗法奠定基础。方法:通过蚀斑实验纯化病毒并测定纯化的NDV7793株的感染力;用乳酸脱氢酶微量释放法测定纯化病毒对人LoVo和Ls174t结肠癌细胞株的杀伤作用并且通过血凝实验测定病毒在不同细胞中的增殖力。结果:NDV7793在感染细胞96h后出现直径约为0.5mm左右的空斑,PFU为1.25×107个/ml,为弱毒株;NDV7793对LoVo和Ls174t人结肠癌细胞株有明显的杀伤作用,而且杀伤作用的强度与病毒作用的时间和病毒的浓度呈正相关的关系;NDV7793可以在肠癌细胞中生长复制,该病毒株在人结肠癌细胞株LoVo的复制能力强于Ls174t。结论:NDV7793具有较强的选择性杀伤人结肠癌细胞的作用,且为弱毒株,这株病毒具备肿瘤生物治疗的潜能。     

NDV7793促进人CD4＋T细胞表达TRAIL的实验研究

目的：研究新城疫病毒（Newcastle disease virus,NDV）弱毒株NDV7793能否促进人CD4＋T细胞表达肿瘤坏死因子相关凋亡因子诱导配体（TNF-related apoptosis-inducing ligand,TRAIL）。方法：首先用免疫磁珠分选法（magnetic activated cell sorting,MACS）分选外周血静止CD4＋T细胞,然后加抗CD3抗体、抗CD28抗体和白细胞介素-2（interleukin-2,IL-2）使之成为能被NDV激活的CD4＋T细胞。用流式细胞技术（flow cytometry, FCM）检测NDV刺激的CD4＋T细胞的TRAIL的表达水平。结果：MACS法分选外周血得到的CD4＋T细胞纯度达到（97.38±0.28）%;能被NDV激活的CD4＋T细胞表面分子CD25和CD69双阳性表达率可达（29.30±1.08）%,与PBS阴性对照组（2.40±1.30）%相比,差异有统计学意义（P〈0.05）;FCM检测结果显示,与对照组比较, NDV7793刺激的CD4＋T细胞TRAIL表达水平均有显著升高,且在NDV7793效价为25 HU时达到最大值。结论：NDV7793可刺激CD3抗体、CD28抗体和IL-2预先活化的CD4＋T细胞表达TRAIL。     

新城疫病毒对人NK细胞表达TRAIL的促进作用

目的:研究新城疫病毒(Newcastle disease virus,NDV)刺激自然杀伤(natural killer,NK)细胞产生肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)的作用机制。方法:采用免疫磁珠分选(magnetic activated cell sorting,MACS)法,分离得到高纯度的NK细胞;乳酸脱氢酶释放法检测NDV激活的NK细胞对人肝癌HepG2细胞的杀伤作用;实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)检测NDV刺激NK细胞4h后对TRAILmRNA表达水平的影响;FCM检测NDV刺激NK细胞16h时TRAIL蛋白的表达情况;ELISA法检测NDV刺激NK细胞后对其分泌干扰素γ(interferon-γ,IFN-γ)的影响;最后采用抗IFN-γ抗体中和IFN-γ后,再采用FCM检测NDV刺激的NK细胞中TRAIL蛋白的表达情况。结果:FCM检测结果显示,分离得到的NK细胞的纯度达(90.60±1.15)%;NDV刺激NK细胞16h后,明显增强NK细胞的杀伤肿瘤作用,NDV效价为204.8HU刺激组的杀伤率达到(22.28±0.84)%;RFQ-PCR及FCM检测结果显示,不同效价的NDV刺激NK细胞后,NK细胞中TRAILmRNA及蛋白表达水平均明显升高;ELISA检测结果显示,NDV刺激NK细胞后IFN-γ的分泌水平明显增加,在NDV效价为25.6HU刺激16h时达到峰值(796.47±37.87)pg/mL;采用抗IFN-γ抗体中和IFN-γ后,NK细胞中TRAIL蛋白表达水平明显下降。结论:NDV刺激NK细胞后可增强IFN-γ的分泌水平,进而通过IFN-γ上调TRAIL的表达,此途径为NDV刺激NK细胞表达TRAIL的机制之一。此外,NDV直接刺激NK细胞表达TRAIL是另一条重要的途径。