新城疫病毒HN基因对肝癌细胞SMMC7721的细胞毒性研究

目的:探讨新城疫病毒HN基因对肝癌细胞SMNC7721的杀伤作用及其机制.方法:以脂质体介导方法在体外转染含新城疫病毒HN基因的质粒pVHN于细胞SMMC7721 24 h后,采用MTT法检测细胞活性;3,5-二羟基甲苯测定其唾液酸含量的变化;丫啶橙/溴化乙锭(AO/EB)染色,荧光显微镜观察细胞形态学改变;以及FCM检测病毒HN基因和HLA-A,B,C的表达.结果:体外转染pVHN能显著地降低细胞表面唾液酸含量(P＜0.05),有效地杀伤肿瘤细胞SMMC7721;荧光显微镜观察可见典型的细胞死亡形态学改变;实验组细胞与对照组比较HN表达差异显著,HLA-A,B,C表达上调.结论:HN基因在体外能够显著降低肿瘤细胞表面唾液酸含量,同时,使其高表达HN抗原,上调HLA-A,B,C表达,从而,可能增强了肿瘤细胞的抗原性和免疫识别,诱导了细胞SMMC7721死亡.     

新城疫病毒HN基因诱导肝癌细胞SMMC7721凋亡的作用机制

目的 探索含新城疫病毒(NDV)HN基因的质粒pVHN诱导人肝癌细胞SMMC7721凋亡的作用及其机制。方法 以脂质体介导方法在体外转染pVHN于SMMC7721细胞24h后,通过MTT方法检测细胞活性状态;采用流式细胞仪(FCM)检测法,通过碘化丙啶(PI)染色测定细胞死亡率;以罗丹明123染色测定线粒体跨膜电位(△ψ)的改变;提取细胞基因组DNA进行电泳,检测DNA有无断裂;用底物颜色反应检测Caspase-3活性。结果 细胞SMMC7721在体外转染pVHN后,死亡率达50．0％(转染空载体质粒对照组为5．2％);细胞基因组DNA呈梯状谱带;线粒体△ψ下降,Caspase-3活性明显升高。结论 新城疫病毒HN基因在体外转染细胞SMMC7721,能明显诱导细胞SMMC7721凋亡,其发生机制可能是由于HN基因的导入引起线粒体△ψ下降,进而激活Caspase-3使细胞凋亡。     

抗肿瘤核酸疫苗的研究进展

抗肿瘤核酸疫苗的研究进展袁玫肿瘤疫苗的组成有两个基本要素：肿瘤抗原（特异或相关）和佐剂。其作用是激活抗原提呈细胞，进而活化Ｔ淋巴细胞，以克服肿瘤抗原性弱的问题。根据这一基本要求，目前已有多种疫苗构建成功，如自身肿瘤细胞加卡介苗（ＢＣＧ）、抗独特型抗体...     

联合应用新城疫病毒及其 HN基因对小鼠黑色素瘤抑制效应的研究

背景与目的:尽管新城疫病毒( Newcastle disease virus, NDV)对多种肿瘤具有抑制作用 ,但其抑瘤机制尚不完全清楚.以前的研究结果表明,该病毒的血凝素神经氨酸酶( hemagglutinin-neuraminidase,HN)基因在该病毒的抗肿瘤作用上发挥重要作用且 HN蛋白表达后能够定位于肿瘤细胞膜上,以 HN蛋白作为肿瘤细胞的外源抗原来研究其抑瘤作用尚未见报道.本研究拟探讨 HN蛋白作为外源抗原在新城疫病毒抗肿瘤的作用以及二者联合应用对小鼠黑色素瘤的抑制效应.方法:在 C57BL/6小鼠右后肢皮下接种 B16黑色素瘤细胞 2× 105个.荷瘤第 2天,左后肢肌肉注射含新城疫病毒 HN基因的重组质粒,荷瘤第 7天,瘤内注射新城疫病毒 2× 109pfu;同时设单独 HN基因或 NDV治疗组及注射 PBS的对照组.通过抑瘤率观察动物体内的抑瘤效果;通过特异性细胞毒 T淋巴细胞( cytotoxic T lymphocyte,CTL)实验, ICAM Ⅰ、 CD48及新城疫病毒 HN蛋白在肿瘤细胞表面表达检测,探讨 HN蛋白所介导的抑瘤作用.结果:联合应用新城疫病毒及该病毒 HN基因对肿瘤的抑制效果明显好于单独 HN基因及新城疫病毒,抑瘤率达 82.8%,特异性 CTL反应增强,对靶细胞的杀伤率为 18.4%;单独 HN基因及新城疫病毒治疗组的抑瘤率分别为 56.6%、 41.0%,特异性 CTL活性分别为 4.4%、 10.1%;瘤内注射 NDV的肿瘤细胞表面检测到 HN分子的表达, ICAM Ⅰ、 CD48分子表达上调.结论:定位于肿瘤细胞表面的 HN蛋白介导机体对肿瘤细胞的特异性杀伤,联合应用新城疫病毒及该病毒 HN基因显著增强了机体对肿瘤细胞的杀伤效应.     

基于SFV RNA复制子核酸疫苗pIRSFV HN的构建及其体内外抑瘤作用

目的:构建基于塞姆利基森林病毒（Semliki forest virus,SFV）RNA复制子和新城疫病毒HN基因的核酸疫苗pIRSFVHN，并探讨其体内外的抑瘤作用。〖HT5W〗方法：基于SFV RNA复制子和新城疫病毒HN基因构建核酸疫苗pIRSFVHN，pIRSFVHN转染人结肠癌细胞HCT116，以West     

基因枪介导的neu基因DNA疫苗抗肿瘤作用的实验研究

目的 探讨DNA疫苗pWRG-neu的皮内免疫,对高表达neu基因的小鼠移植瘤生长和转移的抑制作用。方法 向小鼠黑色素瘤B16F10细胞系转染pcDNA-neu,用有限稀释法筛选一株高表达neu基因的细胞株B16F10-neu。在基因枪介导下,向C57BL／6小鼠导入DNA疫苗pWRG-neu,通过观察免疫动物的生存期,评价DNA疫苗的抗肿瘤作用。分离免疫动物脾细胞,经自体淋巴细胞混合培养实验,分析DNA疫苗体内免疫后机体的CTL应答。结果 筛选到一株高表达neu基因的B16F10-neu细胞株,转基因过程和外源基因的表达没有改变细胞系的增殖特性。用基因枪轰击,进行DNA疫苗pWRG-neu皮内免疫,对小鼠黑色素瘤B16F10-neu进行预防、治疗和抗转移的实验研究,结果表明,DNA疫苗的免疫能够明显推迟移植瘤的生长,延长小鼠生存期,获得明显的抗肿瘤效果。DNA疫苗免疫后可诱导小鼠脾淋巴细胞CTL活性。结论 基因枪介导的DNA疫苗pWRG-neu经皮内免疫,能够有效的诱导机体的细胞免疫应答,预防和治疗小鼠移植瘤的发生,并有一定的预防肿瘤肺转移的作用。     

表达新城疫病毒HN基因重组鸡痘病毒的构建及其抑瘤作用

目的〖HT5”SS〗： 构建表达新城疫病毒（newcastle disease virus, NDV）HN基因的重组鸡痘病毒vFVHN,探讨vFVHN对体外培养人肝癌细胞 SMMC7721的抑制作用和对小鼠荷肝癌模型的体内抑瘤效果。〖HT5W〗方法〖HT5"SS〗： 以野生型鸡痘病毒（fowl poxvirus, FPV）282 E4株为载体,构建表达新城疫病毒HN基因的重组鸡痘病毒vFVHN。采用5溴2脱氧尿苷（5bromo2deoxyribouridine,BrdU）加压法筛选重组体,并通过RTPCR和Western blot等方法对其进行鉴定。采用噻唑蓝（methylthiazoletetrazolium,MTT）染色法检测vFVHN对人肝癌细胞SMMC7721的杀伤率,并通过检测抑瘤率观察其在C57BL/6小鼠荷肝癌模型的抑瘤效果。〖HT5W〗结果〖HT5"SS〗： 成功构建重组鸡痘病毒vFVHN,其对SMMC7721细胞的杀伤率为43.37%,对C57BL/6小鼠肝癌模型的抑瘤率为20.65%。〖HT5W〗结论〖HT5"SS〗： 重组鸡痘病毒vFVHN可有效杀伤体外培养的人肝癌细胞SMMC7721,并对C57BL/6小鼠荷肝癌模型体内的实体肿瘤有一定抑制作用。     

定位表达的新城疫病毒HN蛋白对荷瘤小鼠的抗肿瘤免疫作用

背景与目的 新城疫病毒HN蛋白是新城疫病毒产生溶瘤作用的重要免疫原.在前期体外实验基础 上,比较定位表达细胞不同部位的HN蛋白体内抗肿瘤免疫作用.方法 通过构建荷瘤小鼠,瘤内注射定位表达于 细胞不同部位的新城疫病毒HN蛋白,即胞浆型(Cy-HN)、跨膜型(M-HN)、分泌型(Sc-HN)重组真核表达质 粒,比较荷瘤小鼠的肿瘤生长速度,脾淋巴细胞增殖反应和细胞毒T细胞活性.结果 瘤内注射跨膜型重组真核表达 质粒的荷瘤小鼠肿瘤生长缓慢,与瘤内注射胞浆型和分泌型重组真核表达质粒的荷瘤小鼠相比有统计学差异(第18 天:P=0.022;第21天:P<0.01),同时,该组荷瘤小鼠的淋巴细胞增殖反应和细胞毒T细胞活性也较高[M-HN vs Cy- HN, P=0.019; M-HN vs Sc-HN, P=0.043; M-HN vs pcDNA3.1(+), P<0.01].结论 定位表达于细胞不同部位的HN蛋白体内 抗肿瘤免疫作用存在差异,跨膜型HN蛋白的重组DNA质粒能够提高荷瘤小鼠的特异性细胞免疫应答.     

异位hCGβ-CTP37基因疫苗的抗肿瘤作用

目的 :研究异位hCGβ CTP37基因免疫诱导的肿瘤特异性免疫应答及其抗肿瘤作用 ,为肿瘤的免疫生物治疗寻求新途径。方法 :分离获取异位hCGβ CTP37的编码基因 ,并将 4拷贝基因以头尾串联形式克隆于真核表达载体TR42 1构建重组质粒TR42 1 CTP37.4,以PCR、酶切和DNA序列测定进行鉴定 ;肌内注射法对BALB/c小鼠实施 3次基因免疫 (1 0 0 μg/ 1 0 0 μl) (每只小鼠 ) ,间隔 3周 ,并以空载质粒为对照。ELISA法检测免疫小鼠血清中特异性抗hCGβ IgG抗体 ;免疫小鼠脾细胞经特异性抗原hCGβ刺激后 ,用3H TdR掺入法和同位素释放法分别检测其特异性淋巴细胞增殖活性和CTL细胞毒活性 ;皮下接种SP2 / 0 hCGβ细胞攻击免疫小鼠 ,以成瘤率及实体瘤重量评估体内抗瘤效果。结果 :TR42 1 CTP37.4质粒免疫小鼠产生了较高水平的抗hCGβ IgG抗体 ,与空载质粒组有显著差异 ;hCGβ蛋白刺激TR42 1 CTP37.4质粒免疫小鼠脾细胞生产了高水平的特异性淋巴细胞增殖活性 ,且明显高于空载质粒免疫小鼠 ;特异抗原诱导的TR42 1 CTP37.4质粒免疫小鼠脾细胞对SP2 / 0 hCGβ细胞的CTL活性明显高于SP2 / 0 preS2 /S细胞 P <0 .0 1 ) ;空质粒免疫小鼠接种SP2 / 0 hCGβ细胞后成瘤率为 1 0 0 % ,瘤重达 3 .37g,而TR42 1 CTP37.4质粒免     

Enhancement of Anti-tumor Activity of Newcastle Disease Virus by the Synergistic Effect of Cytosine Deaminase

This study was conducted to investigate enhancement of anti-tumor effects of the lentogenic Newcastledisease virus Clone30 strain (NDV rClone30) expressing cytosine deaminase (CD) gene against tumor cells andin murine groin tumor-bearing models. Cytotoxic effects of the rClone30-CD/5-FC on the HepG2 cell line wereexamined by an MTT method. Anti-tumor activity of rClone30-CD/5-FC was examined in H22 tumor-bearingmice. Compared to the rClone30-CD virus treatment alone, NDV rClone30-CD/5-FC at 0.1 and 1 MOIs exertedsignificant cytotoxic effects (P<0.05) on HepG2 cells. For treatment of H22 tumor-bearing mice, recombinantNDV was injected together with 5-FC given by either intra-tumor injection or tail vein injection. When 5-FCwas administered by intra-tumor injection, survival for the rClone30-CD/5-FC-treated mice was 4/6 for 80 daysperiod vs 1/6 , 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC and saline alone, respectively. When5-FC was given by tail vein injection, survival for the rClone30-CD/5-FC-treated mice was 3/6 vs 2/6 , 0/6 and0/6 for the mice treated with rClone30-CD, 5-FC or saline alone, respectively. In this study, NDV was used forthe first time to deliver the suicide gene for cancer therapy. Incorporation of the CD gene in the lentogenic NDVgenome together with 5-FC significantly enhances cell death of HepG2 tumor cells in vitro, decreases tumorvolume and increases survival of H22 tumor-bearing mice in vivo.     

新城疫病毒对癌细胞的杀伤作用

 目的　探讨新城疫病毒NDV对癌细胞的直接杀伤作用。方法　将NDV与各组细胞的混悬液一起加入细胞培养板中三孔法,同时设不加NDV的对照孔,经8、16、24、36和48小时五个时相观测细胞生长情况和病毒复制情况。结果　成纤维细胞和对照组细胞生长良好,而实验孔的各组癌细胞逐渐减少,出现融合、裂解。48小时癌细胞组的上清液病毒效价较高而成纤维细胞的上清液病毒效价为0。结论　NDV对癌细胞有较强的亲和力,可在癌细胞内大量复制,具有直接杀伤癌细胞的作用,而对正常细胞无明显的杀伤作用。     

Anti-tumor Immunity of Gene Vaccine with Nucleofection Technology

OBJECTIVE To observe enhancement of anti-tumor immunity by gene vaccine using nucleofection technologyMETHODS The technique of nucleofection was used to transfer effectively plasmid DNA into immature dendritic cells (iDCs); we studied immune responses regulated by DNA vaccine using real-time quantitative polymerase chain reaction (PCR) and western-blotting to optimize the follow-up lymphocyte activation. The anti-tumor capacity of lymphocytes primed by DCs was analyzed using lactate dehydrogenase with a non-radioactive cytotoxicity assay.RESULTS Human monocyte-derived dendritic cells (hMoDCs) were induced by interleukin (IL)-4 and granulocyte-macrophage colonystimulating factor (GM-CSF) in vitro from human monocytes for 5 or 6 days. DNA vaccine was transfected to iDCs with high transfection (35.73%) using nucleofection. Compared with the iDC group, the expression of Th1 cell cytokine IL-12, IL-18 and Th2 cell cytokine IL-4 increased after stimulation. CD86 and CD83 were upregulated compared with non-nucleofected groups 48 hours after nucleofection with DC-pVAX-PRA. The result of the cytotoxicity assay showed that DCs-pVAX-PRA primed non-adherent peripheral blood mononuclear cells (PBMCs) exhibit their highest cytotoxicity against target cells.CONCLUSION The results show that DNA vaccine was transfected to iDC with high transfection efficiency using nucleofection, priming autologous lymphocytes for anti-tumor immunity by upregulated expression of co-stimulatory molecules, adhesion molecules and cytokines. These results provided a basis to explore the molecular mechanism of DNA vaccine in vivo.     

Liver Pathology in Rats Treated with Newcastle Disease Virus Strains AF2240 and V4-UPM

Background: Treatment of cancer with chemo-radiotherapy causes severe side effects due to cytotoxic effects towards normal tissues which often results in morbidity. Therefore, developing anticancer agents which can selectively target the cancer cells and cause less side effects are the main objectives of the new therapeutic strategies for treatment advanced or metastatic cancers. Newcastle disease virus strains AF2240 and V4-UPM were shown to be cytolytic against various cancer cells in-vitro and very effective as antileukemicagents. Methods: 45 rats at 6 weeks of age, were randomly assigned to nine groups with 5 rats in each group, both azoxymethane (AOM) and 5-Fluorouracil (5-FU) were given to rats according to the body weight. NDV virus strains (AF2240 and V4-UPM) doses were determined to rats according to CD50 resulted from MTT assay. After 8 doses of NDV strians and 5-FU, tissue sections preparations and histopathological study of rats’ organs were done. Results: In this article morphological changes of rats’ organs, especially in livers, after treatment with a colon carcinogen (azoxymethane) and Newcastle disease virus strains have been recorded. We observed liver damage caused by AOM evidenced by morphological changes and enzymatic elevation were protected by the oncolytic viruses sections. Also we found that combination treatment NDV with 5-FU had greater antitumor efficacy than treatment with NDV or 5-FU alone. Conclusion: We noted morphological changes in liver and other rats’ organs due to a chemical carcinogen and their protection by NDV AF2240 and NDV V4-UPM seems to be most protective.     

NDV7793体外激活的小鼠单核巨噬细胞(MΦ)对小鼠肝癌细胞的杀伤作用及其机制

 目的
初步研究NDV7793激活的小鼠单核巨噬细胞（MΦ）对小鼠肝癌Novikoff细胞的杀伤作用,并探讨其杀伤机制与TNF-
α和TRAIL的关系。 方法从腹腔分离6周龄BALB/C小鼠MΦ, 用NDV7793于体外刺激小鼠MΦ,以ELISA分别测定
NDV7793刺激小鼠MΦ后产生的TNF-α及TRAIL水平;NDV7793体外刺激MΦ后,与小鼠肝癌Novikoff细胞混合培养,
以LDH微量释放法测定小鼠MΦ对小鼠肝癌Novikoff细胞的杀伤效应。同时设立3组实验对照组：IFN-β阳性对照组
、紫外线灭活NDV（UV-NDV）对照组以及空白对照组。 结果与3个对照组相比,NDV7793在体外能提高MΦ分泌TNF-
α、TRAIL的水平;NDV体外刺激后的小鼠MΦ能杀伤小鼠肝癌Novikoff细胞。结论NDV7793在体外能激活小鼠MΦ,
小鼠MΦ被NDV7793刺激后,对小鼠肝癌Novikoff细胞的杀伤作用增强,NDV激活后的小鼠MΦ对小鼠肝癌Novikoff细
胞的杀伤机制可能与TNF-α和TRAIL有关。     

体内IL-2基因和IFN-γ基因转染巨噬细胞抗肿瘤作用的研究

白细胞介素17是来源于活化T淋巴细胞的细胞因子.研究发现白细胞介素17能维持纤维母细胞对CD34~ 造血祖细胞的支持作用,并能介导它们向中性粒细胞增殖分化.目前也认为IL-17是最新发现的T淋巴细胞来源的作用于造血系统的细胞因子.本文对IL-17的来源、结构和功能等方面作一综述.