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。     

6株NDV鄱阳株对肝癌细胞杀伤作用的初步研究

目的:研究从江西鄱阳湖野鸭分离的6株NDV对肝癌细胞的杀伤效应,进一步筛选NDV溶瘤毒株。方法:用MTT法研究6株NDV鄱阳株对两株传代肝癌细胞株Novikoff和HepG-2及一株正常肝细胞株HL-7702的杀伤效应。结果:6株NDV鄱阳株对肝癌细胞株Novikoff和HepG-2有显著杀伤效应,而对人正常肝细胞HL-7702无明显影响;病毒对细胞的杀伤活性与病毒作用剂量和病毒作用时间成正比;病毒在肝癌细胞中有明显复制增殖现象;Novikoff肝癌细胞对NDV敏感性强于HepG-2肝癌细胞。结论:6株NDV鄱阳株对Novikoff及HepG-2肝癌细胞产生显著的杀伤作用,而对正常肝细胞HL-7702未见明显影响。     

6株NDV鄱阳株对肝癌细胞杀伤作用的初步研究

目的：研究从江西鄱阳湖野鸭分离的6株NDV对肝癌细胞的杀伤效应，进-步筛选NDV溶瘤毒株。方法：用MTT法研究6株NDV鄱阳株对两株传代肝癌细胞株Novikoff和HepG-2及一株正常肝细胞株HL-7702的杀伤效应。结果：6株NDV鄱阳株对肝癌细胞株Novikoff和HepG-2有湿著杀伤效应，而对人正常肝细胞HL-7702无明显影响；病毒对细胞的杀伤活性与病毒作用剂量和病毒作用时间成正比；病毒在肝癌细胞中有明显复制增殖现象；Novikoff肝癌细胞对NDV敏感性强于HepG-2肝癌细胞。结论：6株NDV鄱阳株对Novikoff及HepG-2肝癌细胞产生显著的杀伤作用，而对正常肝细胞HL-7702未见明显影响。     

新城疫病毒D817株体外高效杀伤肝癌细胞及其作用机制

 目的 与NDV疫苗株LaSota对比研究一株NDV D817株对肝癌细胞高效特异性的杀伤效应和作用机制, 进一步筛选NDV溶瘤毒株。 方法 用MTT法对比病毒对三株传代肝癌细胞株SMMC-7721、Bel-7404和HepG-2及一株正常肝细胞株 HL-7702的杀伤效应,并用TUNEL法及透射电 子显微镜观察病毒诱导肿瘤细胞发生凋亡作用。 结果 NDV D817株对肝癌细胞株SMMC-7721、Bel-7404和HepG-2杀伤效应高达80％,显著高于疫苗 LaSota株（P<0.01）,而对人正常肝细胞HL-7702无明显影响;病毒在肝癌细胞中明显复制增 殖,对细胞的杀伤活性与病毒作用剂量和病毒作用时间成正比;NDV D817株有效诱导肝癌细胞 发生凋亡。 结论 NDV D817株有效诱导肝癌细胞发生凋亡, 对SMMC-7721、Bel-7404和HepG-2细胞具有高效杀伤 性,而对正常肝细胞HL-7702未见明显影响,推测为溶瘤株。     

新城疫病毒7793株抑制人结肠癌LoVo细胞裸鼠移植瘤的生长及其机制

目的:观察新城疫病毒7793株(Newcastle disease virus 7793 strain,NDV 7793)对人结肠癌LoVo细胞裸鼠移植瘤生长的作用,并探讨其可能机制。方法:建立LoVo细胞裸鼠移植瘤模型,随机分成3组,分别静脉注射PBS、5-FU以及NDV 7793,观察各组裸鼠肿瘤的生长情况,流式细胞术检测移植瘤细胞的坏死率和凋亡率,免疫组织化学法检测移植瘤组织中Bax、Bcl-2蛋白的表达,细胞色素C试剂盒检测移植瘤组织中细胞色素C的含量,ELISA法检测移植瘤组织中TNF-α含量。结果:NDV 7793较5-FU更明显抑制LoVo细胞移植瘤的生长(抑制率50.14%vs 37.14%,P<0.05)。NDV 7793组移植瘤LoVo细胞凋亡率显著高于5-FU对照组[(28.7±1.5)%vs(1.46±0.3)%,P<0.01],且NDV 7793组诱导LoVo细胞的凋亡率和坏死率[(28.7±1.5)%vs(27.80±3.32)%]相当。NDV 7793能促进移植瘤组织中Bax蛋白的表达,对Bcl-2蛋白的表达无影响。NDV 7793可提高移植瘤组织中的细胞色素C含量[(2.28±0.68)vs(0.68±0.13)μg/μl]和TNF-α的水平[(489.6±5.2)vs(167.9±3.9)pg/ml]。结论:NDV 7793可抑制人结肠癌LoVo细胞移植瘤的生长,其机制可能与其上调Bax蛋白、细胞色素C和TNF-α的表达,以及促进肿瘤细胞凋亡有关。     

新城疫病毒Lasota 弱毒株对人肿瘤细胞的体外修饰作用

 目的 研究新城疫病毒（NDV）弱毒株Lasota对体外培养的人肿瘤细胞株免疫功能的影响。方法 以不同病毒滴度作用于肿瘤细胞，采用MTT染色法检测NDV对肿瘤细胞的杀伤作用，同时用NDV处理过的小鼠黑色素B16细胞免疫小鼠，检测其对小鼠NK细胞活性影响。结果 NDV Lasota株对4种癌细胞株均具有较强的杀伤作用，用其处理小鼠黑色素瘤细胞株后，再次接种可提高小鼠体内的NK细胞活性。结论 NDV能够抑制肿瘤细胞生长，诱导其凋亡，免疫接种小鼠后，可提高小鼠的细胞免疫功能。     

新城疫病毒D90毒株致肺癌A549细胞死亡的方式

新城疫病毒(newcastle disease virus, NDV)属副黏病毒,可以引起禽类的新城疫.新城疫病毒在人类癌细胞中的复制效率是在正常细胞中的10 000倍, 因此,NDV可以选择性杀伤肿瘤细胞,这提示人们可将它作为潜在的抗癌因子.目前,已经应用于人癌症治疗的NDV毒株有73-T、MTH-68等溶癌毒株和Μlster等非溶癌毒株, 其中NDV73-T已经被证明可以在体外杀死人的多种肿瘤细胞,同时在体外试验中,该毒株被证明不会杀死正常的、增生性的白细胞或人的正常皮肤纤维原细胞,但是在于人的肿瘤细胞具有明显的杀伤作用.本研究在新城疫病毒D90株明显抑制肺癌A549细胞生长并导致细胞死亡的基础上,对其可能的致死亡作用方式进行了探讨.     

鸡新城疫病毒对人类口腔鳞癌细胞的杀伤作用

目的　检测鸡新城疫病毒 (newcastlediseasevirus ,NDV)对人口腔鳞癌细胞的杀伤作用。方法　利用MTT方法检测NDV对人口腔鳞癌 (oralsquamouscellcarcinoma ,OSCC)颈淋巴结转移癌细胞系 (GNM)及人舌鳞癌细胞系 (TSCCa)细胞株的杀伤性。结果　NDV作用后的人口腔鳞癌细胞株的细胞活性比对照组的细胞活性有显著下降 (P <0 0 1) ,NDV血凝效价值显著升高。结论　NDV能够通过在人口腔鳞癌细胞内增殖杀死癌细胞株。     

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。     

全反式维甲酸对结肠癌不同增殖潜能细胞株增殖的抑制作用研究

目的:研究ATRA对结肠癌不同增殖能力细胞株生长抑制的可能机制,为ATRA应用于结肠癌的治疗提供可靠依据.方法:应用细胞观察、FACS和MTT方法,研究ATRA对结肠癌不同增殖能力细胞株LS174T和CW-2细胞的生长抑制现象.结果:LS174T的生长速度明显比CW-2细胞快.ATRA对体外培养的结肠癌细胞株LS174T和CW-2细胞的生长有明显抑制和诱导细胞分化作用.结论:ATRA对LS174T和CW-2细胞的生长有抑制作用,抑制细胞增殖的程度与ATRA的浓度有关,ATRA对结肠癌细胞有一定的抑制癌细胞增殖和诱导癌细胞分化作用.     

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。     

Host mediated anti-tumor effect of oncolytic Newcastle disease virus after locoregional application

Several strains of the Newcastle disease virus (NDV) have raised considerable interest in recent years for clinical application because of their oncolytic properties. In this study we characterized virological, immunological and anti-tumor properties of some NDV strains. The oncolytic strain MTH-68/H was the most potent interferon-alpha inducer and, after UV light inactivation, it was the only tested NDV strain which induced in human PBMC anti-tumor activity in vitro. Upon systemic application to mice bearing a virus susceptible intradermal tumor, no significant anti-tumor effects were observed with the two oncolytic strains Italian and MTH-68/H while the treatment had significant side effects as seen by loss of body weight. In contrast, when using a locoregional application model for treatment of liver metastases of luciferase transfected CT26 colon carcinoma cells, MTH-68/H showed a significant delay in tumor growth, as well as prolonged survival but no effects on body weight. Surprisingly, this CT26 murine tumor cell transfectant was resistant in vitro to virus infection and oncolysis. These results suggest: i) that locoregional application of oncolytic NDV is more effective than systemic i.v. application; and ii) that oncolytic NDV can mediate effects even against a virus-resistant tumor line. The involvement of host anti-tumor immune responses as an important mechanism in therapies based on oncolytic NDV will be discussed.     

细胞凋亡检测用于肿瘤细胞株对化疗敏感性的研究

目的 评价Annexin-V荧光标记FACScan法、TUNEL法细胞凋亡检测在肿瘤细胞株化疗药物敏感性研究中的应用。方法 将DDP、MMC、5－FU、EPI按血浆峰浓度（PPC）、1／10PPC、1／5PPC、5PPC、10PPC与结肠腺癌LoVo和Ls-174-t细胞温育24及48h，用FACScan法、TUNEL法检测细胞凋亡，用MTT比色法检测细胞生长抑制率，并提取DNA进行琼脂糖凝胶电泳。结果 LoVo与Ls-174-t细胞经化疗药物诱导48h后，FACScan检测的细胞凋亡率在PPC时最高，TUNEL法检测的凋亡率与MTT法检的细胞增殖抑制率呈正的直线相关（P＜0．05）。结论 Annexin-V荧光标记FACScan法检测细胞凋亡既能优选出敏感的药物，又能寻找合适的药物浓度，是目前优选有效化疗药物种类及剂量的一个可探索、选用的方法。     

Antiproliferative effect of newcastle disease virus strain D90 on human lung cancer cell line A549

Newcastle disease virus (NDV) and variants of this virus have oncolytic properties and are potential anticancer agents. The objective of this study was to compare the effect of NDV strain D90 and strain D93 isolated from natural sources on human non-small cell lung cancer (NSCLC) cell line A549. We determined the 50% embryo infective dose (EID50) and 50% tissue culture infective dose (TCID50) of the NDV strains. The MTT assay was used to evaluate the effects of NDV strains on cell viability. We determined the expression of Annexin V and Bcl-2 proteins in NDV-infected cells. Light microscopy and electron microscopy indicated that the D90 strain significantly altered cell morphology and reduced cell viability, while strain D93 had negligible effects. Neither strain had a significant effect on normal cultured fetal liver cells. We used acridine orange staining to show that strain D90 (but not strain D93) induced nuclear fragmentation of A549 cells. An Annexin V-based apoptosis assay indicated that strain D90 (but not strain D93) caused significant apoptosis of A549 cells. Moreover, strain D90 (but not strain D93) significantly repressed the expression of Bcl-2 (an antiapoptotic protein) in A549 cells. Taken together, our results indicate that NDV strain D90 (but not strain D93) had no significant effect on normal cultured cells, but induced apoptosis of cultured NSCLC cells via a caspase-dependent pathway. These results suggest that NDV strain D90 has potential as an anticancer agent.     

Liver metastasis models of colon cancer for evaluation of drug efficacy using NOD/Shi-scid IL2Rgammanull (NOG) mice

To examine the drug efficacy of a novel farnesyltransferase inhibitor (FTI), CH4512600, in vivo, we developed a reliable liver metastasis model of human colon cancer using NOD/Shi-scid IL2Rgamma(null) (NOG) mice. Eleven human colon cancer cell lines were examined for their ability to form diverse metastatic foci in the livers of NOG mice. When inoculated with 10(4) COLO320DM, HCT 116, HT-29, WiDr, LoVo and LS174T cells, liver metastasis was evident in 100% (6/6), 100% (6/6), 88.9% (8/9), 87.5% (7/8), 83.3% (5/6) and 50.0% (3/6) of the NOG mice, respectively. CaCo2, COLO201, LS123, SW48 and SW1417 showed no metastasis when seeded at 10(4) cells even in NOG mice. The mRNA expression levels and genetic mutations of N, H and K-RAS genes, which directly affect the levels of cellular RAS protein that would be molecular target for FTI, were also examined in these six metastatic human colon cancer cell lines for molecular biological and genotypic characteristics. Only three cell lines had a point mutation in the RAS oncogene. LS174T cell line had a point mutation of the K-RAS gene at codon 12 (gly12 --> asp; G12D), and HCT 116 and LoVo cell lines had a point mutation of the K-RAS gene at codon 13 (gly13 --> asp; G13D). Relative gene expression levels of N, H and K-RAS genes in the HCT 116 cell line were 2.6-5.0-fold lower than that of LS174T and LoVo cell lines. We selected HCT 116 cell line from our liver metastasis model for evaluation of FTI CH4512600 efficacy in vivo. Using the NOG mouse liver metastasis model, we demonstrated the effectiveness of FTI CH4512600 to suppress tumor growth in vivo and to prolong mouse survival significantly from 36.9+/-2.9 to 50.3+/-9.4 days.     

靶向干预N-WASP蛋白功能区对大肠癌细胞侵袭转移能力的影响

目的研究靶向干预N-WASP蛋白的VCA功能区对大肠癌细胞侵袭转移能力的影响。方法构建含有N-WASP蛋白CA结构区域信息但缺乏V结构区域信息的重组表达质粒（CA质粒）,脂质体转染法将重组质粒及空质粒导入人高侵袭性大肠癌细胞株LoVo细胞内,G418筛选获得稳定转染的细胞株。采用Transwell侵袭小室和制备裸鼠结肠癌肝转移模型检测转染后LoVo细胞侵袭转移能力的变化。结果Transwell侵袭小室实验结果显示,重组质粒转染组LoVo细胞的穿膜细胞数量较空质粒转染组及未转染组明显减少（P〈0.05）。动物实验结果显示,重组质粒转染组的裸鼠肝脏表面的转移瘤结节数较空质粒转染组及未转染组明显减少,生存率明显提高,差异均具有统计学意义（P〈0.05）;而空质粒组和未转染组两者无明显差别（P〉0.05）。结论靶向干预N-WASP蛋白的VCA功能区能有效抑制大肠癌细胞的侵袭转移能力。     

Synergism of 1-beta-D-arabinofuranosylcytosine and cis-diamminedichloroplatinum in their lethal efficacies against seven established cancer cell lines of gastrointestinal origin

We previously reported that the combination of cis-diamminedichloroplatinum (CDDP) and 1-beta-D-arabinofuranosylcytosine (ara-C) induced a remarkable synergistic killing effect on an established human colon carcinoma cell line, LoVo. The current study investigated whether this effect was LoVo specific or could be extended to other colon cancer cell lines as well as to cell lines of different histological origins, including an estrogen receptor-positive breast cancer cell line (MCF7), an ovarian cancer cell line (OV1225), and an esophageal cancer cell line (Hcu18). The six human colorectal cancer cell lines included in this study represent three biological groups with distinct phenotypic properties. Group 1 (well-differentiated) consisted of LoVo and SW48; group 2 (intermediately differentiated) comprised SW480 and SW620; and group 3 (undifferentiated) was represented by SW403 and SW1116. No significant synergistic cytotoxicity was noted after the breast and ovarian cancer cells were treated. However, synergistic lethal effects were observed in all of the six colon cancer cell lines as well as the esophageal cancer cell line. The synergistic effect on the gastrointestinal cancer cell lines was related to the concentration of ara-C and CDDP during treatment. Our results suggest that the cytotoxic synergism between ara-C and CDDP may be tissue-type specific and that synergism may depend on the histological origin of the cancer.     

Inhibition of metastatic carcinoma cell growth in livers by poly(I):poly(C)/cationic liposome complex (LIC)

The complex of poly(I):poly(C) and a new cationic liposome (LIC) has a potent antitumor activity against many tumor cell lines in vitro, whereas poly(I):poly(C) itself has no such activity. In the present study we tested the sensitivity of 21 human colon and pancreatic cancer cell lines to LIC or Adriamycin in vitro. The growth of most of the cell lines was strongly inhibited by both LIC and Adriamycin in vitro, although a few insensitive cell lines were different. We also studied the in vivo antitumor activity of LIC or Adriamycin in three experimental liver metastasis models in nude mice using a human pancreatic cancer cell line (AsPC-1) and two human colon cancer cell lines (Ls174T and HCC-M1544). The administration of LIC or Adriamycin was started 3 days after the injection of tumor cells. Animals received 0.1 mg/kg LIC IV twice weekly or 5 mg/kg Adriamycin IV every 5 days during the experiments. LIC showed potent antitumor activity in all three liver cancer models. Although Adriamycin had potent antitumor activity in the HCC-M1544 model, it had only a moderate effect in the AsPC-1 model and at most a weak effect in the Ls174T model. At the effective doses LIC did not cause detectable pathological changes in the liver and did not elicit toxicity to mice in these models, whereas Adriamycin did exhibit toxic effects. These results suggest that LIC is a promising candidate drug to treat hepatic metastasis.