新城疫病毒D417株抗小细胞肺癌的体外研究

目的 研究新城疫病毒(Newcastle disease virus,NDV)19417株体外对小细胞肺癌细胞NCI-H446及正常人胚肺成纤维细胞HFL-1的杀伤作用.方法 应用四甲基偶氮唑盐比色法检测NDV D417株对NCI-H446和HFL-1的杀伤效应.D417株感染NCI-H446后,于光镜和电镜下检查细胞病理变化.结果 NDV D417株对NCI-H446有杀伤作用,杀伤作用与病毒剂量和作用时间呈正相关.13417株对HFL-1也有杀伤作用,但比同等条件下对NCI-H446的杀伤作用弱.D417株感染NCI-H446后光镜下可见细胞病变及细胞融合现象,电镜下可见细胞呈凋亡形态.结论 NDV D417株对小细胞肺癌细胞有较强的杀伤作用,对正常细胞影响较小.     

山奈酚诱导人小细胞肺癌H446细胞凋亡及机制

目的探讨山奈酚对人小细胞肺癌H446细胞的抑制作用,并研究其作用机制。方法采用MTT法、DAPI染色、流式细胞术等方法检测山奈酚对H446细胞增殖及凋亡的影响;使用Western blot检测山奈酚处理后H446细胞中p53、bax和bcl-2的表达变化。结果山奈酚抑制人小细胞肺癌H446细胞增殖,促进H446细胞阻滞于S期及G2/M期,诱导该细胞株凋亡,上调p53、bax的表达水平,降低bcl-2的表达水平。结论山奈酚对H446细胞有明显的抑制作用,该抑制作用与山奈酚诱导的细胞周期阻滞和细胞凋亡有关。     

HSV-TK/GCV系统对人肺癌细胞株的杀伤效应

目的观察单纯疱疹病毒胸苷激酶基因HSV-TK／GCV系统对人肺癌细胞H446的体外杀伤作用。方法利用脂质体(1ipofectin)将重组逆转录病毒载体pLXSN-TK导入包装细胞PA317，G418筛选至抗性克隆出现，收获病毒液。加入聚凝胺感染H446细胞，并筛选出含TK基因的H446细胞。观察GCV对H446和不同比例混合的H446、H446／TK的杀伤作用及旁观者效应。结果经电镜及PCR检测证实TK基因已导入PA317、H446细胞。H446／TK细胞对GCV的敏感性明显高于H446细胞；随着TK 细胞比例的增加，存活率进一步减低，提示GCV不仅杀死TK 细胞，也杀死混合培养的未转导TK基因的H446细胞，表明HSV-TK基因转导的H446细胞具有明显的旁观者效应。结论HSV-TK／GCV系统赋予人肺癌细胞对GCV的高敏感性及旁观者效应。     

三种不同IL-15基因转染对NCI—H446肿瘤细胞凋亡的影响

目的探讨不同形式IL-15基因转染对人小细胞肺癌细胞株NCI-H446细胞凋亡的影响。方法用构建的3种转染不同IL-15基因的NCI—H446细胞模型：原型（CIL-15）、成熟肽（CIL-15mp）及以IL-2信号肽替代IL-15信号肽的改型IL—15基因（CIL-2sp—IL-15mp）,以野生株NCI—H446细胞（CW）为对照,流式细胞术检测细胞凋亡率及Fas蛋白表达。结果与CW相比,CIL-15、CIL-15mp和CIL-2sp—IL-15mp凋亡率及Fas蛋白表达依次增加（P＜0．05或＜0．01）。结论3种IL-15基因转染均可致NCI—H446细胞凋亡率明显增加,强度依次为改型＞成熟肽＞原型;这种作用与增加NCI-H446细胞Fas蛋白的表达有关。     

siRNA腺病毒载体沉默MEKK3基因促进TRAIL诱导肝癌HCC-9204细胞凋亡

目的研究抑制丝裂原活化蛋白激酶/细胞外信号调节激酶-激酶3（MEKK3）基因表达促进TRAIL诱导人肝癌HCC-9204细胞凋亡的作用。方法构建人MEKK3基因的siRNA重组腺病毒载体,转导人肝癌HCC-9204细胞,以RTPCR和印迹法检测MEKK3 mRNA和蛋白的表达,筛选并建立稳定沉默MEKK3基因表达的人肝癌HCC-9204细胞株,以流式细胞仪检测TRAIL诱导细胞凋亡的情况。结果①成功构建并鉴定表达人MEKK3基因的siRNA重组腺病毒载体。②建立MEKK3基因有效且稳定沉默的人肝癌HCC-9204细胞株。③500 g/LTRAIL处理MEKK3基因沉默的HCC-9204细胞株,细胞凋亡率较野生HCC-9204细胞株显著提高。结论本研究成功建立有效且稳定沉默MEKK3基因表达的人肝癌HCC-9204细胞株模型,初步证实MEKK3基因表达与TRAIL诱导肝癌细胞凋亡的敏感性相关。     

MDA-7基因的腺病毒载体构建及对肺腺癌A549细胞凋亡的影响

目的构建一种由腺病毒AdMax包装系统表达目的基因——黑色素瘤分化相关基因-7（MDA-7）的复制缺陷型腺病毒,并初步探索其抗肿瘤活性。方法通过基因操作技术将目的基因（MDA-7）插入到5型腺病毒AdMax包装系统的E1A区域,构建复制缺陷型腺病毒Ad.hMDA-7-EGFP;同时构建对照病毒Ad.EGFP。病毒滴度计算按寇化法（Karber法）进行腺病毒感染性滴度（TCID50）测定。应用免疫组化染色法检测MDA-7在感染2种病毒的肺腺癌A549细胞中的表达状态;通过二苯甲亚胺-碘化丙啶（Hoechst33342-PI）双染色法及流式细胞仪检测2种病毒对肿瘤细胞杀伤的差异。结果成功构建携带目的基因的腺病毒载体,经聚合酶链反应（PCR）鉴定正确;MDA-7在感染Ad.hMDA-7-EGFP的肺腺癌A549细胞中表达,以相同感染复数（MOI）值感染Ad.EGFP时肺腺癌A549细胞中未检测到MDA-7表达;Ad.hMDA-7-EGFP对肿瘤细胞株杀伤能力明显高于Ad.EGFP。结论成功构建复制缺陷型腺病毒Ad.hMDA-7-EGFP,并证实MDA-7蛋白在肿瘤细胞中过表达,诱导肺腺癌A549细胞株发生凋亡。     

重组复制型溶瘤腺病毒p53的质量控制方法

建立重组复制型溶瘤腺病毒p53(SG600-P53)的质控检测方法与质量标准。采用限制性内切酶酶切、PCR法对端粒酶启动子、低压缺氧调控元件融合的巨细胞病毒(cytomegalovirus,CMV)启动子、p53基因等重组病毒载体结构进行分析,鉴定结果均与理论值相符。经紫外吸收法(A260)检测,病毒颗粒数为2.0×1011 VP.mL-1;TCID50法测定感染活性为5.0×1010 IU.mL-1。p53蛋白ELISA检测结果表明,重组病毒体外感染人肺癌细胞H1299后,感染组核蛋白和空白对照组A450吸收度之比为5.2。该基因治疗制剂对人肺癌细胞A549体外杀伤的MOIIC50为1.0。以相同MOI感染并经TCID50法检测,重组病毒在人肺癌细胞A549与人表皮成纤维二倍体细胞BJ的增殖比值为398。经阴离子高效液相色谱分析,病毒载体颗粒纯度为99.5%。定量PCR检测表明在1×107 VP的病毒颗粒中,野生型腺病毒基因片段少于1个拷贝。本研究建立的重组复制型溶瘤腺病毒的质量标准与检测方法,可用于该制品的质量控制,同时也为其他溶瘤基因治疗病毒载体质控研究提供参考。     

全反式维甲酸对小细胞肺癌细胞的分化诱导作用研究

目的探讨全反式维甲酸(all-trans-retinoic acid,ATRA)对小细胞肺癌细胞的分化诱导作用。方法选择NCI-H446细胞株MTT法检测细胞体外增殖能力,电镜下观察形态,流式细胞仪进行细胞周期分析。RT-PCR法分析维甲酸受体亚型基因。结果全反式维甲酸诱导小细胞肺癌NCI-H446细胞株,细胞增殖能力下降,凋亡增加,更多的细胞被阻止于G1/G0期。结论全反式维甲酸诱导分化能有效抑制小细胞肺癌NCI-H446细胞株的增殖,促进凋亡。     

山奈酚对人小细胞肺癌H446的增殖抑制和诱导凋亡作用的影响

目的 探讨山奈酚对人小细胞肺癌H446细胞生长的抑制作用和诱导凋亡作用.方法 采用MTT 法检测生长的抑制作用;流式细胞仪检测细胞凋亡;Western Blotting技术检测凋亡相关蛋白.结果 不同浓度的山奈酚能抑制人小细胞肺癌H446细胞生长,且随浓度增加抑制作用增强(P<0.01).流式结果 显示不同浓度山奈酚组...     

塞来昔布对肝癌HepG2 HepG3细胞的免疫增敏作用

目的探讨选择性环氧合酶-2（COX-2）抑制剂塞来昔布（Celecoxib）对人肝癌HepG2和HepG3细胞株对肿瘤坏死因子相关的凋亡诱导配体（TRAIL）的增敏作用及其可能机制。方法以人HepG2 HepG3肝癌细胞为研究对象,以塞来昔布和TRAIL作为干预手段,采用四甲基偶氮唑蓝（MTT）法检测塞来昔布对肝癌细胞株的增殖抑制作用,采用流式细胞术,检测塞来昔布联合TRAIL对HepG2,HepG3细胞的凋亡及DR4,DR5的表达。塞来昔布与不同效靶比的细胞因子诱导的杀伤细胞（CIK）联合作用于人肝癌细胞株,经LDH检测法检测其杀伤作用。结果塞来昔布及TRAIL对人肝癌HepG2和HepG3细胞株均有显著抑制作用,塞来昔布联合TRAIL可显著增敏TRAIL对HepG2和HepG3的杀伤,其差异具有统计学意义（P〈0.05）,经过塞来昔布预处理的肝癌细胞更易被CIK细胞杀伤。结论塞来昔布对人肝癌HepG2和HepG3细胞株具有明显细胞毒性,联合TRAIL及CIK可显著增敏后者对肝癌细胞的杀伤效应,其机制可能与塞来昔布上调DR4,DR5有关。     

Cadmium uptake by a human hepatic cell line (WRL-68 cells)

A hepatic human cell line (WRL-68 cells) was employed to investigate the uptake of the toxic heavy metal cadmium. Cd accumulation in WRL-68 cells is a time-, temperature- and concentration-dependent process. A rapid initial phase of uptake was followed by a second slower phase. The transport does not require energy and 55% of Cd transport occurs by temperature-insensitive processes, possibly by diffusion. The rest of Cd transport (45%) occurs by temperature-sensitive processes, probably ion channels and carriers, that involve interaction with sulfhydryl groups. The calcium channel blockers nifedipine and verapamil inhibit the uptake of cadmium, with an inhibition of 35% after 30 min incubation with 100 μM verapamil and 10 μM Cd. These data suggest that about one third of the Cd enters WRL-68 cells through the calcium channels. The toxic metals appear to use the transport pathways that exist for biologically essential metals. Our results in human hepatic cells are very similar to those reported in cultured rat hepatocytes. It appears that transport pathways available for Cd uptake are similar and independent of the species of hepatocyte origin. Moreover, the WRL-68 cell line seems to be an excellent in vitro model to study the mechanism of cell damage due to Cd.     

紫花牡荆素抑制小细胞肺癌NCI-H446细胞系肺癌干细胞样细胞侵袭和转移的研究

目的探讨紫花牡荆素(CAS)抑制人小细胞肺癌NCI-H446细胞系肺癌干细胞样细胞(LCSLCs)侵袭和转移作用。方法体外培养人小细胞肺癌NCI-H446细胞系细胞和LCSLCs。流式细胞术(FCM)检测CD133+表达。Trawell法检测LCSLCs侵袭和转移能力。Trawell法测定CAS对LCSLCs侵袭、转移能力的抑制作用。结果 LCSLCs呈现典型非黏附三维球状,并高表达CD133+。Trawell法结果显示LCSLCs具有很强的体外侵袭和转移能力,而CAS能显著抑制LCSLCs的体外侵袭和转移能力。结论 CAS能显著抑制LCSLCs的体外侵袭和转移能力。     

Antitumor activity of a novel bis-aziridinylnaphthoquinone （AZ4） mediat- ing cell cycle arrest and apoptosis in non-small cell lung cancer cell line NCI-H460

AIM: The cytotoxic activities of a series of bis-aziridinylnaphthoquinone, AZ1 to AZ4, on human lung carcinoma cell lines, H460, and normal lung cells fibroblast cell line, MRC-5, and the mechanisms of H460 cells induced by AZ4 were investigated. METHODS: The MTT assay was used to determine the cell proliferation. Cell cycle was analysed by FACS. The activity of caspase 3, 8 and 9 was determined by cell-permeable fluorogenic detection system. Western blot assay was used to evaluate the regulation of cyclin B, Cdc-2, p53, p21, and the Bcl-2 protein. RESULTS: AZ1 to AZ4 displayed various cytotoxicity activities against H460 and MRC-5 cells. Compared to those compounds, AZ4 was with the most effective agent among the 5 tested analogues at reducing H460 cell viability with an IC(50) value of 1.23 micromol/L; it also exhibited weak cytotoxicity against MRC-5 cells with an IC(50) value of 12.7 micromol/L. The results show that growth arrest on the G2-M phase of H460 cells induced by AZ4 for 24 h was discovered, and this might be altered with the reduced Cdc-2 protein expression of 47% at 2.0 micromol/L AZ4, but not with cyclin B protein expression. The AZ4 treated cells were then led to apoptosis after 48 h. This was associated with the activation of apoptotic enzyme caspase 3 and mediated by caspase 8, but not caspase 9 at various concentrations of AZ4 after being cultured for 48 h and 30 h, respectively. The anti-apoptotic protein (Bcl-2) expression in H460 cells altered by 39% with downregulation, and the p53 protein by 25% with upregulation after being cultured with 2.0 micromol/L AZ4 for 48 h. In a time-dependent manner, the expression of the p53 and p21 proteins were increased to the maximum at 24 h, and then decreased at 48. CONCLUSION: AZ4 represents a novel antitumor aziridinylnaphthoquinone with therapeutic potential against the non-small cell lung cancer cells.     

肿瘤坏死因子相关凋亡诱导配体诱导人脑胶质瘤细胞凋亡作用的研究

目的研究肿瘤坏死因子相关凋亡诱导配体（TRAIL）受体在不同级别胶质瘤中的表达及TRAIL对原代培养的胶质瘤细胞凋亡的诱导作用。方法应用原位杂交的方法检测TRAIL受体在45例胶质瘤和5例正常脑组织中的转录表达；分别对其中22例和3例进行原代培养，用流式细胞仪检测TRAIL作用后细胞的凋亡率。结果在45例胶质瘤中，DR4、DR5、DcR1、DcR2的mRNA的表达例数分别为38例、41例、40例、2例。成功培养出18例胶质瘤细胞和3例正常胶质细胞，TRAIL作用后凋亡率在80％以上、50％～80％、50％以下的例数分别为5例、9例、4例，正常胶质细胞无明显凋亡发生。结论TRAIL受体的表达与肿瘤的恶性程度无关；TRAIL能有效地选择性杀死胶质瘤细胞。     

疫苗生产用细胞基质外源病毒检查的阳性对照病毒感染复数的筛选

目的 筛选阳性对照病毒在不同细胞基质中的最适感染复数（multiplicity of infection,MOI）,以优化水痘减毒活疫苗外源病毒因子检查法（细胞培养法）。方法 致细胞病变观察：将水痘-带状疱疹病毒以0.005、0.010、0.020的MOI分别接种至Vero、2BS和MRC-5细胞,培养7 d后观察细胞病变,病变较典型时对应的MOI即为最适MOI。血细胞吸附观察：将牛副流感病毒3型以0.1、0.2、0.4的MOI分别接种至Vero、2BS、MRC-5细胞,培养7 d后分别于2~8 ℃和20~25 ℃放置30 min,观察血细胞吸附,血细胞吸附较典型时对应的MOI即为最适MOI。结果 对Vero、2BS、MRC-5细胞,当水痘-带状疱疹病毒MOI分别为0.020、0.010、0.005时,30%~50%细胞出现典型病变;对Vero、2BS、MRC-5细胞,当牛副流感病毒3型MOI分别为0.1、0.2、0.4时,30%~50%细胞出现典型血细胞吸附。结论 水痘-带状疱疹病毒感染Vero、2BS和MRC-5细胞的最适MOI分别为0.020、0.010、0.005;牛副流感病毒3型感染Vero、2BS和MRC-5细胞的最适MOI分别为0.1、0.2、0.4。     

P-glycoprotein enhances TRAIL-triggered apoptosis in multidrug resistant cancer cells by interacting with the death receptor DR5

The death-inducing cytokine TRAIL is a promising agent for anticancer therapy since it preferentially kills cancer versus normal cells; however, some cancer cells are TRAIL-resistant. We initially explored whether overexpression of the MDR1 gene product P-glycoprotein (P-gp), which causes multidrug resistance (MDR) in cancer cells, also contributes to TRAIL-resistance. Surprisingly, our results revealed that P-gp-overexpression enhances TRAIL-induced apoptosis not only in neoplastic cells transfected with the MDR1 gene but also in MDR variants selected with cytotoxic anticancer agents. Mechanistic analysis of TRAIL-induced apoptosis in the MDR1-transfected MCF-7 breast cancer cell line BC-19 revealed that TRAIL-triggered significantly more apoptosis in these cells compared with parental MCF-7 cells by binding to the TRAIL receptor DR5. DR5 but not DR4 engagement by TRAIL attenuated cellular ATP levels by robustly stimulating P-gp ATPase activity, and thus triggered P-gp-dependent apoptosis by depletion of the cellular ATP pool. In addition to hyperactive P-gp-mediated ATP hydrolysis, TRAIL-induced, P-gp-potentiated apoptosis was associated with activation of caspases-6, -7, -8, and -9; Bid cleavage; and mitochondrial depolarization. P-gp interacted with the TRAIL receptors DR4, DR5, and DcR1 in plasma membranes and enhanced TRAIL binding to DR5. Interestingly, the decreased level of the decoy TRAIL receptor, DcR1, in BC-19 cells further sensitized these cells to TRAIL. Therefore, both extrinsic and intrinsic apoptosis pathways are involved in this process. These findings for the first time reveal that TRAIL treatment preferentially causes apoptosis in P-gp-overexpressing MDR cells, and suggests significant clinical implications for the use of TRAIL in treating neoplasms that have failed chemotherapy.     

Notoginsenoside R1 up-regulates microRNA-132 to protect human lung fibroblast MRC-5 cells from lipopolysaccharide-caused injury

BackgroundPneumonia is a common lung disease in children with high fatality rate. Notoginsenoside R1 (NGR1) is the main active component extracted from the roots of Panax notoginseng (Burk.) F.H. Chen (Araliaceae). Here, we carefully explored the potential anti-inflammatory and protective effects of NGR1 on lipopolysaccharide (LPS)-induced lung fibroblast MRC-5 cell injury.MethodsViability and apoptosis of MRC-5 cells after different treatment or transfection were respectively assessed using CCK-8 assay and Annexin V-FITC/PI staining. The expression levels of microRNA-132 (miR-132), IL-1β, IL-6 and TNF-α in MRC-5 cells were measured using qRT-PCR. MicroRNA transfection was conducted to reduce the expression level of miR-132. Western blotting was used to analyze the protein expression levels of key factors involving in cell proliferation, apoptosis, NF-κB pathway and JNK pathway.ResultsLPS treatment caused MRC-5 cell proliferation inhibition, apoptosis and over-production of inflammatory cytokines. NGR1 treatment had no significant effects on MRC-5 cell proliferation, apoptosis and production of inflammatory cytokines, but protected MRC-5 cells from LPS-caused cell proliferation inhibition, apoptosis and over-production of inflammatory cytokines. In addition, NGR1 increased the expression level of miR-132 in MRC-5 cells. Knockdown of miR-132 reversed the protective effects of NGR1 on LPS-treated MRC-5 cells. Furthermore, NGR1 attenuated LPS-activated NF-κB and JNK pathways in MRC-5 cells via up-regulation of miR-132.ConclusionThis research confirmed the protective roles of NGR1 in lung fibroblast cell inflammatory injury. NGR1 protected MRC-5 cells from LPS-caused inflammatory injury through up-regulating miR-132 and then inactivating NF-κB and JNK pathways.     

和厚朴酚及厚朴酚抗肺癌药理作用及机制的研究进展

和厚朴酚及厚朴酚是疏水性烯丙基联苯酚类结构的同分异构体,均有抗肺癌药理作用。和厚朴酚能抑制肺癌A549细胞、H1299细胞和Lewis细胞移植瘤在小鼠体内的生长和转移,而厚朴酚能预防乌拉坦诱导小鼠发生肺癌和抑制A549细胞移植瘤在小鼠体内生长。和厚朴酚及厚朴酚在体外能浓度相关地抑制人小细胞肺癌N446细胞、H446细胞,非小细胞肺癌中的肺腺癌A549、H1299、H441、H1975、Calu3、A2、PC、SPC-A-1、95D细胞,大细胞肺癌H460细胞,人肺鳞癌H226、H520、CH27细胞以及小鼠肺癌Lewis细胞增殖,并诱导其凋亡和坏死。和厚朴酚及厚朴酚抑制肺癌的主要机制是：（1）抑制I型去乙酰化酶的表达和酶活性,上调死亡受体-5（DR5）的表达,激活肿瘤坏死因子相关的凋亡诱导配体（TRAIL）信号通路;（2）激活与半胱天冬酶无关的凋亡通路;（3）阻滞癌细胞中的微管聚合,破坏微管结构;（4）阻滞蛋白激酶B（AKT）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路,诱导癌细胞自噬;（5）阻滞磷脂酰肌醇-3激酶（PI3K）/AKT信号通路而抑制癌细胞转移。