共查询到18条相似文献,搜索用时 140 毫秒
1.
HyTK基因转移联合ganciclovir对瘢痕成纤维细胞的体外杀伤作用 总被引:1,自引:0,他引:1
增生性瘢痕和瘢痕疙瘩的过度增生主要是由于高度增生活性的成纤维细胞的数量异常增多及细胞外基质合成增加所致.用逆转录病毒载体介导单纯疱疹病毒胸苷激酶基因(HSVtk)的转移,随后应用药物9-(1,3-二羟基-丙氧基-甲基)鸟嘌呤(ganciclovir,GCV)可选择性地杀死增生细胞.采用组织块贴壁法在体外原代培养成功增生性瘢痕病人的成纤维细胞(FB).重组逆转录病毒GTK转染FB细胞后,用hygromycinB筛选出阳性细胞克隆(FB/GTK),经PCR方法检测证明HyTK基因已成功地导入FB中,但不含可复制的辅助病毒.分别用不同浓度的GCV作用于FB/GTK及FB,光镜下观察不同时间后细胞形态变化及MTT法检测细胞活性.结果表明,GCV浓度大于0.1μmol/L时即对FB/GTK有显著的杀伤作用,且具有强有力的“旁观者效应” 相似文献
2.
利用微丝(microfilament,MF)解聚药物细胞松驰素B(cytochalasinB,CB)处理G_0期小鼠C_3H_(10)T_(1/2)成纤维细胞,对G_0至S期DNA合成,胸腺嘧啶核苷激酶(thymidinekinase,TK)活性、TK基因表达、钙调素(calmodulin,CaM)水平和一些细胞周期早期基因的表达进行了观察,G_0期细胞经3mg/LCB处理2h,促MF解聚增强了血清对S期细胞TK活性、TK基因表达和DNA合成的刺激作用,并促进细胞提前进入S期.血清刺激G_0期细胞进入晚G_1期和S期时,CaM水平明显升高,而CB预处理则使CaM含量进一步增加,特别是CB处理促使S期CaM增加向核内转移.CB处理明显增强血清对c-jun、c-fos和c-myc基因表达的刺激作用,而PKC抑制剂H_7则抑制CB处理对这些基因转录的刺激作用,说明CB使G_0期细胞MF解聚刺激c-jun、c-fos和c-myc的转录活性与PKC的作用有关.结果表明G_0至S期早期MF的重组可促进细胞进入S期,增强DNA合成. 相似文献
3.
腺病毒载体介导的肝癌细胞专一性自杀基因表达 总被引:5,自引:0,他引:5
构建由肝癌细胞专一的afp基因表达调节元件控制自杀基因HSV-tk的穿梭质粒,将它与缺陷型腺病毒载体重组,得到AdrAFPTK病毒。经PCR及Southern杂交等证实它们含afp元件和tk基因。空斑形成试验表明病毒效价达1×1015pfu/L。同时构建由CMV启动子控制tk基因的类似载体作为对照。将这两个重组腺病毒分别感染AFP阳性(HepG2)或阴性(HeLa,BRL-3A)细胞株(m.o.i.=100),以丙氧鸟苷(ganciclovir,GCV)处理后,用MTT法测定杀伤细胞的效应。结果,AdCMVTK感染这三种细胞后,GCV半杀伤浓度分别为1.3、2、<1μmol/L;但是,AdrAFPTK感染的HeLa和BRL-3A细胞的GCV半杀伤浓度都>1000μmol/L,而对HepG2细胞只有<1μmol/L,表现出极高的细胞专一性。重组腺病毒AdrAFPTK可望用于肝癌的专一性基因治疗 相似文献
4.
用逆转录病毒载体将单纯疱疹病毒胸苷激酶基因(HSVtk)导入恶性肿瘤细胞,随后可应用药物9-(1,3-二羟基-丙氧基-甲基)鸟嘌呤(ganciclovir,GCV)选择性地杀死肿瘤细胞.将HyTK基因替换逆转录病毒载体GlNa中的neo基因,构建成重组逆转录病毒载体GTK,转染混合包装细胞(双噬性PA317细胞和单噬性GP+E-86细胞),通过“乒乓效应”获得高滴度重组病毒.用该重组病毒转染小鼠恶性黑色素瘤细胞系B16细胞,用hygromycinB筛选出阳性细胞克隆(HyTK+),经PCR方法检测证明HyTK基因已成功地导入肿瘤细胞中,且不含可复制的辅助病毒.分别用不同浓度的GCV作用于HyTK-及HyTK+的B16细胞,光镜下观察24h和48h后细胞形态及进行活细胞计数.结果表明,GCV浓度大于0.1μmol/L时即对B16/HyTK+细胞有显著的杀伤作用 相似文献
5.
细胞松弛素B促微丝解聚对DNA合成的作用 总被引:2,自引:0,他引:2
利用微丝(MF)解聚药物细胞松弛素B(CB)处理G0期小鼠C3H10T1/2成纤维细胞,对G0至S期DNA合成,胸腺嘧啶核苷激酶(TK)活性、TK基因表达、钙调素(CaM)水平和一些细胞周期早期基因的表达进行了观察。G0期细胞经3mg/LCB处理2h,促MF解聚增强了血清对S期细胞TK活性、TK基因表达和DNA合成的刺激作用,并促进细胞提前进入S期。血清刺激G0期细胞进入晚G1期和S期时,CaM 相似文献
6.
目的和方法:将可产生含有HSVTK基因逆转录病毒的包装细胞与BEL7402肝癌细胞混合后,接种于裸鼠皮下,复制肝癌模型。确定HSVTK基因转移后,给予动物注射GCV,观察HSVTK/GCV系统基因治疗实验性肝癌的在体疗效。结果:在体条件下,逆转录病毒可将HSVTK基因部分转导至肝癌细胞;GCV作用后,TK+中瘤细胞的生长受到明显抑制;电镜观察发现,GCV杀伤TK+肿瘤细胞的作用主要体现在细胞核;虽然组化染色分析表明TK基因转移效率只有10%~30%,但测量肿瘤体积显示HSVTK/GCV系统杀伤肝癌细胞的效果依然明显,故其作用机制可与“旁观者效应”有关。结论:HSVTK/GCV“自杀”基因系统有可能成为基因治疗肝癌的有效方法 相似文献
7.
胸苷激酶基因治疗胃癌的体外实验 总被引:3,自引:0,他引:3
将单纯疱疹病毒胸苷激酶基因(HSV-tk)导入恶性肿瘤细胞,随后可应用药物丙氧鸟苷(ganciclovir, GCV)选择性杀死肿瘤细胞.构建了含胸苷激酶与潮霉素磷酸转移酶(hph)融和基因(HytK)的真核表达载体LXpsp-HytK.以脂质体(lipofectin)为介导,将这种质粒与仅含潮霉素B基因的质粒LXSH 分别转染胃癌细胞系BGC-823,用60 U/m l潮霉素B进行筛选,得到了可稳定传代的阳性克隆,分别命名为BGC-HytK 和BGC-Hy.三种细胞的生长曲线无明显差别.用不同浓度的GCV 分别作用于BGC-HytK, BGC-Hy 及BGC-823,0.02~200 μg/m l 的GCV 对BGC-HytK 细胞有明显的杀伤作用(IC50= 0.02 μg/m l),而对另外两种细胞几乎无毒性作用(IC50> 200μg/m l).20 μg/m lGCV 作用96 h 后,仅存在20% 的BGC-HytK 就可使周围的大部分HSV-tk- 的肿瘤细胞死亡,说明存在较显著的“旁观者效应” 相似文献
8.
抗菌肽与碱性成纤维细胞生长因子基因的融合及其在酵母中的表达 总被引:8,自引:1,他引:8
从重组质粒rBS上切下柞蚕抗菌肽D基因片段,切去终止密码后连接到重组穿梭质粒pVT-GF上碱上成纤维细胞生长因子cDNA的5′端,使密码框正确排列,构建成融合 组质粒pVT-CDGF,转化到酵母中进行表达。转化子酵母蛋白粗提物用E.coliK12D31作指示菌进行抑菌圈测试,初步检出具有换菌活性,用ELISA检测证明其具有碱性成纤细胞生长因子的抗原性。 相似文献
9.
从重组质粒rBS上切下柞蚕抗菌肽D基因片段,切去终止密码后连接到重组穿梭质粒pVT-GF上碱性成纤维细胞生长因子cDNA的5′端,使密码框正确排列,构建成融合基因重组质粒pVT-CDGF,转化到酵母中进行表达。转化子酵母蛋白粗提物用E.coliK12D31作指示菌进行抑菌圈测试,初步检出具有抑菌活性,用ELISA检测证明其具有碱性成纤维细胞生长因子的抗原性。 相似文献
10.
MTT法测定眼镜蛇毒细胞毒素的抗癌活性 总被引:5,自引:1,他引:5
目的测定眼镜蛇毒细胞毒素(Cobravenomcytotoxin,CV-CTX)对体外培养的人癌细胞的杀伤作用。方法溴化二苯四偶氮盐(MTT)比色法。结果CV-CTX对SGC-7901,Bel-7402,K562和U9374种人癌细胞有很强的杀伤作用,量效关系明显,IC50分别为4.10,2.08,0.29和0.17μg/ml。结论MTT法检测CV-CTX的抗癌活性,操作简便、快速和准确。 相似文献
11.
为构建单纯疱疹病毒I型胸苷激酶(HSV1TK)的真核表达载体pcDNA3.1-EGFP/HSV1TK,鉴定其在真核细胞中的表达和功能.以pORF-HSV1TK为模板,PCR扩增的目的基因HSV1TK片段与pMD18-T载体相连接构建重组克隆pMD18-T/HSV1TK.再双酶切出HSV1TK片段,插入pcDNA3.1-EGFP多克隆位点,构建pcDNA3.1-EGFP/ HSV1TK真核表达载体并进行酶切、测序鉴定[1].分别用荧光显微镜观察和RT-PCR方法检测脂质体介导pcDNA3.1-EGFP/ HSV1TK在卵巢癌细胞SKOV3的表达;分别用MTT法和光镜检测胸苷激酶/丙氧鸟苷(HSV1TK/GCV)系统对SKOV3体外杀伤作用及旁观者效应.结果表明,重组载体酶切鉴定结果与预期结果一致,基因序列与GenBank上报道的HSV1TK基因序列完全一致.荧光显微镜观察转染后的细胞发出绿色荧光;RT-PCR结果表明HSV1TK基因能在SKOV3内有效表达.MTT和光镜结果显示转染HSV1TK基因的SKOV3细胞,加入前体药物丙氧鸟苷(GCV)处理后对其有明显的杀伤作用和旁观者效应.成功构建的真核表达载体pcDNA3.1-EGFP/ HSV1TK能在SKOV3细胞中稳定表达,且HSV1TK对卵巢癌细胞株SKOV3体外有强大的杀伤作用和旁观者效应. 相似文献
12.
Drake RR Wilbert TN Hinds TA Gilbert KM 《The Journal of biological chemistry》1999,274(52):37186-37192
The therapeutic combination of the herpesvirus simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and the prodrug, ganciclovir (GCV), has found great utility for the treatment of many types of cancer. After initial phosphorylation of GCV by HSV-1 TK, cellular kinases generate the toxic GCV-triphosphate metabolite that is incorporated into DNA and eventually leads to tumor cell death. The cellular and pharmacological mechanisms by which metabolites of GCV lead to cell death are still poorly defined. To begin to address these mechanisms, different mutated forms of HSV-1 TK at residue Gln-125 that have distinct substrate properties were expressed in mammalian cell lines. It was found that expression of the Asn-125 HSV-1 TK mutant in two cell lines, NIH3T3 and HCT-116, was equally effective as wild-type HSV-1 TK for metabolism and sensitivity to GCV, bystander effect killing and induction of apoptosis. The major difference between the two enzymes was the lack of deoxypyrimidine metabolism in the Asn-125 TK-expressing cells. In HCT-116 cells expressing the Glu-125 TK mutant, GCV metabolism was greatly attenuated, yet at higher GCV concentrations, cell sensitivity to the drug and bystander effect killing were diminished but still effective. Cell cycle analysis, 4', 6'-diamidine-2'-phenylindoledihydrochloride staining, and caspase 3 activation assays indicated different cell death responses in the Glu-125 TK-expressing cells as compared with the wild-type HSV-1 TK or Asn-125 TK-expressing cells. A mechanistic hypothesis to explain these results based on the differences in GCV-triphosphate metabolite levels is presented. 相似文献
13.
14.
Human herpesvirus 8-encoded thymidine kinase and phosphotransferase homologues confer sensitivity to ganciclovir
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Human herpesvirus 8 (HHV-8) sensitivity to the nucleoside analog ganciclovir (GCV) suggests the presence of a virally encoded kinase that catalyzes the initial phosphorylation of GCV. Analysis of the HHV-8 genome identified two candidate kinases: proteins encoded by open reading frame (ORF) 21, with homology to the herpesvirus thymidine kinases (TK), and ORF 36, with homology to the herpesvirus phosphotransferases (PT). Experiments presented here show that both ORF 21 and ORF 36 encode GCV kinase activities as demonstrated by GCV phosphorylation and GCV-mediated cell death. In both regards the PT homologue ORF 36 was more active than the TK homologue ORF 21. ORF 21, but not ORF 36, weakly sensitized cells to killing by penciclovir. Neither ORF sensitized cells to killing by (E)-5-(2-bromovinyl)-2'-deoxyuridine. 相似文献
15.
健脾化瘀中药提高胞嘧啶脱氨酶/单纯疱疹病毒胸苷激酶基因治疗肝细胞癌的实验研究 总被引:2,自引:0,他引:2
目的:观察健脾化瘀中药提高胞嘧啶脱氨酶/单纯疱疹病毒胸苷激酶基因治疗肝细胞癌的作用。方法:脂质体lipofectamine将含有双自杀基因的腺病毒载体pAd-CD/TK导人293细胞,收集病毒上清转染人肝癌细胞BEL7402,MTT法测定BEL7402细胞存活率。裸鼠人肝癌模型转染CD/TK双自杀基因后,给予5-FC500mg/kg,GCV 100mg/kg腹腔注射,同时予健脾化瘀中药960复方灌胃。观察肿瘤生长情况。结果:给予前体药物5-FC和GCV后,CD/TK转染细胞被杀死。并表现出较强的旁观者效应。转染细胞比例达到10%即表现出较强的杀伤作用(P<0.01)。健脾化瘀中药960复方具有提高旁观者效应作用,1.67ml/kg和2.5ml/kg960复方含药血清组细胞存活率显著低于对照组(P<0.01)。转染基因组应用5-FC和GCV治疗后,裸鼠肝癌的生长明显受到抑制(P<0.05),抑瘤率39.42%,单用中药组抑瘤率18.04%,中药与CD/TK 5-FC/GCV联合运用组,较单纯CD/5-FC/HSV-tk/GCV对裸鼠肿瘤模型的生长抑制作用更加明显(P<0.05),抑瘤率55.10%。结伦:腺病毒介导CD/TK自杀基因可有效地杀死人肝癌BEL7402细胞,健脾化瘀中药960复方具有显著提高CD/TK双自杀基因对人肝癌细胞的抑杀作用。 相似文献
16.
17.
Hayashi K Lee JB Maitani Y Toyooka N Nemoto H Hayashi T 《The journal of gene medicine》2006,8(8):1056-1067
BACKGROUND: The most extensively investigated strategy of suicide gene therapy for treatment of cancer is the transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of antiviral prodrugs such as acyclovir (ACV) and ganciclovir (GCV). The choice of the agent that can stimulate HSV-TK enzymatic activity is one of the determinants of the usefulness of this strategy. Previously, we found that a diterpenoid, scopadulciol (SDC), produced a significant increase in the active metabolite of ACV. This suggests that SDC may play a role in the HSV-TK/prodrug administration system. METHODS: The anticancer effect of SDC was evaluated in HSV-TK-expressing (TK+) cancer cells and nude mice bearing TK+ tumors. In vitro and in vivo enzyme assays were performed using TK+ cells and tumors. The phosphorylation of ACV monophosphate (ACV-MP) was measured in TK- cell lysates. The pharmacokinetics of prodrugs was evaluated by calculating area-under-the-concentration-time-curve values. RESULTS: SDC stimulated HSV-TK activity in TK+ cells and tumors, and increased GCV-TP levels, while no effect of SDC was observed on the phosphorylation of ACV-MP to ACV-TP by cellular kinases. The SDC/prodrug combination altered the pharmacokinetics of the prodrugs. In accord with these findings, SDC enhanced significantly the cell-killing activity of prodrugs. The bystander effect was also significantly augmented by the combined treatment of ACV/GCV and SDC. CONCLUSIONS: SDC was shown to be effective in the HSV-TK/prodrug administration system and improved the efficiency of the bystander effect of ACV and GCV. The findings will be considerably valuable with respect to the use of GCV in lower doses and less toxic ACV. This novel strategy of drug combination could provide benefit to HSV-TK/prodrug gene therapy. 相似文献
18.
Balzarini J Liekens S Solaroli N El Omari K Stammers DK Karlsson A 《The Journal of biological chemistry》2006,281(28):19273-19279
Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least approximately 4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5'-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK). 相似文献