CEA启动子控制HSV-TK基因表达对人结直肠癌细胞的杀伤作用

构建了以CEA启动子控制的HSV-TK基因表达质粒pCEA-TK。转染pCEA-TK的人结肠癌细胞LoVo对GCV的敏感性提高了1300倍。同样条件下,人宫颈癌细胞HeLa对GCV的敏感性仅提高8倍,且对低于血药浓度(20μmol/L)的GCV不敏感。以上结果显示在GCV存在时,CEA启动子控制下HSV-TK基因的表达使CEA阳性的人结直肠癌细胞获得专一性杀伤。此外,DNA片段分析和电镜观察表明GCV诱导转染pCEA-TK的LoVo细胞发生凋亡可能是这个系统杀死肿瘤细胞的机制之一。本工作还讨论了癌胚抗原(CEA)基因启动子用于人结直肠癌专一性自杀基因治疗的可能性。     

组织专一性表达自杀基因治疗人结肠癌的研究

构建了以癌胚抗原(CEA)基因启动子控制的HSV-TK和ECCD的表达质粒PCEA-TK和pCEA-CD. 将它们分别与pSV2-neo共转染人结肠癌细胞株LoVo和人宫颈癌细胞株HeLa. G418筛选得到细胞克隆LoVo/CEA-TK、toVo/CEA-CD、HeLa/CEA-TK和HeLa/CEA-CD. 与野生型LoVo细胞相比, LoVo/CEA-TK和LoVo/CEA-CD形态无明显改变, 生长曲线也相似, 但对GCV或5-FC的细胞毒的敏感性分别提高了2000倍或700倍.而HeLa/CEA-TK(或HeLa/CEA-CD)仍对低浓度GCV(或5-FC)不敏感. 以上结果显示了应用组织专一性表达的自杀基因治疗人结肠癌的可能性.     

Selective gene therapy for human lung adenocarcinoma by tumor-specific expression of herpes simplex virus thymidine kinase gene

According to the fact that CEA gene expressed only in lung adenocarcinoma but not in normal lung cells, a retroviral expression vector (pCEATK) of the herpes simplex virus thymidine kinase (HSV-TK) gene regulated by CEA promoter was constructed and introduced into CEA-producing human lung adenocarcinoma cells GL and non-CEA-producing HeLa cells. The expression of pCEATK and Ganciclovir (GCV) sensitivity of the transfected cells were tested in vitro and in vivo . pCEATK expressed only in CEA-producing GL cells but not in non-CEA-producing HeLa cells. The sensitivity to GCV of pCEATK-transfected GL was 992 times higher compared with that of the parental cell line and there was obvious "bystander effect" in vitro. HeLa cells transfected wtih pCEATK were still resistant to GCV. Injection of GCV resulted in significant regression of pCEATK-transfected GL tumors in nude mice. In addition, all mice with any fraction of GL cells expressing HSV-TK exhibited a significant reduction in tumor growth, including mice     

转TK基因的人结肠癌细胞对多种原药敏感性的研究

构建了含有单纯疱疹病毒胸苷激酶基因（HSV-TK）的重组逆转录病毒载体LTKSN．经PA317细胞包装后,感染人结肠癌细胞株LoVo．用G418筛选到稳定表达HSV-TK基因的细胞克隆LoVo／LTKSN．LoVo／LTKSN与野生型LoVo细胞相比,生长曲线无明显差异,细胞形态亦无改变．细胞毒试验证明LoVo／LTKSN对GCV的敏感性很高,半杀伤浓度IC50为0.5μmol/L,比野生型细胞提高了4000倍以上．三种不同的原药GCV,ACV和BVDU对LoLo／LTKSN具有效果不等的杀伤作用．BVDU和GCV联合作用效果更好．旁杀伤效果十分明显,低浓度GCV就可以将合10%LoVo／LTKSN的混合细胞中的大部分肿瘤细胞杀死．     

腺病毒载体介导CEA基因元件调控自杀基因治疗

构建以CEA启动子控制HSV-TK基因表达的复制缺陷型腺病毒载体(AdCEATK).纯化的重组腺病毒滴度达1×10¹²pfu/ml.CEA阴性的HeLa细胞感染AdCMVTK后对丙氧鸟苷(GCV)很敏感,而感染了AdCEATK后不被GCV杀伤.与此相反CEA阳性的LoVo细胞中AdCMVTK和AdCEATK都有很好的表达活性,说明CEA启动子有良好的细胞专一性.AdCEATK/GCV系统还有明显的旁杀伤效应.此载体将有助于实现对CEA阳性肿瘤的专一性自杀基因治疗.     

重组含HSV—TK基因腺病毒的构建及对肿瘤细胞的杀伤作用

High-titer replication-defective recombinant adenovirus expressing HSV-TK gene was constructed. Firstly, shuttle plasmid pAdCMVTK containing HSV-TK gene and CMV promoter was constructed and then recombined with right arm of adenovirus DNA. Secondly, the positive plaques containing recombinant adenovirus were identified and selected out by PCR and Southern blotting after infection into human embryo kidney 293 cells. The titer of recombinant adenovirus AdCMVTK was determined by plaque forming assay and it was as high as 10(12) pfu/ml. Tumor cells were infected with AdCMVTK and then treated with GCV. Cytotoxic effects were assayed with MTT method. HeLa, A549 and LoVo cells infected with AdCMVTK (M. O. I. = 100) became sensitive to the prodrug GCV, with IC50 less than 4 mumol/L. Significant bystander effect was observed. Results here show that the AdCMVTK/GCV system might be potential in the gene therapy for cancer.     

The anti-tumor effect of human monocyte-derived dendritic cells loaded with HSV-TK/GCV induced dying cells

Herpes simplex virus thymidine kinase (HSV-TK) gene and dendritic cells (DC) have been used as the pioneering in cancer therapy. HSV-TK gene can induce apoptosis and necrosis in tumor cells in the presence of the non-toxic prodrug ganciclovir (GCV). We investigated the anti-tumor effect of DC vaccination by introducing dying cells from HSV-TK gene treatment as an adjuvant. HepG₂-TK cell line was established by transfecting human hepatoma cell line HepG₂ (HLA-A₂ positive) with HSV-TK gene. Dying tumor cells were generated by culturing HepG₂-TK cells with GCV. After engulfed dying cells efficiently, immature DCs (imDC) derived from human monocytes were fully matured and elicited marked proliferation and cytotoxicity against HLA matched HepG₂ cells in autologous peripheral blood mononuclear cells (PBMC). It also implied that HepG₂ specific CTLs played an important role in the cytotoxicity which was primarily depended on Th1 responses. Given the feasibility of inducing dying cells by HSV-TK/GCV in vivo, our results suggest an effective method in clinical human hepatocellular carcinoma (HCC) treatment by an in vitro model of applying HSV-TK gene modified human tumor cells integrated with DC vaccination.     

PBI-SUR-TK载体靶向介导HSV-TK自杀基因诱导肝癌细胞凋亡

目的:构建Survivin启动子调控的表达载体,并检测在启动子调控下HSV-TK自杀基因对肝癌细胞HepG₂和正常肝细胞HL-7702凋亡的影响。方法:合成含TK基因的质粒PBI-SUR-TK,利用脂质体Lipofectamine 2000将其导入肝癌细胞和肝细胞。然后分别运用RT-PCR和Western blot特异性检测基因和蛋白的表达情况;利用CCK8方法检测细胞增殖情况,流式细胞仪上机检测细胞凋亡情况。结果:肝癌细胞转染组有更多的TK基因表达产物,增殖情况减弱,凋亡情况明显。结论:Survivin启动子驱动的HSV-TK/GCV自杀基因系统对肝癌可能有一定的治疗作用。     

DLC-1基因表达对结肠癌细胞侵袭转移能力的影响

目的:研究DLC-1基因对结肠癌细胞侵袭迁移能力的影响.方法:将DLC-1 shRNA(短发夹状RNA,short hairpin RNA)序列克隆到质粒pGCsi-U6/Neo载体,采用脂质体介导的转染方法将构建的DLC-1 shRNA表达质粒转入结肠癌细胞系LoVo细胞.采用RT-PCR技术和Western Blot技术分别检测LoVo细胞中DLC-1mRNA和蛋白表达水平的变化.Transwell小室人工重组基底膜侵袭转移实验观察LoVo细胞侵袭迁移能力的改变.结果:结肠癌细胞系LoVo细胞表达DLC-1分子.所构建质粒表达载体能有效地干扰LoVo细胞DLC-1 mRNA和蛋白质表达水平;Transwell小室人工重组基底膜侵袭转移实验结果显示,转染后LoVo细胞侵袭转移能力明显增强(p＜0.05).结论:结肠癌细胞系LoVo细胞表达DLC-1基因,应用RNAi技术可特异性降低其表达.DLC-1的表达水平与结肠癌细胞侵袭转移相关.     

癌胚抗原(CEA)启动子在肿瘤细胞中的特异表达及介异bak基因诱发凋亡

构建由癌胚抗原 (CEA)启动子控制报道基因增强型绿色荧光蛋白 (EGFP)表达的重组表达质粒pCEA EGFP .用转染细胞后检测荧光的方法对CEA阳性细胞进行简便、直观的检测 ,并结合流式细胞计数对CEA启动子在人结直肠腺癌细胞LS 1 74T、结肠癌细胞SW 4 80、肺腺癌细胞A5 49、人宫颈癌细胞HeLa和人喉癌细胞HEp 2中的活性进行了分析 ,发现其在SW4 80、LS 1 74T、A5 49中活性较强 ,而在HeLa和HEp 2中无活性 .构建由CEA启动子控制凋亡基因bak表达的重组表达质粒pCEA bak ,转染HeLa及SW 4 80细胞 ,用Hoechst332 5 8染色及PI染色 流式细胞计数分析的方法证明 ,pCEA bak转染能够特异性引起SW 4 80细胞的凋亡 .结果表明 ,CEA启动子具有很好的特异性 ,CEA介导bak基因的方法可望用于CEA阳性癌细胞的靶向性基因治疗 .