Angiostatin基因真核表达载体的构建及对B16黑色素瘤体内生长抑制作用

Angiostatin是一种新发现的对肿瘤生长有特异抑制作用的抗血管生成因子,实验已证实其对多种肿瘤有明显的抑制作用.本文报告构建了含Angiostatin基因的真核表达载体pAG3,通过建立荷瘤小鼠模型来研究Angio-statin对人黑色素瘤B16的原位生长,植入及与化疗药物DTIC的联合作用等来探讨Angiostatin裸DNA肌肉注射的体内抗瘤效应.实验结果表明Angiostatin可明显抑制C57荷瘤小鼠的肿瘤生长;人黑色素瘤B16细胞植入前5天肌肉注射pAG3能显著阻止正常C57小鼠新肿瘤的形成;但在pAG3与DTIC联合化疗实验中,两者未表现出明显的增强效应.本实验为拓展非病毒介导的Angiostatin抗血管生成基因治疗途径奠定了基础.     

逆病毒载体介导双耐药基因在脐血CD34+细胞中的表达和抗性研究

为探讨转染醛脱氢酶基因(ALDHl)和多药耐药基因(MDRl)的人脐血CD34+细胞能否同时增强对活性环磷酰胺(4-HC)和MDRl基因靶药的抗性,构建了同时含ALDHl和MDRl双耐药基因的逆转录病毒表达质粒GlNa-ALDHl-IRES-MDRl,经LipofectAMINE介导转染GP+E86和PA317包装细胞,采用含长春新碱(VCR)和4-HC的培养基克隆选择后收集重组病毒上清于单向型GP+E86与双嗜型PA317包装细胞行乒乓交互感染,获得PA317重组病毒生产细胞(最高滴度达5.6×105CFU／ml),将含ALDHl和MDRl双耐药基因重组病毒的上清在细胞生长因子刺激下重复感染人脐血CD34+细胞,用PCR、RT-PCR、Southernblot、Northernblot、FACS和MTT等方法检测外源ALDHl与MDRl基因在CD34+细胞中的转移和表达。结果显示逆转录病毒载体介导的双耐药基因已经整合人转染靶细胞基因组并获得有效表达,同时传递不同的耐药表型。经双耐药基因修饰的脐血CD34+细胞对4-HC和VCR药物同时产生抗性,其IC50值分别比未转染细胞高4倍和7.2倍,本研究为开展肿瘤基因治疗的临床研究奠定了实验基础。     

逆病毒载体介导双耐药基因在脐血CD34^＋细胞中的表达和抗性研究

To explore whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH1) and multidrug resistance gene (MDR1) increase resistance to 4-Hyaroxycyclophosphophamide (4-HC) and P-Glycoprotein Effluxed Drugs, a bicistronic Retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed. The vector was transduced into the packaging cell lines GP + E86 and PA317 by LipofectAMINE. Using the medium containing VCR and 4-HC for cloning selection and pingponging supernatant infection between ecotropic producer clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfectced repeatedly with supernatant of retrovirus containing human ALDH1 and MDR1cDNA under stimulation of hemopoietie growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT method analyses show that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4- to 7.2-folds stronger resistance to cyclophospsphamede and P-Glycoprotein Effluxes drug in comparison with the nontransduced cells. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial.     

逆转录病毒载体介导MGMT和MDR1基因增强人脐血CD34＋细胞对联合化疗抗性的研究

To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) and multidrug resistance gene (MDR1) increase resistance to 1,3-Bis(2-Chloroethy1)-1-Nitrosourea (BCNU) and P-glycoprotein effluxed drugs, the present authors obtained a full length cDNA fragment encoding MGMT from liver tissue of a patient with cholelithiasis by RT-PCR. A bicistronic retroviral vector G1Na-MGMT-IRES-MDR1 cDNA was constructed and transfected the packaging cell lines GP + E86 and PA317 by electric performation method, using the medium containing VCR and BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, cord blood CD34+ cells were enriched with a high-gradient magnetic cell sorting system (MACS), and then transfected repeatedly with supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hemapoietic growth factors. PCR, RT-PCR, Southern blot, Northern blot, Western blot, FACS and MTT assay were used to evaluate the transfer and expression of the double genes in cord blood CD34+ cells. The cDNA encoding MGMT was verified by DNA sequencing and the bicistronic retroviral vector was confirmed by restriction endonuclease analysis. The purity of cord blood CD34+ cells was approximately 92% and recover rate was 75%, the highest titer of recombinant amphotropic retrovirus in the supernatant was up to 5.8 x 10(5) cfu/ml. The efficiency of gene transduction was 18% and 20% tested by colony formation and PCR, respectively. No helper virus was found by both nested PCR and rescue assay. The results showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The MTT analysis showed a 4.5 to 7.8-fold increase of resistance of transducted cells to BCNU and P-glycoprotein effluxed drug as compared with the nontransduced cells. This study provided a foundation for ameliorating combination chemotherapy toxicity in tumor clinical trial.     

逆转录病毒载体介导MGMT和MDR1基因增强人脐血CD34+细胞对联合化疗抗性的研究

为探讨转染六氧甲基鸟嘌呤DNA甲基转移酶基因(MGMT)和多药耐药基因(MDR1)的人脐血CD34+细胞能否同时增强对卡氮芥(BCNU)和MDR1基因靶药的抗性,应用逆转录一聚合酶链反应(RT-PCR)从人肝组织中获得编码六氧甲基鸟嘌呤-DNA-甲基转移酶(MGMT)cDNA,构建双顺反子逆转录病毒载体G1Na-MGMT-IRES-MDR1,以电穿孔介导的基因转移法导入GP+E86和PA317病毒包装细胞,采用含BCNU和长春新碱(VCR)的培养基克隆选择后收集重组病毒上清于单向型GP+E86与双嗜型PA317包装细胞行乒乓交互感染,将含MGMT和MDR1双耐药基因重组病毒的上清在细胞生长因子刺激下重复感染经免疫磁珠分离系统(MACS)分离纯化后的人脐血CD34+细胞,用PCR,RT-PCR,Southern blot,Northern blot,FACS和MTT等方法检测外源MGMT与MDR1基因在CD34+细胞中的转移和表达.结果显示,DNA测序及酶切鉴定证实MGMTcDNA克隆和双顺反子逆转录病毒载体构建的正确性,MACS分离纯化后的人脐血CD34+细胞纯度平均达92%,回收率为75%,含双耐药基因重组病毒的上清最高滴度为5.8×105 cfu/ml,逆转录病毒载体介导的双耐药基因已整合入转染靶细胞中基因组并获得有效表达,同时传递不同的耐药表型,应用集落计数、PCR方法测定基因转导效率分别为18%和20%,巢式PCR及补救分析均未检测到辅助病毒存在,经双耐药基因修饰的脐血CD34+细胞对BCNU的IC50较对照组提高4.5倍,对VCR,柔红霉素(DNR)和秋水仙碱(COL)的IC50较未转染细胞分别高7.8,6.6和5.5倍.本研究对降低联合化疗骨髓毒性作用的肿瘤临床研究奠定了实验基础.     

Angiostatin基因真核表达载体的构建及对B16黑色素瘤体内生长抑制作用

Angiostatin是一种新发现的对肿瘤生长有特异抑制作用的抗血管生成因子,实验已证实其对多种肿瘤有明显的抑制作用。本文报告构建了含Angiostation基因的真核表达载体pAG3,通过建立荷瘤小鼠模型来研究Angiostation对人黑色素瘤B16的原位生长,植入及与化疗药物DTIC的联合作用等来探讨Angiostation裸DNA肌肉注射的体内抗瘤效应。实验结果表明Angiostation可明显抑制C57荷瘤小鼠的肿瘤生长;人黑色素瘤B16细胞植入前5天肌肉注射pAG3能显著阻止下C57小鼠新肿瘤的形成;但在pAG3与DTIC联合化疗实验中,两者未表现出明显的增强效应。本实验为拓展非病毒介导的Angiostation抗血管生成基因治疗途径奠定了基础。