目的探讨肺癌患者痰标本中FH IT、P16、MGMT、RASSF1A和APC等抑癌基因启动子异常甲基化及其联合检测在肺癌筛查及早期诊断中的价值。方法采用甲基化特异性PCR(methylation specific PCR,MSP)法,检测47例肺癌组织及对应的痰标本FHIT、P16...目的探讨肺癌患者痰标本中FH IT、P16、MGMT、RASSF1A和APC等抑癌基因启动子异常甲基化及其联合检测在肺癌筛查及早期诊断中的价值。方法采用甲基化特异性PCR(methylation specific PCR,MSP)法,检测47例肺癌组织及对应的痰标本FHIT、P16,MGMT、RASSF1A和APC基因启动子区甲基化状态。24例肺良性疾病患者痰标本作为对照。结果 47例肺癌组织标本中FHIT、P16、MGMT、RASSF1A、APC基因启动子区甲基化检出率分别为40.4%(19/47)、53.2%(25/47)、36.2%(17/47)、21.3%(10/47)和38.3%(18/47);对照的痰标本中五者甲基化检出率分别为38.3%(18/47)、48.9%(23/47)、36.2%(17/47)、17.0%(8/47)和29.8%(14/47),两组甲基化检出率存在着一致性[P>0.05;κ(0.8～1.0)]。24例肺良性病变痰标本中未检测到任何异常甲基化,与肺癌组比较差异有统计学意义(P<0.05)。五项指标联合检测可明显提高肺癌检测的灵敏度(80.9%)和特异度(100.0%)。FHIT和P16基因痰标本甲基化检出率与患者吸烟指数有相关性(P<0.05)。结论痰标本中多个肺癌相关基因甲基化联合检测有望成为肺癌筛查、早期诊断简便有效的指标。展开更多
Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O^6-methylguanine-DNA-methyltrans (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-1-Nitros...Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O^6-methylguanine-DNA-methyltrans (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT- PCR method from total RNA of fresh human liven the fragment was cloned into PGEM-T vector and further subcloned into GINa retrovirus vector. Then the GINaMGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8x105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT- cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.展开更多
目的探讨O6-甲基鸟嘌呤-DNA甲基转移酶(O6-m ethylguan ine-DNA m ethyltransferase,MGMT)基因不同位点的甲基化与结、直肠癌发生、进展的关系。方法应用DNA微阵列技术对20例结、直肠癌标本及癌旁组织进行MGMT基因启动子区5种高甲基化...目的探讨O6-甲基鸟嘌呤-DNA甲基转移酶(O6-m ethylguan ine-DNA m ethyltransferase,MGMT)基因不同位点的甲基化与结、直肠癌发生、进展的关系。方法应用DNA微阵列技术对20例结、直肠癌标本及癌旁组织进行MGMT基因启动子区5种高甲基化模式的定量检测分析。结果20例样本中有17例无论是肿瘤组织还是癌旁组织其各位点均有不同程度的甲基化,且肿瘤组织启动子区各CpG位点甲基化水平均高于其对应的癌旁组织(P<0.05),其中CpG13-15、20-23位点甲基化水平明显增高。MGMT基因各CpG位点的甲基化水平在不同的病理分级和肿瘤淋巴结转移中差异无显著性(均P>0.05)。结论结、直肠癌中存在高频MGMT基因启动子高甲基化,且其甲基化模式不止一种。MGMT基因表型遗传性失活可能在结、直肠肿瘤发生过程中起重要作用。MGMT基因启动子的异常甲基化与肿瘤发展水平并无直接相关性。展开更多
文摘目的探讨肺癌患者痰标本中FH IT、P16、MGMT、RASSF1A和APC等抑癌基因启动子异常甲基化及其联合检测在肺癌筛查及早期诊断中的价值。方法采用甲基化特异性PCR(methylation specific PCR,MSP)法,检测47例肺癌组织及对应的痰标本FHIT、P16,MGMT、RASSF1A和APC基因启动子区甲基化状态。24例肺良性疾病患者痰标本作为对照。结果 47例肺癌组织标本中FHIT、P16、MGMT、RASSF1A、APC基因启动子区甲基化检出率分别为40.4%(19/47)、53.2%(25/47)、36.2%(17/47)、21.3%(10/47)和38.3%(18/47);对照的痰标本中五者甲基化检出率分别为38.3%(18/47)、48.9%(23/47)、36.2%(17/47)、17.0%(8/47)和29.8%(14/47),两组甲基化检出率存在着一致性[P>0.05;κ(0.8～1.0)]。24例肺良性病变痰标本中未检测到任何异常甲基化,与肺癌组比较差异有统计学意义(P<0.05)。五项指标联合检测可明显提高肺癌检测的灵敏度(80.9%)和特异度(100.0%)。FHIT和P16基因痰标本甲基化检出率与患者吸烟指数有相关性(P<0.05)。结论痰标本中多个肺癌相关基因甲基化联合检测有望成为肺癌筛查、早期诊断简便有效的指标。
文摘Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O^6-methylguanine-DNA-methyltrans (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT- PCR method from total RNA of fresh human liven the fragment was cloned into PGEM-T vector and further subcloned into GINa retrovirus vector. Then the GINaMGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8x105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT- cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.
文摘目的探讨O6-甲基鸟嘌呤-DNA甲基转移酶(O6-m ethylguan ine-DNA m ethyltransferase,MGMT)基因不同位点的甲基化与结、直肠癌发生、进展的关系。方法应用DNA微阵列技术对20例结、直肠癌标本及癌旁组织进行MGMT基因启动子区5种高甲基化模式的定量检测分析。结果20例样本中有17例无论是肿瘤组织还是癌旁组织其各位点均有不同程度的甲基化,且肿瘤组织启动子区各CpG位点甲基化水平均高于其对应的癌旁组织(P<0.05),其中CpG13-15、20-23位点甲基化水平明显增高。MGMT基因各CpG位点的甲基化水平在不同的病理分级和肿瘤淋巴结转移中差异无显著性(均P>0.05)。结论结、直肠癌中存在高频MGMT基因启动子高甲基化,且其甲基化模式不止一种。MGMT基因表型遗传性失活可能在结、直肠肿瘤发生过程中起重要作用。MGMT基因启动子的异常甲基化与肿瘤发展水平并无直接相关性。
