目的探讨O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)在膀胱移行细胞癌(transitional cell carcinoma of bladder,TCCB)中的表达及意义。方法采用免疫组织化学法检测80例膀胱移行细胞癌组织,30例癌旁组织及20例正常膀胱组织中的MGMT表达,分析其...目的探讨O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)在膀胱移行细胞癌(transitional cell carcinoma of bladder,TCCB)中的表达及意义。方法采用免疫组织化学法检测80例膀胱移行细胞癌组织,30例癌旁组织及20例正常膀胱组织中的MGMT表达,分析其与临床病理的相关性及意义。结果MGMT在膀胱移行细胞癌组织中的阳性表达率为32.5%(26/80),显著低于癌旁组织(76.7%,23/30)和膀胱正常组织(65.0%,13/20);膀胱移行细胞癌分化Ⅰ级的阳性表达率为(48.6%,18/37),显著高于Ⅱ级(23.1%,6/26)和Ⅲ级(11.8%,2/17);但MGMT表达与膀胱癌临床分期、患者性别、患者年龄、肿瘤数量、发病次数均无显著相关。结论 MGMT可能成为膀胱癌诊断及判断预后的标志物之一。展开更多
Objective Using template switch mechanism at the 5’ end of mRNA technique (SMART) to construct a full length cDNA library of human normal bladder tissue. Methods The novel procedures used the template switchin...Objective Using template switch mechanism at the 5’ end of mRNA technique (SMART) to construct a full length cDNA library of human normal bladder tissue. Methods The novel procedures used the template switching activity of powerscript reverse transcriptase to synthesize and anchor first strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo(dT) primer and an anchor primer to enrich the full length cDNA population with 1.0 g human normal bladder poly(A) + RNA, then double strand cDNA was synthesized. After digestion with sfiI and size fractionation by CHROMA SPIN 400 columns, double strand cDNA was ligated into λ TripIEx 2 vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results The titer of the cDNA library constructed was 2.1×10 6 pfu·mL -1 , and the amplified cDNA library was 6×10 11 pfu·mL -1 , the percentage of recombination clones was 99%. Conclusion Using SMART technique helps us to construct full length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.展开更多
文摘Objective Using template switch mechanism at the 5’ end of mRNA technique (SMART) to construct a full length cDNA library of human normal bladder tissue. Methods The novel procedures used the template switching activity of powerscript reverse transcriptase to synthesize and anchor first strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo(dT) primer and an anchor primer to enrich the full length cDNA population with 1.0 g human normal bladder poly(A) + RNA, then double strand cDNA was synthesized. After digestion with sfiI and size fractionation by CHROMA SPIN 400 columns, double strand cDNA was ligated into λ TripIEx 2 vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results The titer of the cDNA library constructed was 2.1×10 6 pfu·mL -1 , and the amplified cDNA library was 6×10 11 pfu·mL -1 , the percentage of recombination clones was 99%. Conclusion Using SMART technique helps us to construct full length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.
