| Objectiv: Recently, melanoma particularity antigen-1( MAGE-1), the BAGE, GAGE and the LAGE/ NY- ESO-1 the cancer / testicle antigen gene( cancer- testis antigen, CTA) authenticates the enunciation, its common characteristics expresses in various tumors organize, but testicle with placenta excepted of normal organizes to all have no the expression.Tumor/ testicle antigen has the tumor particularity, the gene's outcome is the gene of target that a kind of tumor reject the antigen, can induce the lymphoid cell( CTL) in T in toxicity in cell particularity identify with kill the tumor cell, this characteristics may make the CTA become the tumor immunity treatment, and the earlier period in tumor examines a patient and the under medical treatment has the important meaning.The cancer- associated gene( CAGE) is a new gene of CTA that 2002 Cho for someone the detection, erupting the CAGE to all have the excessiveness expression in stomach cancer organize, temple neck cancer organize, lung cancer organize and various tumors cell , among them colorectal cancer cell department 50% expresses, but they to should of normal organizes and did not see the CAGE expression.But did not do the colorectal cancer organizes and the adenoma studies, the current domestic not yet discovers research concerning CAGE and report.The RT- PCR( reverse transcription- PCR) method examination CAGE of this research application half fixed amount compare its the difference of expression in colorectal cancer, adenoma and normal expression in the mucosa in large intestines;The applied MSP( methylation- specific PCR) method examination the circumstance of CAGE promoter methylation in colorectal cancer, adenoma and normal .To understand CAGE is in earlier period in cancer in large intestine occurrence, development and cell propagate of function.The antigen that CAGE code is a new tumor/ testicle antigen.The detection between new tumor in conduct and actions in this antigen particularity antigen and its coding gene, the research offering the new path with the tumor immunity diagnosis and therapy. CAGE may become a new biomarker that earlier period in cancer, also may become a kind of new path that tumor cure.Materials and methods:RT- PCR Tissue Samples:Forty-six specimens were obtained from patients who underwent colorectalscopy at PLA202 hospital.Eath tissue sampl was immediately frozen in liquid nitrogen and stored at -70℃ until use for analyses by RT-PCR.RNA Exteaction and RT-PCR Analysis The total RNA withdraws to press TRIZOL manual proceeds. CDNA was generated using avian myeloblastosis virus reverse transcriptase.RT were performed in a total volume of 20μ l of reaction mixture. 37℃ 1h,90℃ 5min. CAGE PCRs were performed in a total volume of 25 μ l. 30 cycles,94℃ 30s；57℃ 30s；72℃ 60s；72℃4min.The sequences of these PCR primer were as follows: CAGE(300 bp) sense primer 5 ′- GGT GCC GAT ACT CCC ACT AT-3′; antisense primer 5 ′- TTG CTT CAG ATT CCC CGT TT-3′. GAPDH (230 bp), sense primer 5 ′- TGA TTT TGG AGG GAT CTC GC-3′, antisense primer 5 ′- ACG GAT TTG GTC GTA TTG GG-3′.In the next step,10μ l of PCR product were electrophoresed on 2% gelose gels and stained with ethidium bromide.The PCR products were scanned by densitometry. MSP( methylation- specific PCR) Tissue Samples:Forty-six specimens were obtained from patients who underwent colorectalscopy at PLA202 hospital.Eath tissue sampl was immediately frozen in liquid nitrogen and stored at -70℃ until use for analyses by MSP.Proceed the DNA modifier according to the Herman method.MSP were performed in a total volume of 50μ l. 95℃ 5min,30cycles,94℃30s；57℃30s；72℃ 60s；72℃ 4min。The sequences of these PCR CpG island methylation primer were as follows: sense primer 5 ′- TTT TAT ACG ATT CGG AAT TCG AC-3′, antisense primer 5 ′- CAA ATC TAC GAC CTA TTT CCC G-3′ ;CpG island demethylation primer sense primer 5 ′- GTT TTT TAT ATG ATT TGG AAT TTG AT-3′, antisense primer 5 ′- AAT TCA AAT CTA CAA CCT ATT TCC CA-3′.In th... |
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