| IntroductionTwo hit hypothesis (Knudson, 1971) is a scientific prophecy about the mechanism of tumorigenesis which is generally accepted by cancer geneticists, and has directed the study of molecular mechanism of tumorigenesis for morethan 20 years. Mutation refers to those of tumor related genes-oncogenes andtumor suppressor genes (TSG). Till now, more than 100 oncogenes and 20 TSGs have been found. In different developmental stages, different oncogenes and TSGs are involved in various tumors. Tumorigenesis is a complex multi-stages process in which multi-genes are involved. Therefore, the detection of oncogenes and TSGs in different tumors is the basic prerequisite to understand the mechanism of tumorigenesis and to conduct gene diagnosis and gene therapy. Cloning and location of pathogenetic genes including tumor and other diseases are one of the major contents in human genome project. Because of the advantage of patients' resources in our country, cloning and location of pathogenetic genes according to our developmental status of genetics are of hot spots.As the majority of head and neck cancer, laryngeal carcinoma, which is common in upper respiratory tract, accounts for 1%-8. 4% of human malignant tumors. Clinical data showed a tendency of increasing incidence in Northeast part of China these years, which may relate to smoking, drinking and cold weather. Although the patients' pain can be released in some degree by surgery, radiotherapy and chemotherapy, the malfunction and local deformity after treatment and the high recurrent rate threaten the health of patients physically and mentally. There are few data about molecular genetics study in laryngeal cancer at present. Although some authors reported that laryngeal cancer was related to oncogenes ( ras, c-myc, EGFR, PRAD, Int-2, etc ) and TSGs ( p53, Rb,p 16, etc) , no clear report about gene cloning related to laryngeal carcinoma was reported. It is important for us to clone and locate laryngeal cancer-related genes by taking full advantage of patients'resources in our country and participating in human genome project so as to fill in the gaps in this field.In our study, we used cDNA-Representational Difference Analysis method to look for and identify novel genes related to laryngeal squamous cell carcinoma, and it would provide target genes for gene diagnosis and gene therapy.Materials and methodsSamplesMatched normal and tumorous tissues from patients who suffered from laryngeal carcinoma were pathologically confirmed. Manual microdissection confirmed that the cancerous cell accounted for more than 80% of total cells. cDNA-Representational Difference Analysis, Cloning and SequencingTotal RNAs were extracted from the samples mentioned above by TRIzol reagent ( GIBCO BRL Co. ) , then mRNAs were isolated by mRNA purification kit (Promega). Double strands cDNA were synthesed, then cutted by Mbo I (Promega) , ligated to R-Bgl-12/24 adaptor after purification. Oligonucleotides were annealed to each other, then incubated for 12 h at 12 - 16 C. Multiple PCR reactions were set up to generate the initial representations after dilution. The R-adaptors ( derived from cancerous tissues) were removed from the representations with same endonuclease, and the digest was phenol extracted and ethol precipitated to form driver, and the tester ( derived from normal tissues) was ligated to the J-Bgl-12/24 adaptor in the manner described above. The first subtractive hybridization was carried out at the ratio of 1: 100, in 41 EPPS buffer at 61 C for 20 h, then the diluted hybridization mix was amplified, 18 cycles (3 min, 95 C;1 min 72 C) ,the 24 bp adaptors acted as primers, then got the first differential products (Dp1). The latter two rounds of subtractive hybridization were performed at the ratio of 1:400 and 1 : 80000 respectively. Dp3 was composed of four fragments and they were purified and cloned into pGEM-T vec-tor. The four clones were then sequenced by ABI 377 Genetic analyser or artificial sequencing after confirmed by c...
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