| Tumor is one of the diseases threating human life and health , and the incidence of the disease is increaing year after year. In our country , malignant tumors lead to the first death of all kinds of diseases. So, oncologists have been studying the causing of neoplasms. Now, the theory of oncogenes activity and tumor suppressor genes (TSG) inhibition can lead to cancer have been accepted. In the 1970s, oncologist bought up "two hit hypothesis" when they studied the genetic tumors , and found RB gene lost in all Retinoblastoma patients. From then on , the study on TSG is in the ascendant. It is the TSG that protects human from the threat of cancer.Ovarian cancer is one of the three malignant tumors of female reproduction tract. It shows no obvious syndrome in the early stage because the two ovaries are in deep pelvic cavity. When they are found and diagnosed, 70% of patients had been in late stage. Studing on ovarian cancer not only accounts for the mechanism of its being but also guarantees female health gready. Presendy , the study of ovarian cancer is mainly in three fields: genetic factor , environmental factor and endocrinic factor. The genetic study on ovarian cancer is mostly focused on some known gene which is expressed or lost in tissues while the studies about ovarian cancer genes cloning is very few at home. If we had efficient ways to separate and clone genes concerning ovarian cancer for further study , they would contribute to the genes diagnosis and therapy of ovarian cancer.In our study, we used cDNA - Representational Difference Analysis method to look for and identify genes related to ovarian cancer, and it would provide target genes for genes diagnosis and therapy.Materials and methodsSamples:Matched normal and tumor tissues from the same patient who suffered from serous cystadenocarcinoma in early stage were pathologically confirmed. The normal tissues also can be get from the patients who resected one side of the ovaries because of other diseases.cDNA - Representational Difference Analysis, Cloning and Sequencing.Total RNAs were extracted form the samples mentioned above by TRIzol reagent ( GIBCO BRL Co. ) , then mRNAs were isolated by mRNA purification kit (Promega). Double strands cDNA were synthesized, then cut by Dpn II (NEB) ; ligated to R - 12/24 adaptor after purification . Oligonucleotides were annealed to each other, then incubated for 12h at 12 -16C. 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 tissue) was ligated to the J - 12/24 adapjpr in the manner described above . The first subtractive hybridization was carried out at the ratio of 1:100, in 4uJ EPPS buffer at 67C for 20h, then the diluted hybridization mixure was amplified, 18 cycles(3min, 95C ; 1min 12C ) ,the 24bp adaptors acted as primers, then got the first differential products ( DP I ) . The latter two rounds of subtractive hybridization were performed at the ratio of 1 ;400 and 1:80,000 respectively. DP III was composed of three fragments which were purified and cloned into pMD - 18 vector. The three cloning were then sequenced by Jikang company after confirmed by cleaving with EcoR I and Hind III endonucleases.RT-PCR test:There are 5 pairs of sample, including the sample of this experiment, to be test for RT - PCR, and it proved that the different fragments originated in mRNA.ResultscDNA - Representational Difference Analysis and cloning, identifying and sequencing.We obtained three different cDNA fragments( DPIII -1 DPIII -2 DPIII -3) ,which then were purified and cloned into pMD - 18 vector. The recombi-nated vector was transformed into Top10 competent cells. The positive clones were sequenced, then we analysis the similarity of nucleic acid and amino acid with Blast similarity searching system in NCBI. The sequen...
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