| It is a multiple steps, multiple phases, progressing procession of the tumourgenesis, and it is a type of gene disease involved in activation of oncogenes and inactivation of tumour suppressors. However, it is not a single gene which can determine the tumourgenesis and maintain the malignant phenotype,many genes and the regulation and control among them involve in this procession.Following the implementation of Genome Project and the development of technology in molecular biology,people peered more secrets and deeper understanding about gene sequence ,but the exactfunction of genes are unclear.Therefore,it is a very important subject in new era for scientists to pay much more attentions on exploring genes and to conduct the genes how to organize,how to regulate during developing and how to expression in special time and space structure, and so on.ING1 (inhibitor of growth 1), A new candidate tumour suppressor, was found and located in the subtelomeric region of human chromosome 13q33-34,whose suppression is associated with increased proliferation and immortalization. This growth inhibitor participates in cell cycle regulation and overexpression of ING1 from transfected DNA constructs efficiently decreasing S-phase fraction by blocking the entry of cell into S-phase and inhibiting normal cell growth. The repression of ING1 expression frequently accompanies tumour development of breast cancer, stomach cancer, lymphoma, and so on. It was ' reported that the growth -inhibitory effects of P33 ING1, the expression product of ING1, directly cooperates with P53 protein in vivo. However, the expression of ING1 gene in tissue of pulmonary carcinoma is unknown. The P53 protein plays a core role in the regulation of cell proliferation, apoptosis and celluar aging, so there is a most closely relationship between P53 and human tumour. Some of the investigations showed that the mutation of p53 gene is anearly molecular event in the occurence of lung cancer, it is very important of P53 for early forecasting of lung cancer. However, there still have been about 50% cases of lung cancer in which the mutation of p53 gene has not been appeared. Studies on P53 protein showed that its function of suppressing tumour growth is regulated and controled by other genes or gene's functional products. Now, it is still unclear that the complicated regulation network around P53 protein, so any efforts of discovering one of those links will really do benefit to deeper understanding of the occurrence of lung cancer. For this reason, Western blot was used to detect the protein expression of P33 ING1 in lung cancer tissues firstly and the tissues neighbored to lung cancer cube, and to unveil the relationship between P33 ING1 and P53 protein. Methods and materials:1. Collection of samples: Three groups, tumour tissues(A), tissues which were 3cm(B) and 5cm(C) from tumour cubes, were obtained from patients with lung cancer. All tissues were frozen in liquid nitorgen immediately after surgery and then stored at -80 until the extraction of protein.2. Tissues protein extraction: Weighing 0.1g-0.5g lung tissue, washed with ice cold PBS buffer, grinded immediately in lysis buffer at 0 ,then added 2 X SDSsample buffer with equal volume to lysis buffer, heated in . boiling water for 10min,sonicated for disjunction of DNA, the supernatant was collected after centrifugation at 10,000rpm for 10min at room temperature, the extracted protein was in supernatant, and stored at -20.3. Protein separation and membrane Blot: Protein samples were mixed with an equal volume of sample loading buffer, samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) and transfered to a nitrocellulose membranes.4. Hybridization and staining: The membrane was blocked . with 20% fetal bovine serum in PBS and incubated with primary antiboy at room temperature for 1 hour. Protein bands in membrane were stained with a horseradish peroxidase-conjugat... |
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