| Lung cancer is one of the most malignant cancers. Its incidence rate and death rate have the tendency of increasing and the increasing extent is the most rapid among cancers. Its development is the process involved in multiple genes, multiple phases and multiple factors, and it is the consequence of the failure of controlling of the cell apoptosis and the cell proliferation. More than one gene takes part in this process and they form the network to regulate and control each other and finally lead to the carcinogenesis.NF-kB (Nuclear transcription factor-KB) is one of the most important transcription factors that exist in the cytoplasm of eukaryote. It is a protein family composed of complicated polypeptide subunits and including at least 5 members: RelA (P65X RelB, C-Rel, NF-kB 1 (P50/P105), NF-kB 2 (P52/P100) . NF-icB may suppress cell apoptosis, promote cell proliferation or directly adjust the expression of oncogene and thus lead to the carcinogenesis. It also connects with the sensitivity of tumor cells to chemotherapy. NF-kBP65 is an important member of the NF-KB family,which molecular weight is 65KD. High expression of NF-KBP65 has been found in large intestine cancer, liver cancer, osteosarcoma, pancreatic cancer, and so on. But its role and protein expression in lung cancer hasn't been well known.HSP70 (Heat shock protein 70) is the main protein that was generated when cell was in stress. The regulation of HSP70 expression mainly takes place at transcription level. Nowadays many studies found that HSP70 took part in the carcinogenesis and cell apoptosis procession, and it could also combine to the antigen peptide on tumor surface and take part in the immunization of tumor. High expression of HSP70 has been found in pancreatic cancer, larynx cancer, stomach cancer, and so on.It's already shown that NF-kB is relationship with HSP70 in stress, inflammation and other disorders. But there was no report about the relationship between them and their roles in the procession of lung cancer. Western blot was used in this study to detect the protein expression of NF-kBP65 and HSP70 in lung cancer tissues and the matched normal tissues, and the differential expression in different tissues types and their relationship with each other were analyzed. Methods and materials:1. Collection of samples: 43 cases of tumor tissues(C) and the matched normal tissues (N) were collected. Among them, 18 cases were squamous carcinoma, 16 cases wereadenocarcinoma, 5 cases were adenosquamous carcinoma, 3 cases were small cell lung cancer and 1 case was large cell lung cancer. All tissues were frozen in liquid nitorgen immediately after surgery and then stored at -80 ℃ until the extraction of protein.2. Extraction of tissues protein: Weighing 0.1g-0.5g lung tissue, washed with ice-cold PBS buffer, grinded immediately in lysis buffer at 0℃,then added equal volume of 2x SDS sample buffer to lysis buffer, heated in boiling water for 1 0min,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. Using SDS-PAGE to separate protein: Protein samples were mixed with an equalvolume of sample loading buffer, and then separated by 12% sodium dodecyl sulfate polyacrylamide gel eletrophoresis.4. Western blot: The protein in the gel was transfered to a nitrocellulose membrane. After that, the membrane was blocked with 0.05% Tween-20 in TBS, incubated with primary antibody, second antibody in turn, and at last stained with 3',3 diaminobenzidine(DAB).5. Imaging: Bio Imaging System was used to capture pictures and then GeneTools software was used to analyze the protein bands.6. Statistics methods: Data of the experiment was processed by SPSS software (10.0) . Results:1. Expression of NF-kBP65 protein in lung cancer tissuesIncreased expression of NF-KBP65 was found in 74.4%(32/43) of the lung cancer tissues. Significant difference of NF-KB...
|
PDF Full Text Request