| Cervical cancer is a malignant tumor that occurs in the vagina and cervix of the uterus.In China, cervical cancer is more common in multiple gynecological malignancies, ranking first, and the incidence of groups increasingly younger. A large number of studies show that cervical cancer is caused by the human papilloma virus(HPV), the virus can direct spread through skin to skin contact, and can be latent in the body for more than a decade, so early in the absence of clinical signs. With the continuous progress of the disease, the patient can show abnormal vaginal bleeding symptoms. The treatment is mainly to surgery, radiotherapy, chemotherapy and traditional Chinese medicine and Western medicine combined with treatment of comprehensive treatment, but in advanced and recurrent and metastasis in patients with therapeutic effect is unsatisfactory. Postoperative recurrence, metastasis, patient survival rate is not ideal, but also to bring harm and pain to the patient. Therefore, it is an important and urgent task for us to research a new way and method of cervical cancer. In recent years, with the continuous development and progress of biological science and technology, proteomics has been developed rapidly. Proteomics is the study of the cell or tissue from the whole level, in particular space and time, the composition of all proteins and the law of the interaction between them. According tothe content of proteomics research, it can be divided into two types: cell mapping proteomics and expression proteomics. The former focuses on the study of the interaction between proteins in order to understand the complex mechanism of intercellular signal transduction pathways. The latter is mainly to study the expression and changes of all proteins in cells or tissues under different body states. The emergence of proteomics suggests that the study of biology has entered the post genomic era. In the post genomic era, proteomics is the main research object. From the whole point of view, the composition and the dynamic changes of the cellular protein and the regularity of its activities are analyzed.Then in a normal protein expression on the basis of fingerprint, through the protein in serum and serum of normal group were compared and analyzed to screen out of specific proteins associated with the disease. These specific proteins can not only be used as a protein marker for early diagnosis of some diseases, but also can be used as a molecular target for drug development. In addition, through the study of the structure and function of the specific protein markers, we can also find the pathophysiological mechanism of the occurrence and development of the disease.Therefore, it is an effective way to screen specific protein markers and to explore the effective methods of drug molecular targets. In the previous study, relying on surface enhanced laser desorption ionization time of flight mass spectrometry(SELDI-TOF-MS) in Wilms tumor in children with serum and normal control group serum were tested and screened Wilms tumor serum in children with normal serum differential proteins. Screening of these differential proteins by gel electrophoresis(TRICINE-SDS-PAGE) technique. By matrix assisted laser desorption ionization tandem time of flight mass spectrometry(MALDI-TOF/TOF) technology for the identification of isolated and purified proteins, and ultimately identified as 6455.5Da M/Z peptide. This experimental research focuses on the Role and influence of the 6455.5Da M/Z peptide on cervical cancer cells. By using the indirect immunofluorescence technique of cells to track the location of the 6455.5Da M/Z peptide in cervical cancer cells. By with the technique of cytotoxicity test to evaluate the intervention effect of the 6455.5Da M/Z peptide on cervical cancer. Through the use of Western blot to analysis the target protein in cervical cancer cells expression variation,whichare extracted of cervical cancer cells after the the 6455.5Da M/Z peptide intervention.Cervical cancer cell cycle and apoptosis were determined by flow cytometry. The study will lay a foundation of further research for monitoring and evaluating mechanism of the treatment effect of the 6455.5Da M/Z peptide on cervical cancer, provides the new research methods and ideas for the follow-up treatment method of cervical cancer.Objective To study and identify the role and influence of the 6455.5Da M/Z peptide on cervical cancer, and to lay the foundation for the further research of the mechanism and effect of the 6455.5Da M/Z peptide in the abdominal solid tumors.Materials and Methods Materials Cervical cancer cell: Hela cell line, Siha cell line. Normal cells: 7702 liver cell line.These cell lines were purchased from the Shanghai cell bank of the Chinese Academy of Sciences. Methods 1.Indirect immunofluorescence technique Hela cells, Siha cells and 7702 cells were treated with different concentrations of fluorescent the 6455.5Da M/Z peptide with FITC, and the cell morphology was demonstrated by indirect immunofluorescence technique. 2.Cytotoxicity test--CCK8 test By using different concentrations of the peptide to deal with Hela cells, Siha cells, through Counting Kit-8 Cell(referred to as CCK-8) to detect and describe the cell growth curve, evaluating the effect of the 6455.5Da M/Z peptide on cervical cancer.3.Flow cytometry The Hela and Siha cells were prepared into single cell suspension respectively, then staining with a fluorescent dye(pyridine iodide(PI).The DNA content of cervical carcinoma cells were analysised.The cell population was divided into three subpopulations(G1 phase, S phase and G2).The content of DNA of cell can reflect the cell ploidy state, non ploidy cells are closely related to the degree of tumor malignant.By using the flow cytometry to calculate the proportion of tumor cells on cervical cancer that at the different stages.The flow cytometry can calculate the proportion of the tumor cells at the different stages.After the intervention of the 6455.5Da M/Z peptide,we can study the change of the proportion of the tumor cells.Therefor,we can monitor the therapeutic effect of the 6455.5Da M/Z peptide and guide the follow-up treatment. Combined membrane associated protein V isothiocyanate fluorescein annexin V-FITC with PI staining, we can distinguish the early apoptosis and late apoptotic cells and necrotic cells.According to the change of the proportion of the three types of cells that after the intervention of the 6455.5Da M/Z peptide,we can lay the foundation for the further research of the mechanism of the 6455.5Da M/Z peptide for cervical cancer. 4.Western Blot By using the technology of the polyacrylamide gel separation,transferred the target protein to nitrate fiber membrane, then combined specific antibody and second antibody with the nitrate fiber membrane,finally imaging pictures with hair chromogenic substrate. Through the technology of western blot,we can obtain the expression of the target protein in the cervical cancer cells and analysis the expression of target protein from the location and staining of the staining of the nitric acid fiber film,in order to learn the effect of the 6455.5Da M/Z peptide.Results 1.Indirect immunofluorescence results of cervical cancer cells The content of the 6455.5Da M/Z peptide into the Hela cells and Siha cells respectively in different concentration and time specific effects are obviously different, and in a concentration dependent effect relationship(P<0.05). While under the same condition,the 6455.5Da M/Z peptide are not effect on 7702 cells(P>0.05). 2.CCK-8 results of cervical cancer cells The 6455.5Da M/Z peptide has inhibitory effect on the proliferation of Hela cells and Siha cells at different concentration and specific action time, and showed a time concentration dependent effect relationship(P<0.05). 3.The results of cell cycle and apoptosis of cervical cancer cells In the cell cycle, with the increasing the 6455.5Da M/Z peptide concentration, curb the proliferation of cervical cancer cells in S phase, with the concentrations of the 6455.5Da M/Z peptide showed a concentration dependent effect(P<0.05); In the detection of apoptotic cells, with increasing the 6455.5Da M/Z peptide concentration,the early apoptotic cells gradually increasing and the 6455.5Da M/Z peptide concentration was concentration dependent effect(P<0.05). 4.The results of the expression of the target protein in Western blot With the increase of concentration of the 6455.5Da M/Z peptide, the expression of PCNA in Hela and Siha cells reduce gradually, and it is negatively correlated with the concentration of the 6455.5Da M/Z peptide(P<0.05).Conclusions This experiments show that the 6455.5Da M/Z peptide can inhibit the growth of cervical cancer cells and induce the apoptosis of cervical cancer cells. So there are have more important reference guiding significance for the further study of the 6455.5Da M/Z peptide and its subsequent treatment. |
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