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Inhibitive Effect of Camellia on the Growth of CNE-2 Cells in Vitro and the Gene Expression of VEGF-C \VEGFR-3

Author: ShenJie
Tutor: ZhuHua
School: Guilin Medical College,
Course: Pathology and Pathophysiology
Keywords: The golden camellia alcohol extracts Of people poorly differentiated nasopharyngeal carcinoma cells Cell cycle Apoptosis rate Caspase-3 VEGF-C \\ VEGFR-3
CLC: R285
Type: Master's thesis
Year: 2011
Downloads: 43
Quote: 0
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Abstract


Objective: Golden Camellia (Camellia chrysantha (Hu) Tuyama, CCT) is a kind of high medicinal value of natural herbs. trace elements. Golden Camellia in lowering blood lipids, blood sugar, anti-cancer, anti-cancer, with special effects. Its role in tumor suppression mechanism already discussed, the study found the golden camellia obvious inhibitory effect in rats [3]. But reported no relevant inhibition of nasopharyngeal carcinoma cell research at home and abroad. The experiment by observing the golden camellia alcohol Extract in poorly differentiated human nasopharyngeal carcinoma line CNE-2 cell proliferation, cell cycle and apoptosis and lymphangiogenesis factors and their receptors VEGF-C \\ VEGFR-3 protein expression impact of Camellia chrysantha mechanism for inhibiting the growth of tumor cells to lay the theoretical foundation for further study found. Golden camellia alcohol extract method: 1. MTT assay was used to detect different concentrations (25μg/ml, 50μg/ml, 100μg/ml, 200μg/ml, 400μg/ml) at different times (24h, 48h, 72h) under CNE-2 cells in vitro inhibitory effect. 2. Different concentrations observed under an inverted phase contrast microscope (50μg/ml, 100μg/ml, 200μg/ml) the golden camellia alcohol extract role CNE-2 cells, changes in cell morphology. CNE-2 cells. Hoechst33342 staining observed under a fluorescence microscope at different concentrations (50μg/ml, 100μg/ml, 200μg/ml) Camellia role of alcohol extract apoptotic morphology. 4 Flow cytometric analysis of cell cycle changes and apoptosis rate in different concentrations (50μg/ml, 100μg/ml, 200μg/ml) of the golden camellia alcohol extract role CNE-2 cells. Application protein immunoblotting (Western Blot) method to detect different concentrations (50μg/ml, 100μg/ml, 200μg/ml) Camellia role of alcohol extract of CNE-2 cells after intracellular Caspase-3 protein expression. 6 enzyme-linked immunosorbent assay (ELISA) to detect different concentrations (50μg/ml, 100μg/ml, 200μg/ml) the golden camellia alcohol extract role CNE-2 cells after cell supernatant lymphatic endothelial growth factor and its receptor (VEGF-C \\ VEGFR-3) protein expression. Results 1 the golden camellia alcohol extract of the CNE-2 cells with growth and proliferation inhibition, the inhibitory effect of time and dose dependent, with the extension of time and the increase of the concentration, the inhibitory effect of gradually increased. Concentration greater than 50μg/ml the golden camellia alcohol extract compared with the negative control group, the difference was highly significant with significance (p lt; 0.01). Inverted phase contrast microscope observed with increasing drug concentration, the CNE role of the golden camellia alcohol extract-2 cells after 48h by growth arrest, and gradually the cell shrinkage smaller rounded off, and finally shows a large number of cells shrinkage, fragmentation, exfoliated cells were suspended in the culture medium. Apoptotic cells under a fluorescence microscope, different concentrations of the golden camellia alcohol extract role CNE-2 cells 48h observed strong blue fluorescent stain revealed a dense, nuclear condensation, nuclear cleavage, nuclear chromatin aggregation form, as the concentration increased apoptotic cells gradually increased. 4. Function of Camellia nitidissima and alcohol extract CNE-2 cells 48h after the G1 phase of the cell concentration increase in the proportion of G2, S phase cells have different degrees of reduction cells arrest in G1 phase . The same time as the concentration increased, the rate of apoptosis increased in comparison with the negative control group differences were statistically significant (p lt; 0.05). Western Blot experiments: With the increase of the concentration of the drug, the activation of Caspase-3 protein bar band gradually deepened and enhanced expression. Gel image processing system determination of integral optical density values, the differences were statistically significant (p lt; 0.05) compared with GAPDH bands and the integrated optical density value of the negative control group. 6. ELISA experiments: With the increase of the concentration of the drug, CNE-2 cell culture supernatant of VEGF-C \\ VEGFR-3 protein expression levels down. Conclusion 1. The golden camellia alcohol extracts in vitro can inhibit the proliferation of CNE-2 cells, and time-effect relationship exists and the dose-effect relationship. The mechanism may inhibit the growth of nasopharyngeal carcinoma cells to promote apoptosis by inducing G1 arrest and Caspase-3 protein upregulation. 3. ELISA test showed that VEGF-C \\ VEGFR-3 protein expression may play a certain role in the prevention and treatment of human nasopharyngeal carcinoma metastasis.

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