| Objective: Cervix lesion is one of the most common suffering in women, and it can develop to cervical carcinoma. In female carcinoma, the incidence of cervical carcinoma is the second only to mammary cancer. In our country, cervical carcinoma is very a common malignant tumor. It may take ten years of more from cervix lesion developing to cervical cancer. So we believe that it was a kind of cancer could be prevented and cured if diagnosed and properly treated early. It is very important to prevent it's developing in early time. It is well known that HPV(human papilloma virus)is the initiation factor in the occurrence of the cervical cancer. Up to now, HPV cannot be cultivated in vitro, because of which, the research about preventing cervical cancer is falled behind. Based on previously study, we want to study the effect of allacin on gene expression of HPV18-E6, hTERT and P53 of Hela cell in vitro. The purpose of this study is to provide a new way to prevent and cure the cervical cancer in clinic.Methods1 Cell culture: Hela cells freezed in liguid nitrogen were revived in routine method, and inoculated in RPMI-1640 blended with 10% foetus cattle serum, cultured in culture bottle. The bottle was put in the incubator with the condition of 37℃,5%CO2. The growth of cells was observed every day. 70% of the cells confluence was digested with EDTA and then was made into cell suspension.2 The growth suppression of cell was estimated by MTT: adjusted deuterium of cells to 3×104 / ml, mixed them completely and inoculated them to cultivation Plate of 96–pore, 200μl in every pore. Cells were put to incubator and cultured to logarithm phrase. Then they were got out , the superior liquor was got rid of and 200μl culture medium including drug was added. The concentration of drug are 25mg/L, 50 mg/L, 100 mg/L. Then they were put back to incubator, watched everyday. 20μl MTT was added in every pore after cultured 24,48,72 hours, cultured in incubator again for 4 hours. Then, all liquor was got rid of. 200μl di–methyl–Sulfoxide was added, shocked for 8 minutes. And then the number of A and calculated the inhibition rate were measured. The sequences were done for 3 times in the same way.3 Observation by inverted microscope. Hela cell suspensions were seeded in 24~well culture plates at a density of 3×104 cells/ml (1ml/well) in culture medium. Experimental treatment of cells was same as above. 24 and 72 h later, cells were observed and shot by inverted microscope. Observation after Giemsa staining by light microscope. Hela cell suspensions were seeded in 24-well culture plates at a density of 3 x 104 cells/ml (1ml/well) in culture medium with a small piece of cover glass in each well. Experimental treatment of cells was same as above. 72 h later, the glass was taken out and stained with Giemsa stain for 10 to 15 min. Cells were observed by light microscope.4 Observation by electron microscope. Hela cell suspensions were seeded in 100ml culture flasks. Experimental treatment of cells was same as above. 72 h later, the cells were harvested by treatment with EDTA(0.02%) and fixed by glutaral(4%) at 4 ℃. After desiccation, penetration, embedding, section and staining, cells were observed by electron microscope (JEM-1230, Japan).5 Assayed by TUNEL Test:Experimental treatment of cells was same as above. The cells were treated according to instruction of TUNEL Test Kit. The cells whose nucleuses were stained by brown were apoptosis cells, and the blue are negative. 300 cells were counted in every section, and then calculated the apoptosis rate of cells.6 With PCR and RT-PCR, the expression change of HPV18E6 mRNA, p53 mRNA as well as hTERT mRNA were studied in the process of Hela cell apoptosis in vitro.Results 1. Observation after Giemsa staining by light microscope:After Giemsa staining, cytoplasm of Hela cells in untreated group was pink and nucleus the number of which was two or three were royal blue. Cell membrane and nucleus were integrated. More karyokinesis and few chromatin margi...
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