| Objective : As malignant tumor, cervical cancer threatens the life and healthy of the women. The frequency of this disease has increased throughout the young span of women over the past few years. Traditional therapy surgical operation and radiation-therapy may make the ovary function declined or lost, so that the life quantity of the patients will descend significantly. The application of HRT(Hormone Replacement Therapy) maybe solve the problem, but the trouble is whether it has bad effects on the original disease and the potential cancer-causing problem. This study aimed at the effects of estradiol and progesterone on proliferation and apoptosis of human cervical cancer cell line Hela in vitro, so as to determine whether the women with cervix cancer may apply HRT.Methods: 1.Cell culture:Refrigerated Hela cells were anabiosised and grown in 100ml culture flasks in RPMI 1640 supplemented with 10% fetal bovine serum(FBS ), 100 [mu]g/ml streptomycin and 100 IU/ml penicillin at 37℃ under a humidified 5% CO2/95% air atmosphere. Cells were digested and subcultured with 0.02% ethylenediaminetetra-aceticacid (EDTA ).2. Immunocytochemistry for ER and PR: Hela cells were made into single cell suspensions and were droped on the carry sheet glasses. After airing, the cells were fixed by cold acetone at 4℃. ER and PR contents within cultured cells were determined using immunocytochemistry according to the immunocytochemical assay for the detection of ER and PR. Expression levels of ER and PR were evaluated using the quantitative H score whose reliability had been demonstrated previously: H score =[SIGMA]Pi (i + 1), where i is the intensity of staining from 0 (none) to 3 (strong), and Pi is the percentage of stained cells for each given i (0-100%).3. Cell proliferation: Hela cells were seeded in 96-well culture plates at a density of 3 x 104 cells/ml (200 [mu]l/well) in culture medium. After having stayed over, the cells were incubated at 4℃ for 1h so as to come to synchronization. The cells were divided into four groups and their culture media were as follows: [1] E2 group: basic RPMI1640 media added with E2 ( solubilized in 100% ethanol, then diluted in RPMI1640 to a final ethanol concentration of ≤0.03%),the concentration of which was 0.50,1.00,5.00,10.00nM respectively; [2] P group: basic media added with P the concentration of which was 0.01,0.10,1.00,10.00 [mu]M respectively; [3] E2 plus P group : basic media added with E2 and P. The concentration of E2 was same as that of E2 group and the concentration of P was same as that of P group. [4] blank control group: basic media added with ethanol thefinal concentration of which was 0.03%. Each concentration of all the groups had eight wells. After 72 h, 20[mu]l/well MTT (5mg/ml)was added to Hela cells for 4h at 37℃. Then the liquid was aspirated, 200 [mu]l DMSO was added into each well. 96-well culture plates were put on the oscillator for 10 min and absorbance (A) of cells in each well was detected with spectrophotometer when wavelength was 492nm. Each assay was performed three times independently.4. Cell cycle and apoptotic percentage: Hela cell suspensions were seeded in 100 ml culture flasks at a density of 3 x 104 cells/ml in culture medium. After having stayed over, the cells were treated for synchronization and replaced culture medium. Each study group added with the largest concentration of drugs, that is to say, the concentration of E2 was 10.00nM in E2 group, the concentration of P was 10.00[mu]M in P group and the concentration of E2 and P in E2 plus P group was as above respectively. In control group, ethanol was added in basic media and the final concentration of ethanol was 0.03%. After 72h, the cells were harvested by treatment with EDTA (0.02%) and fixed by cold ethanol(70%) at 4℃. The cells were stained with propidium iodide (PI) later, cell cycle and apoptotic percentage were detected by flow cytometer (FCM).5. Cell morphology observation: ①Observation by inverted microscope. Hela cell suspensions were...
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