| IntroductionCalcium is a most important second - messenger in cell, the change of intracellular Calcium concentration closely correlates with cell proliferation, gene mutation and the development of tumor. Its reported that low calcium is inclined to induce the development of malignant tumor, and that VD3 can not only enhance the absorption of calcium ,thus to act as a synergist of calcium to play the anticancer role; but also change the rate of gene transcription by binding with VDR, thus to regulate the proliferation, differentiation and apoptosis of cell. Our experiments are to detect the effect and regulatory mechanism of calcium and VD3 on the proliferation of cervical cancer by MTT and ARC.Materials and MethodsMaterials-Hela cell line is generously provided by the department of cell biology of China Medial University. Human cervical cancer tissue is ob-tained from the First Affiliated hospital of China Medical University. [ Methyl - H ] Thymidine, nuclear emulsions are purchased from China nuclear - energy institute; RPMI1640 medium is purchased from life technology USA; 96 - well microplates and MTT are obtained from Huamei Biological Engineer Company; Small bovine serum is obtained from Tianjin Biochemical Product Factory. Methods-1. Cell experimentMTT assay are performed to detect the effect of Calcium and VD3 on the Hela cell proliferation.2. Clinical material experimentFresh and aseptic cervical cancer tissue is incised into 1 x 3mm pieces and treated with different drugs for 72hours, then one - pulse labeling method is performed to label the tissue with 3 H - TdR, and then the specimen are embedded in paraffin and sectioned into 7u,m, the 7 fim tissue sections are mounted on glass slides, dehydrated and dipped in nuclear track emulsion, then the slides are packed in air -tight boxes containing desiccant. After 21 - day exposure time at 4T!, the slides are developed, fixed, and stained with hematoxylin and eo-sin. After stained, the sections are covered with Coverslips and sealed with permout. Slides are examined by light microscopy to determine the labeled rate.Results1. The effect of different drugs on the proliferation of Hela cells by MTT assay.Fig. LA shows, the proliferation rate of Hela cells treated with dif-ferent drugs for 6 hours displays no statistic difference with that of control group. After treated for 24 hours, lOmM, 20mM calcium reduce the proliferation rate of Hela cells, the inhibition rates are 10% , 15% respectively, moreover, the inhibition rates are time - dependent. When supplemented with VD3(100nM) (Fig. IB) , all the groups reduce the proliferation rate of Hela cells after 24 hours, the inhibition rates are 5% , 20% , 34% respectively. Also the inhibition rate is time - dependent. As Fig. 1C shows, the proliferation rate of Hela cells treated with calcium and VP for 6, 24, 48, 72hours displays no statistic difference with that of control group, Which suggests that VP may antagonize the inhibition effect of calcium on the proliferation rate of Hela cells.From Fig. 2 (two curves ) , we can know that the inhibition effect of calcium on proliferation rate of Hela cells is concentration - dependent and that VD3 can act as its synergist.Fig. 3 shows that 20mM Ca2 + reduces the proliferation rate of He-la cells; and VD3 is its synergist. VP can antagonize the effect of calcium , but shows no effect on that of VD3, which suggests that extracellular calcium influxing is a main pathway of the high concentration calcium - induced inhibition effect on Hela cell proliferation rate, but not of VD3, that is to say, there are other mechanisms of VD3 - induced inhibition effect on Hela cells proliferation rate.2. The effect of different drugs on the cell - labeled rate of cervical cancer tissue by ARG.As the pictures show, when compared with control group (pic. 1) , 20mM calcium (pic. 2) reduces the cell -labeled rate, however 100nM VD3 ( pic. 3) shows no statistically significant effect on cell -labeled rate of cervical cancer ti... |
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