The Influence On The Gene Expression Of P73 Gene And P53 Gene Of V79 Cell Which Is Caused By The Chrysotile From Different Areas In China And The Substitute Chrysotile Fibers

Objective:This experiment uses the chrysotiles from the four areas in China (Mangya in Qinghai province, Xinkang in Sichuan province, Aksu in Gansu province and the southern part of Shanxi province) and the foursubstitute chrysotile fibers (glass fibers, ceramic fibers, wollastonite, rock wool) on the Chinese Hanster Lung Cells which is also called V79 cells, to investigate the toxic effects of the dust on the cells and to compare the different toxic effects between chrysotiles and substitute chrysotie fibers, moreover, to investigate the expression of cell injury in p73 gene and p53 gene which is caused by the dust and also to investigate the relationship between the expression and genes.Methods:Choose the four chrystioles from differnent areas and the four substitute chrysotile fibers, and each set up the dust suspension in five types of concentration (25μg/ml,50μg/ml,100μg/ml,200μg/ml,400μg/ml) to act on V79 cell. Set up a black control group. After 24 hours, use cell counting kit-8 (CCK-8) to test the cellproliferation. Compare the cell survival rate of each group. Choose the dust suspension of 50μg/ml to act on V79 cell in 24hours, each dust set up three parallel control groups and a black control group. Observe the cell morphology by Wright-Giemsa staining; extracte RNA, reverse transcription to cDNA, use real-time quantitative PCR to detecte p73 gene and p53 gene expression.Results:(1) The results of Wright-Giemsa staining:the majority of chrysotile and substitute fiber groups can be found in bare nucleus, vacuoles, and the phenomenon of dark brown heavy particles. (2) The cell survival rate:after different concentrations of dust suspension act on V79 cells in 24h, the majority of cells in 50μg/ml have a fastest breeding, most cells in chrysotile groups doubled, and the proliferation of chrysotile groups are generally greater than the substitute groups. (3) Use real-time quatitative polymerase chain reaction (real-time Q-PCR) to investigate p73 gene and p53 gene.①The group of p73 gene:Aksu group, glass fiber group, rock wool group and Southern Shanxi group, each compare with the black control group. The results are:the expression of Aksu group, glass fibers group, rock wool group and Southern Shanxi group is obviously higher than the black control group and each shows significent difference (P<0.05); whereas, there is no obvious difference between the black control group and Mangya group, Xinkang group, ceramic fibers group, wollastonite group (P>0.05). Other dust groups compare with eachother, and there are no significant difference (P>0.05).②The group of p73 gene:the black control group compare with each dust group and each dust group compare with eachother, and there are no significant difference (P>0.05). Conclusion:(1) Most of chrysotile and alternative fiber group showed cell toxicity. (2) The expression of p73 gene in Aksu group, glass fiber group, rock wool group and Southern Shanxi group is obviously higher than the black control group. This result suggests that these groups of the chrysotile and the substitute chrysotile fibers may lead to malignant changes in cells and the p73 gene may be regulated its expression to play a role of cancer gene when the cells malignat changes occur. Aksu group, glass fiber group, rock wool group and Southern Shanxi group are more likely to lead to cancer, while the possibility of the other dust groups lead to cancer is not clear. (3) There is no significant difference in each group of the p53 expression. This suggests that there is no clear role of p53 when the dusts damage the cells.