| ObjectivesCispltin (CP) is one of the most popular antitumor drugs. However, its cytotoxicity (kidney toxicity, ear toxicity, nerve toxicity) affects the application of CP in the area of tumor treatment. Therefore, it is important to understand the cytotoxic mechanisms of CP. In this study, based on mice embryonic stem cells (ESC), microarray method is applied, gene expression profile was studied. Different genes were selected after CP treatment compared with control. Biological functions of these genes are analyzed in order to understand the molecular functions of cisplatin-induced cytotoxicity on the mouse embryonic stem cells. It will lay the foundation on researching cisplatin’s cytotoxicity.Methodologys1. ESC culturingThe primary mouse embryonic fibroblast (MEF) is prepared, passaged3to5generations and then treated with10μg/ml mytomycin C for2.5hours. After mytomycin C treatment,1×105ml cells are seeded in flask as feeders. The feeder cells are cultured with the ESC culture media. After12hours,1.5×105/ml ESC are seeded on the top of feeders, cultured at37℃,5%CO2. The culture media are replaced everyday and cells are passaged every2to3days.2. Effect of CP on cell activity of ESC.5mg/ml CP is dissolved in culture media and diluted in different concentration. After mytomycin C treatment,1×107ml MEFs were used as feeders.1.5×105ml ESC are cultured on the top of feeders in96-well plates. Different concentrations of CP are added into cells culturing for48hours, replace media everyday. Cell activity is measured by MTT method. Inhibition ratios are calculated with SPSS11.5software and IC10is calculated.3. Microarray experiment of ESCCP IC10is used in the following experiments. ESC is treated with CP IC10for48hours, medium is changed every day. Cells are collected and total RNA is isolated. Microarray protocols are followed. DNA-Chip Analyser (d-Chip) of Affymetrix (version2009) is used. The intensity of signals and ratio are calculated and analysed, differentially expressed genes are screened and compared with control. These genes go GO explanation, and KEGG (Kyoto encyclopedia of genes and genomes) database is used for Pathway analysis, biological functions and biological processesin order to identify the effects of these genes in the prcess of cisplatin-induced cytotoxicity on the mouse embryonic stem cells.Results1.The37℃thermal digestion is a better method to prepare mouse embryonic fibroblast cells. The cells present the shuttle or irregular triangular in shape with bigger nucleus, clear cell boundary, better refraction and3-dimension appearance.2. MTT results show CP concentration in ICio is0.5471μg/ml.3. Microarray results indicate2686genes have changed their expression more than2folds, compared with control.1718among them are up regulated genes and968are down regulated genes. Compared with control,37gene expressions are up regulated4fold high.Conclusion1. This method which is explored in this experiment to prepare the mouse fibroblast2. Mouse ESC may serve as a ideal platform for the study of cisplatin-induced cytotoxicity on the mouse embryonic stem cells.3. The ESC cytotoxicity can be induced with the different concentration of CP for48hours.4. The analysis of104-fold high expression genes that are induced by cisplatin suggest inflammation, oxidative stress, apoptosis are the mainly molecular mechanism associated with the cisplatin-induced cytotoxicity on the mouse embryonic stem cells. In view of the synergistic effect of endotoxin in cisplatin cytotoxicity, nosocomial infection should be monitored and prevented at any time in order to avoid aggravating the cisplatin-induced cytotoxicity in the treatment of cancer. |
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