| Objective:To explore G1/S phase testing point function changes and relationship between dose and time response to DNA damage in the process of vinyl chloride carcinogenic with detecting different phase distribution of cell cycle inrat liver and DNA damage of peripheral blood lymphocytes in rats by sub-chronically VCM;to explore the role of mi RNAs in chloride-induced the G1/S checkpoint injury, and from the molecular level to further clarify the chloride-induced liver tumors pathogenesis withdetecting the expression of related mi RNAs in rat liver.Methods:Ninty-six healthy SD(Sprague Dawley) rats(male) were randomly divided into a control group and three vinyl chloride dose groups include low dose(5mg/kg), medium dose(25mg/kg) and high dose(125mg/kg),and 24 rats each group.Intraperitoneal injection every other day exposure, three times a week for 12 weaks. Eight rats in each group will be killed at the end of 6, 8 and 12 weaks respectively.1. The study of vinyl chloride induced liver genetic toxicity: comet experimental was applied to detect DNA damage levels on peripheral blood lymphocyte;flow cytometry technology was applied to detect the changes of hepatocyte cell cycle.2. The expression of liver cell mi R-199a-3p, mi R-221, mi R-222 in liver cell: take its liver,extract total RNA extraction and run reverse transcription reaction, apply real-time fluorescent quantitative PCR to detect the mi-RNA expression in liver tissue and the 2-△△Ct method for data analysis.Results:1. Rat general, changes in body weight and hepatopancreas somatic indices(HSI)Rats eat normally,and had normal activities, each dose group had no obvious poisoned symptoms after rats exposed to VCM, weight was normal growth trend, but the difference was not statistically significant(P>0.05). After 8 weeks and 12 weeks,HSIof dose groupswere below than the control group, but there was no statistically significant difference(P>0.05).2. Comet experimental study on the level of DNA damage in peripheral blood lymphocytesAfter eight weeks of VCMinfection, compared with the control group, the high-dose group(125mg/kg) was obviously higher, the difference was statistically significant(P<0.01).After twelve weeksof VCMinfection, compared with the control group, Olive tail moment and tail length of each dose group were significantly increased, the difference was statistically significant(P<0.05); mid-dose group(25mg/kg) in the percentage of tail DNA content was lower than the control group, the difference was statistically significant(P<0.05).3. Detect changes of the cell cycle distribution of changes in rats by flow cytometryAfter six weeks of VCMinfection, G1 phase distribution percentage of rats liver cells increased upward with the dose of each exposure group, and G1 phase distribution percentage in high dose group(125 mg/kg) was significantly higher than the control group,the difference was statistically significant(P<0.05).After eight weeks of VCMinfection, G1 phase distribution percentage on low dose group(5 mg/kg) was obviously lower than the control group, the difference was statistically significant(P<0.05); S phase distribution percentage on low dose group was higher than the control group, the difference was statistically significant(P<0.05).After twelve weeks of VCMinfection, S phase distribution percentage increased upward with VCM exposed dose, and S phase distribution percentage inhigh dose groupwas obviously higher than the control group, the difference was statistically significant(P<0.05).4. Real-time PCR detect the expression of mi R-221 and mi R-222 mi R-199a-3p in rat liver cellsAfter six weeks of VCMinfection, the expression levelsofrelative mi RNA-221 and the mi RNA-222 in rat liver tissue decreased with the increasing dose, and the expression of mi RNA-221 and the mi RNA-222 on high dose group was significantly lower than the control group, the difference was statistically significant(P<0.05); Compared with the control group, the expression of the mi RNA-199a-3p on medium dose and high dose were lower than the control group, the difference was statistically significant(P<0.05).After eight weeks of VCMinfection, the expression of mi RNA-221 and mi RNA-222 on the low dose group were higher than control group, but there was no statistically significant difference(P>0.05); The mi RNA-199a-3p relative expression was increased with the increasing dose, but there was no statistically significant difference(P>0.05).After 12 weeks of VCMinfection, mi RNA-221 relative expression were increased with the increasing dose, expression of mi RNA-221 on low doses group was lower than the control group(P<0.05); The mi RNA-222 expression were increased with the increasing dose, but compared with the control group, there was no statistically significant difference(P>0.05); the expression of the mi RNA-199-a-3p were increased with the increasing dose,expression of mi RNA-199a-3p on low and medium dose group were lower than the control group, and difference was statistically significant(P<0.05).5.The correlation analysis between the rat liver cell cycle and the expression level of mi R-199a-3p、mi R-221、mi R-222.After six weeks of VCMinfection, the expression level of mi RNA-221, mi RNA-222 and mi RNA-199a-3p in rat liver cells were negatively correlated with the percentage of G1 phase distribution, and the correlation coefficient were 0.6、0.44 and 0.51 respectivel,and it was statistically significant(P<0.05).After six weeks of VCMinfection, the expression level of mi RNA-221, mi RNA-222 and mi RNA-199a-3p in rat liver cells has no correlated with percentage of G1 phase distribution(P>0.05).After 12 weeks’ VCM infection, the expression level of mi RNA-221, mi RNA-222 and mi RNA-199a-3p in rat liver cells has no correlated with percentage of G1 phase distribution(P>0.05). the expression level of mi RNA-222 in rat liver cells has correlated with percentage of S phase distribution,the correlation coefficient r was 0.41 and it was statistically significant(P<0.05); the expression level of mi RNA-222 and mi RNA-199a-3p has no correlated with percentage of S phase distribution(P>0.05).Conclusions:1. VCM can cause DNA damage of rat peripheral blood lymphocytes.2. VCM may affect G1/S checkpoint function of the rat liver cell, early the phenomenon of G1 phase arrest occurred and then G1 phase disappears, and cause S phase arrest in the late。3. The lower expression of mi RNA-221, mi RNA-222 and mi RNA-199a-3p may affect theregulation of cell cycle G1/S checkpoint in the liver of rat, and be related to cell cycle G1 arrest in the liver of rat, and later the raise expression of mi RNA-221 may be related to the phenomenon that VCM-reduced G1 phase retardationdisappeared and S phase arrest appeared. |
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