| Objective:to exploer the role of hyperglycemic state in the senescence process of fibroblast and its possible mechanism.Methods:(1)We establish a high sugar model to observe the cellular morphology changes.NIH3T3(murine embryonic fibroblast)and WI-38(human embryo lung fibroblast)were cultured in DMEM and MEM with 1%NEAA medium.The glucose concentration of Hyperglycemic group and Control group were adjusted to 33.33mmol/L and 5.56mmol/L.The two groups of cells were cultured in the cell culture incubator with 37? and 5% CO2.We replaced the bath solution every 72 hours,and observed the cellular morphology with a photo taken everyday.When the cell density reached 90%,we passage these cells.(2)We use the ?-gal Cell aging staining method to measure the senescence of fibroblasts.We took part of the 10 th day,20,30 days,45 days,60 days of cell to dissociate,planting on 6-well plates.The second day,we took these cells to do the ?-gal cell aging dyeing,and take count of parts of the hot spots with photos taken.Then we calculated the proportion of senescent cells,and used SPSS20.0 software for two independent sample t-test,and set statistical standards for ? < 0.05.(3)We used the MTT cell proliferation method to measure the activity of fibroblast.We took part of the 10 th day,20,30 days,45 days,60 days of cell to dissociate,planting on 96-well plates,adding MTT,DMSO,etc.Then we used the enzyme coupling detector to tested the absorbance in OD490 nm,and used SPSS20.0 software for two independent sample t-test,and set statistical standards for ? < 0.05.(4)We used the q RT-PCR method(quantitative reverse transcription polymerase chain reaction)to measure the expression of p16 m RNA.Take part of the 10 th,30th,60 th day of cells,training on 6 well-plates.The second day,we used the Trizol RNA extraction reagent to extract RNA,and then we proceed the q RT-PCR test,according to the formula 2-?Ct=2-(target gene Ct-housekeeping Ct)to calculate the relative expression of p16 gene.Use SPSS20.0 software for two independent sample t-test,and set statistical standards for ? < 0.05.(5)We used the Western blot to test the expression of p16 protein.Take part of the 10 th,30th,60th day of cells,training on 6 well-plates.The second day,we used the Western and IP cracking liquid cracking cells and extract the total protein and then we proceed the Western blot experiment to calculate the grey level of the image.Use SPSS20.0 software for two independent sample t-test,and set statistical standards for ? < 0.05.Result:(1)The morphological changes of cells: the cells of Hyperglycemic group had a bigger size than the Control group with time,the richer pigmentation,a bigger cell nucleus which had more lobulation.(2)The ?-gal Cell aging staining:the difference of the proportion of senescent cells increases with time in these fibroblasts.The proportion of the Hyperglycemic group is higher than the Control group in the 45th?60th day of NIH3T3 and in the 30th?45th?60th of WI-38.Difference is statistically significant,p < 0.05.(3)The MTT cell proliferation experiment:the proliferation of Hyperglycemic group was lower than the Control group in the 30th?45th?60th day.Difference is statistically significant,p < 0.05.(4)The q RT – PCR experiment: the p16 gene transcription level is higher in the Hyperglycemic group than the Control group.Difference in the 30th? 45 th ? 60 th day is statistically significant,p < 0.05.(5)The Western blot experiment: p16 protein expression levels is higher in the Hyperglycemic group than the Control group.Difference in the 30th?45th?60th day is statistically significant,p < 0.05.Conclusion:(1)Hyperglycemic state can induce the senescence of fibroblasts.Because of the only difference is the glucose concentration,the whole difference is caused by the glucose concentration.(2)Hyperglycemic state can induce the expression of the p16 gene.(3)The upregulation of the p16 gene expression may be a important mechanism in the senescence of fibroblasts induced by the hyperglycemic state. |
PDF Full Text Request