|
Objective: The purpose of this experiment is to be provided by in vitro cultured human fibroblast cells, effector cells to a certain time by applying different concentrations of mitomycin C (MMC) was observed after mitomycin C to inhibit its normal fibroblasts proliferation ability, induced apoptosis, and to explore the mechanisms of molecular biology. Method: 1. Vitro with MEM culture medium 10% fetal bovine serum, cultured human skin fibroblasts and returns Rev. 5-8 substituting, respectively, with MEM containing 0,0.001,0.01,0.05,0.1,0.2,0.3 mg / ml MMC species in the fibroblast cells in the 96 well plates after 12h, and then added in each hole CCK-8 reagent 100ul culture fluid processing is detected using a microplate reader, placed in a cell incubator for 1 hour with different concentrations of MMC-treated group in the 450/630nm the absorbance value at calculated MMC on fibroblast growth inhibitory effect; 2 is in the logarithmic growth phase of fibroblasts species after disinfection evenly placed on circular coverslips in 6-well plates, with 0 , 0.001,0.01,0.05,0.1,0.2 mg / ml MMC treating cells climbing film after 12 hours, washed with cold PBS culture staining the liquid Hoechest33342 nucleus, apoptotic nuclear morphology was observed under a fluorescence microscope and using Image Pro-Plus software. count each group the percentage of apoptotic cells; Press dosing cultured cells 12h, the control group, 0.2 mg / ml MMC cultured cells 12h, the PI3K/Akt inhibitor LY294002 100nmol / L and MEK / ERK inhibitor PD98059 100nmol / L, respectively treatment of cells for fresh culture medium after 1h 12h divided into 4 groups, culture solution washed with ice-cold PBS, the bottle cells are added 100ul cell lysate, 4oC 12000rpm cleavage after 20 minutes of centrifugation cytoplasmic protein, Western bolt immunoblotting detection of caspase-3, p-ERK1 / 2, p-Akt and Bad expression levels, flow cytometry assay apoptosis rate. Results: different concentrations of MMC inhibit the proliferation of human fibroblasts with drug concentrations were positively correlated in 0.2mg/ml the MMC cell activity inhibition rate was 50.21%; the 0.3mg/ml up 80.71%; Hoechest33342 nuclear staining with MMC concentration increased apoptotic nuclei characteristics chromatin condensation, margination, bright blue nuclear ratio gradually increased image pro-plus software counting apoptotic cells 0.05,0.1,0.2 mg / ml MMC treated withered The death rate was significantly higher than other low-concentration group, the difference was statistically significant (p lt; 0.05). Western blot analysis of cell-associated apoptosis signaling pathways protein expression results: 0.2mg/ml MMC group, PD98059 group, LY294002 caspase-3 expression were significantly higher than that of the control group, p-ERK1 / 2, p-Akt expression compared with The control group reduce 0.2mg/ml MMC group Bad expression level was significantly higher than the other three groups. The results show that the loss of cell apoptosis rate measured 0.2mg/ml MMC group apoptosis rate was significantly higher and inhibitor pretreatment group. Conclusion: 1. Certain concentration of MMC fibroblasts by inducing apoptosis and inhibit the normal proliferation;. Effect ways can be explained in part by lowering of ERK1 / 2 and Akt phosphorylation level of Bad expression intracytoplasmic levels lead to the execution of apoptosis protein caspase-3 expression levels increase, induced fibroblast to turn apoptosis by normal proliferative state to reduce the formation of collagen fibers, thereby inhibiting scar formation. 3.MMC inhibition of epidural scar adhesion molecular biology mechanism one is to make a lot of migration to wound inflammation center as anticancer drugs mitomycin in postoperative and are subject to a variety of inflammatory mediators and stimulate cell growth factor fibroblast activity is suppressed, apoptosis, reduces the effective number of fibroblasts to generate large amounts of scar tissue, fibroblast cells can not produce excessive collagen fiber caused by scar tissue and subdural adhesions few late, thus can solve the the postoperative adhesions lower lumbar failed back surgery syndrome.
|