| ExperimentI The study on the bactriostasis of MINO against AaObjective:Detect MIC of MINO on Aa, to avoid the incapability of clearing the infection caused by Aa and the resistance due to the inadequate dose.Methods:First recovery the strain, then spread plate. After those, logphase and growthcurve of Aa were measured. MIC was determined by agar dilution method.Results:After bacterination for 24h, the log-linear relationship was presented between growth concentration of bacteria and OD value.The growth trends among different periods were different.During the first to 24h, it proliferated slowly, then actively during 48h to 84h. But it became downward in 96h. MIC was 8mg/L. Conclusion:The bactriostasis of MINO on Aa was efficient, and MIC was 8mg/L.ExperimentⅡBiological effect of MINO on hPDLFObjective:Explore culture methods for hPDLF in vitro and the effective concentration range of MINO for periodontal local administration.Methods:Periodontal membrane tissue was used as original tissue for the primary culture.The tissue was from premolor teeth which needed to be extracted because of orthodontics. Three methods, including explant method, enzymatic digestion method and cover-glass, were combined and used for hPDLF primary culture. Vimentin filament, keratin and S-100 factor dealed by immunofluorescence were used for cell identification. The osteogenic property was researched after calcium nodule dyeing induced by mineral solution.The cell growth curve was plotted by MTT method.Steps were as follows:Different concentrations of MINO (0,10,20,40,100,200mg/L) were on the 5th generation hPDLF. After incubation for 1,4,7,10,14d, cell proliferation were measured by MTT method. After incubation for 4d, the change of cell cycle was dected by flow cytometry.Cells total protein was dected by using BCA assay and COLI, ALP, OCN of gene expression and protein expression were assayed by PCR and Western-blot method. The SPSS 13.0 software package was used for statistics analysis.Results:The immunofluorescence results of vitro culture were positive for vimentin filament, negative for keratin and S-100 factor. These were confirmed to be hPDLF. hPDLF could form mineralization nodules in the induction of mineral solution, and performed osteogenic property. For the 5th generation hPDLF, MINO could accelerate proliferation, and mitosis when its concentration interval was 10～200 mg/L and the difference from other interval was statistically significant. The maximum effect concentration was 100mg/L.for concentration interval 20～200mg/L, MINO could significantly improve total protein and COLI expression on gene and protein aspect(P<0.05). Though 10mg/L group had higher total protein and COLI expression on gene and protein aspect than the control group, the difference was not statistically significant(P>0.05). For concentration interval 40～200 mg/L, MINO inhibited the ALP and OCN gene expression and protein expression(P<0.05).For concentration interval 10～20 mg/L, MINO didn't inhibite the ALP and OCN gene expression and protein expression(P>0.05).Conclusion:A large quantities of high-purity hPDLF could be obtained by the primary culture method for hPDLF combined with explant method, enzymatic digestion method and cover-glass.Besides the large quantities, the cells had high activity and multiplication capacity. hPDLF expressed osteogenic property by the induction of mineral solution.10～20mg/L, MINO could accelerate proliferation and mitosis, improve total protein and COLI gene expression and protein expression, but have no inhibition effect on ALP and OCN. General considerating according to the finding 10～20mg/L could be the effective concentration range of MINO for periodontal local administration.
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