| Part I Construction of rats PPARy gene Tet-on lentiviral vector, determine the effect of lentiviral vector on adipogenic and osteogenic differentiation of rat BMSCs in vitroObjective To isolate and culture Rat BMSCs. To construct Rats PPARy gene shRNA Tet-on lentiviral vector, and transfect it into BMSCs, we determine its silencing effects induced by tetracycline and the effect on adipogenic and osteogenic differentiation of rat BMSCs.Methods Rat BMSCs were isolated and cultured with adhesion method, identificated by SABC method. Designed from the rat PPARy gene specific sequence, according to plasmid recombinanting, virus packaging and sequencing, construction of pTRIPZ/PPAR?-shRNA lentivirus vector. We Set blank control group (Control), nonspecific plasmid transfection control group(shNTC) and shPPARy lentiviral plasmid transfection group (shPPARy). The lentivirus vectors were transfected into rat BMSCs. To observe the silencing effect induced by tetracycline and the best tetracyclines concentration. Cells were cultured in osteogenic or adipogenic medium for 14 days, we determine the adipogenic gene C/EBP?? ADD1 mRNA and protein levels by RT-PCR and Western blot assays, determine the osteogenic gene collagen I and Cbfal/Runx2 mRNA and protein levels by RT-PCR and Western blot assays,the differentiation of BMSCs were analyzed by Oil red O staining, alkaline phosphatase (ALP), Alizarin red S staining and calcium deposition.Results The purified rat BMSCs were obtained by adhesion method, the surface of BMSCs expressing of CD44, CD54, CD71, but not expression of CD34, CD45 by SABC method. The rats PPARy gene shRNA Tet-on lentiviral vector was consisitent with the design sequence, the titer is 3.6×10E8 TU/ml,when transfected into rat BMSCs, the red fluorescent protein can be observed, transfection efficiency above 70%. The silencing effect can reach 92% induced by 20?g/ml tetracycline. After adipogenic induction for 14 days, shPPARy group compared with Control group, the expression of the ADD1 and C/EBPa were significantly decreased as measured by RT-PCR and Western blot assay(.P< 0.01), the lipid droplets accumulation was significantly reduced, and adipogenic differentiation was blocked. There were no significant difference between the shNTC group and Control group(.P> 0.05). After osteogenic induction for 14 days, shPPARy group compared with Control group, the expression of the osteogenic genes encoding collagen I and Cbfal/Runx2 were siginificantly increased as measured by RT-PCR and Western blot assay(P< 0.05). the activity of alkaline phosphatase and the amount of calcium deposition were siginificantly increased as measured by Alizarin red S staining(P<0.05). There were no significant difference between the shNTC group and Control group(P>0.05).Conclusion The rats PPARy gene shRNA Tet-on lentiviral vector was successfully constructed, when transfected into rat BMSCs,the stable silencine effect can be obtained induced by tetracycline. The lentiviral vector decreases adipogenic differentiation and promotes osteogenic differentiation in a rat BMSCs model system in vitro.Part ? Pharmacokinetics of tetracycline in the rat femurObjective To investigate the metabolic rule of tetracycline in bone tissue and themarrow tissue of rat femur.Methods 36 rats were randomly divided into 12 groups, one control group and 11 time points group. The control group were given saline orally, others given tetracycline. The bone tissue sample and the marrow tissue sample of rat femur were extracted using protein precipitation at different time points. With metronidazole as internal standard, using HPLC/MS detect tetracycline in each sample, drawing the time-concentration curves. All rat femurs were treated by NaOH solution, the fluorescence were observed by fluorescent microscope, drawing the time-fluorescent intensity curves.Results We detect tetracycline'drug peak in the bone tissue sample and the marrow tissue sample of rat femur by HPLC/MS, draw the standard curves, the limit of quantification is 2.5?g/kg, the accuracy and recoveries were satisfied with the requirement of sample measurement. Tetracycline can reach the peak 48-72h after orally in bone tissue, and will not increase. Tetracycline can reach peak concentration 12h after orally in marrow tissue, and the concentration will decrease gradually. The highest fluorescent intensity was observed at 72h after orally in bone tissue by fluorescent microscope, and will not increase.Conclusion Tetracycline can reach the maximum concentration at 48-72h after orally in bone tissue, and to be stable. It can reach the peak concentration at 12h after orally in marrow tissue, and the concentration will decrease gradually.Part III Tetracycline induced targeting pTRIPZ/PPARy-shRNA lentiviral vector for the treatment of glucocorticoids induced osteoporosisObjective To establish rat GIOP model, investigate tetracycline induced targeting pTRIPZ/PPARy-shRNA lentiviral vector on the treatment of glucocorticoids-induced osteoporosis.Methods 32 male SD rats were randomly divided into 4 groups,8 rats in each group. The control group (Control group) given subcutaneous injection of normal saline 5mg/kg/d,5 times a week; The hormone group (Met group) subcutaneous injection of methylprednisolone 5 mg/kg/d,5 times a week, to establish GIOP model; Tetracycline orally group (Tet group):given tetracycline 100 mg/kg/d orally based on Met group, two times a week; Gene therapy group (shPPARy group):givenTetracycline 100 mg/kg/d orally on the basis of Met group, injection of TRIPZ/PPARy-shRNA lentiviral vector into the medullary cavity of femur after 12h, two times a week. After 8 weeks, rats weight was measured, serum samples were obtained for biochemical analysis, the femur and L3 BMD were measured, the microstructure were observed by Micro-CT and HE staining, comparison of bone histomorphometry and biomechanical parameters in each group.Results(1) Weight:There is no significant differences in each group before the experiment, after 8 weeks, the weight in Met group was siginificantly lower than that in Control group (P< 0.01); the difference of Met group and Tet group were not siginificant(P> 0.05); shPPARy group was significantly higher than Met group(P< 0.01);(2) Serum biochemical analysis:The total cholesterol, serum calcium in Met group was significantly higher than in Control group(P< 0.01), serum ALP was significantly lower than in Control group(P<0.01); Calcium in Tet group were significantly lower than in Met group (P< 0.01), but there were no significant difference of total cholesterol,serum ALP between Tet group and Met group (P> 0.05); The total cholesterol and serum calcium in shPPARy group were significantly lower than in Met group(P<0.05), serum ALP was significantly higher than in Met group (P< 0.05); The serum phosphorus in Met group Tet group and shPPARy group were siginificantly lower than those in Control group (P< 0.05), but no significant difference between the three groups(P>0.05).(3)Femur and L3 BMD:The BMD in Met group was significantly lower than in Control group (P< 0.01); The Tet group and shPPARy group significantly higher than in Met group (P< 0.05); And shPPARy group was significantly higher than in Tet group (P< 0.05).(4) Micro CT analysis:Compared with Control group, the trabeculaer bone in Met group were more less, BMD of Femur and L3 were significantly decreased measured by Micro CT (P< 0.01); There were no siginificant difference between Tet group and Met group in femur, (P> 0.05), but Tet group significantly increased in L3CP< 0.01); Compared with Met group and Tet group, the trabecular bone and BMD in shPPARy group were siginificantly increased(P< 0.01).(5) HE staining:Compared with Control group, the cells number of trabecular bone in Met group decreased, most cells arranged disorderly, network connections were destroyed; the cells number of trabecular bone in Tet group is increased compared with Met group, a part of network structures arranged regular; Compared with Met group, trabecular bone in shPPARy group arranged obviously closely and orderly, the number of trabecular bone increased, and most network structures were basically recovered.(6)Bone histomorphometry analysis:Met group siginificantly decreased%Tb.Ar, Tb.N, Tb.Th compared with Control group, while the Tb.Sp siginificantly increased (P< 0.01); Tet group of%Tb.Ar, Tb.N siginificantly increased compared with Met group, while Tb.Sp siginificantly decreased (P< 0.05), Tb.Th, Tet group and Met group showed no significant difference (P> 0.05);%Tb.Ar,Tb.N, Tb.Th, shPPARy group was siginificantly higher than in Met group, but Tb.Sp was siginificantly lower(P< 0.01); And there were siginificant difference among all the indexes between Tet group and shPPARy group (P< 0.05).(7) Biomechanical parameters:The max load, max deflection and the max stress in Met group was significantly lower than in Control group (P< 0.01); there were no significant difference between Tet group and Met group (P> 0.05); three indexes in shPPARy group were siginificantly higher than that in Met,group and Tet group (P< 0.05).Conclusion GIOP model can be established when subcutaneous injection of methylprednisolone 5 mg/kg/d,5 times a week. Tetracycline induced targeting pTRIPZ/PPARy-shRNA lentiviral vector has a therapeutic effect on GIOP rats, the effect is better than only use of tetracycline. |
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