ABSTRACT
Objective·Giving the ovariectomized (OVX) mice exogenous estrogen replace therapy (ERT) so as to explore the inflammatory mechanism of estrogen in the pain modulation of OVX mice.Methods·Twenty-four 12-week-old female C57BL/6J mice were randomized into three groups:sham group (S),ovariectomy+placebo group (P),ovariectomy+estrogen group (E).The ERT began from one week after OVX and last for four weeks.Paw withdraw threshold (PWT) and paw withdraw latency (PWL) were measured at one day before OVX,and three days,one week,two weeks,three weeks,four weeks and five weeks after OVX.Concentrations of inflammatory cytokines in serum were measured at three days and five weeks postoperatively.Five weeks after operation,the lumbar intumescentia of spinal cord was taken and the expression of p38 was analyzed by Western blotting.Results·All the OVX mice's PWTs or PWLs were decreased after operation compared with non-operation mice;PWTs and PWLs were significantly different from group S separately from one week and three days after OVX (P＜0.01).The inflammatory cytokines of OVX mice were significantly higher than group S (both P＜0.01) at three days after OVX.Western blotting results showed that the p38 of group P was significantly higher than that of group S after five weeks postoperatively (P＜0.01).By receiving ERT,both PWTs and PWLs of group E increased slightly and were significantly different from group S (P＜0.01) at four and five weeks after OVX.At five weeks after OVX,inflammatory cytokines of group E were significantly lower than group P (P＜0.01) but still much higher than group S (P＜0.01).Meanwhile the content of p38 in group E was less than that in group P (P＜0.01).The p38 in group E was still more than group S,however it was no longer significantly different from group S (P＞0.05).Conclusion·Estrogen may reduce concentrations of inflammatory cytokines through the p38 MAPK pathway and plays an important role in the pain modulation of OVX mice.
ABSTRACT
Objective To construct and identify adenovirag (Ad-RUNEP) carrying human B-endorphin(B-EP) genes which can be regulated by mifepfistone (RU486)-inducible system and to evaluate the effects of different concentrations of RU486 on the transgene expression in adenovirus in vitro.Methods The shuttle plagmid pDC312一RUNEP carrying B-EP genes which can be regulated by RU486-inducible system was constructed and was combined with adenovims to form a recombinant Ad-RUNEP using AdMAXTM system.The recombinant Ad.RUNEP was then amplified and purified.The titers of the adenovirus were determined and the adenovirus vector was checked.After being infected by Ad-RUNEP for 24 h,A431 cell line was incubated in liquid culture media containing RU4860,10-10,10-9,10-8,10-7,and 10-6 mol/L respectively(group R0-5) for 48h,and Was then transferred to liquid culture media containing no RU486.The liquid culture media were obtained on 1st.2nd and 4th day of incubation and centrifuged.The supematant Was collected for determination of B-EP concentration by ELISA.Results The analysis of enzyme-incision demonstrated that RU486 regulating system and B-EP were cloned directly into pDC312-RUNEP.The titer of Ad-RUNEP was 2.25×1010 pfuml.The expression of B-EP was significantly higher in group R1-s than in group R0 and was significantly lower in group R1-3 than in group R4 (P<O.05).The expression of B-EP wag significantly higher on 2nd than on 1st and 4th day.Conclusion The adenovims carrying B-EP genes which can be regulated by RU486 (Ad-RUNEP) Wag successfully constructed.