CAJ | 학술논문

目的观察雷帕霉素与低频电磁场联合运用对大鼠骨髓间充质干细胞(BMSCs)成骨效应的影响.方法选取成年SD大鼠15只,采用体外分离法培养大鼠BMSCs,传代3次后接种至细胞培养板中,将其分为对照组、雷帕霉素组、电磁场刺激组和联合应用组.对照组应用达尔伯改良伊格尔培养基(DMEM)进行培养；雷帕霉素组在DMEM中加入浓度为5 nM的雷帕霉素；电磁场刺激组则在DMEM基础上给予频率为15 Hz、强度为1 mT的脉冲电磁场刺激；联合应用组在加入浓度为5 nM雷帕霉素的DMEM基础上给予频率为15 Hz、强度为1 mT,每天1h的脉冲电磁场刺激.分别于干预后7d、14d和21d,提取细胞的总RNA和总蛋白,采用茜素红染色后置于显微镜下观察,分析比较不同时间点各组的成骨效应.结果 7d后,联合应用组的早期成骨效应优于雷帕霉素组,雷帕霉素组的早期成骨指标家禽相关转移因子-2(Runx2)基因表达量明显高于对照组(P＜0.05),电磁场刺激组Runx2基因表达量与对照组差异无统计学意义(P＞0.05)；14 d及21 d后,雷帕霉素组及电磁场刺激组的中期成骨指标骨唾液酸蛋白(BSP)和晚期成骨指标骨桥蛋白(OPN)的基因表达量均显著高于对照组(P＜0.05),联合应用组BSP和OPN基因表达量明显低于雷帕霉素组(P＜0.05).结论单独应用雷帕霉素和电磁场刺激具有良好的促成骨效应,二者联合应用时,促成骨效应减弱.
목적 관찰뢰파매소여저빈전자장연합운용대대서골수간충질간세포(BMSCs)성골효응적영향.방법 선취성년SD대서15지,채용체외분리법배양대서BMSCs,전대3차후접충지세포배양판중,장기분위대조조、뢰파매소조、전자장자격조화연합응용조.대조조응용체이백개량이격이배양기(DMEM)진행배양；뢰파매소조재DMEM중가입농도위5 nM적뢰파매소；전자장자격조칙재DMEM기출상급여빈솔위15 Hz、강도위1 mT적맥충전자장자격；연합응용조재가입농도위5 nM뢰파매소적DMEM기출상급여빈솔위15 Hz、강도위1 mT,매천1h적맥충전자장자격.분별우간예후7d、14d화21d,제취세포적총RNA화총단백,채용천소홍염색후치우현미경하관찰,분석비교불동시간점각조적성골효응.결과 7d후,연합응용조적조기성골효응우우뢰파매소조,뢰파매소조적조기성골지표가금상관전이인자-2(Runx2)기인표체량명현고우대조조(P＜0.05),전자장자격조Runx2기인표체량여대조조차이무통계학의의(P＞0.05)；14 d급21 d후,뢰파매소조급전자장자격조적중기성골지표골타액산단백(BSP)화만기성골지표골교단백(OPN)적기인표체량균현저고우대조조(P＜0.05),연합응용조BSP화OPN기인표체량명현저우뢰파매소조(P＜0.05).결론 단독응용뢰파매소화전자장자격구유량호적촉성골효응,이자연합응용시,촉성골효응감약.
Objective To study whether Rapamycin and a low frequency pulsed electromagnetic field (LFPEMF) have synergistic effects on osteogenesis.Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated from 4-week-old Sprague-Dawley rats (male,60-80 g) and cultured.The passage three BMSCs were transplanted into six well plates.The wells were divided into a control group in which the cells were cultivated in Dulbecco's modification of Eagle's medium (DMEM),a rapamycin group in which the cells were cultivated in DMEM containing 5 nM rapamycin,an electromagnetic field group in which the cells were cultivated in DMEM in a LFPEMF,and a combined treatment group in which the cells were cultivated in DMEM containing rapamycin in a LFPEMF.Proteins and RNA were extracted and the calcium disposition was stained using alizarin red after 7,14 and 21 days of incubation.Real-time PCR or western blotting was then used to detect any differences in the expression of the RUNX2,BSP,OPN or BMP2 genes.Results After 7 days of incubation the mRNA expression of the early osteogenesis indicator RUNX2 in the rapamycin group was significantly higher than that in the control group,while the expression of RUNX2 in the LFPEMF group did not differ significantly from that among the controls.The mRNA expression of the middle and late osteogenesis indicators BSP and OPN in the LFPEMF and rapamycin groups were significantly higher than in the control group in the middle (14 d) and late (21 d) periods,but the combined treatment group showed a much weaker effect than in the LFPEMF and rapamycin groups,as shown by significantly lower expression of BSP and OPN.Conclusions A LFPEMF does not have an obvious osteogenic effect in the early stimulating period,but is effective in the middle and late periods.LFPEMFs have no synergistic effect with rapamycin on osteogenesis.