CAJ | 학술논문

背景:以往实验证实杜仲能促进骨髓基质细胞向成骨细胞分化,但对促进成骨分化的分子机制报道很少.目的;观察中药杜仲水,醇提取物对大鼠骨髓基质细胞骨桥蛋白和骨保护素表达的影响方法:盐杜仲2 g,分别加蒸馏水或甲醇至15 mL,室温振荡1 h,静置过夜后,15 000r/min离心10 min,弃沉淀.水浸上清不经处理即得水提取物;甲醇浸上清经真空浓缩,再加水至15 mL溶解剩余物后,得醇提取物水、醇提取物,用0.22 μm滤膜过滤后于-20℃保存.取2月龄SD大鼠6只,体外培养至第3代SD大鼠骨髓基质细胞进行杜仲水,醇提取物诱导,诱导物分为1×10~(-2),1 ×10~(-3),1×10~(-4)和1 ×10~(-5)4个稀释度:阴性对照组加入PBS;诱导培养6 d.以免疫细胞化学法测定诱导6 d的骨髓基质细胞骨桥蛋白和骨保护素表达.应用反转录一聚合酶链反应方法测定1×10~(-3),1×10~(-4)稀释度诱导3 d的骨髓基质细胞骨桥蛋白和骨保护素的基因表达.结果与结论:细胞免疫化学显示,杜仲水,醇提取物对大鼠骨髓基质细胞骨桥蛋白的表达均有显著的刺激作用,以1×10~(-4)的稀释度作用最强,且醇提取物的作用大于水提取物;骨保护素的表达却无显著变化.反转录-聚合酶链反应显示,水提取物对诱导3 d后大鼠骨髓基质细胞骨桥蛋白的基因表达量显著高于醇提取物,骨保护素未检测出表达.证实杜仲水,醇提取物诱导骨髓基质细胞成骨分化与刺激骨桥蛋白表达相关,与骨保护素无关;水提取物促进骨髓基质细胞骨桥蛋白表达早于醇提取物.
배경:이왕실험증실두중능촉진골수기질세포향성골세포분화,단대촉진성골분화적분자궤제보도흔소.목적;관찰중약두중수,순제취물대대서골수기질세포골교단백화골보호소표체적영향방법:염두중2 g,분별가증류수혹갑순지15 mL,실온진탕1 h,정치과야후,15 000r/min리심10 min,기침정.수침상청불경처리즉득수제취물;갑순침상청경진공농축,재가수지15 mL용해잉여물후,득순제취물수、순제취물,용0.22 μm려막과려후우-20℃보존.취2월령SD대서6지,체외배양지제3대SD대서골수기질세포진행두중수,순제취물유도,유도물분위1×10~(-2),1 ×10~(-3),1×10~(-4)화1 ×10~(-5)4개희석도:음성대조조가입PBS;유도배양6 d.이면역세포화학법측정유도6 d적골수기질세포골교단백화골보호소표체.응용반전록일취합매련반응방법측정1×10~(-3),1×10~(-4)희석도유도3 d적골수기질세포골교단백화골보호소적기인표체.결과여결론:세포면역화학현시,두중수,순제취물대대서골수기질세포골교단백적표체균유현저적자격작용,이1×10~(-4)적희석도작용최강,차순제취물적작용대우수제취물;골보호소적표체각무현저변화.반전록-취합매련반응현시,수제취물대유도3 d후대서골수기질세포골교단백적기인표체량현저고우순제취물,골보호소미검측출표체.증실두중수,순제취물유도골수기질세포성골분화여자격골교단백표체상관,여골보호소무관;수제취물촉진골수기질세포골교단백표체조우순제취물.
BACKGROUND: Previous studies demonstrated that eucommia bark can promote bone marrow stern cells (BMSCs) differentiated into osteoblasts, but relative mechanism is poorly understood. OBJECTIVE: To investigate the effects of eucommia bark water/methanol extracts on expressions of osteopontin (OPN) and osteoprotegedn (OPG) in rat BMSCs. METHODS: Totally 2 g eucommia bark powder were added into water or methanol to 16 mL and oscillated for 1 hour at room temperature. After soaked overnight, both extracts were centrifuged at 15 000 r/min for 10 minutes. Water extract was obtained from supernatant in water soaked powder. In methanol soaked powder, methanol extracts was obtained by concentrated supernatant in vacuo and resolved using 16 mL water. Water and methanol extracts were then filtered by 0.22 μm membrane, and conserved at -20℃. Six SD rats, aged 2 months, were selected, and the 3~(rd)passage of BMSCs were induced by water or methanol extracts with dilution of 1 × 10~(-2), 1 × 10~(-3), 1 × 10~(-4) and 1 × 10~(-5), respectively. PBS was added in the negative control group. All cells were cultured for 6 days. Expressions of OPN and OPG was measured by immunocytochemistry at 6 days with induction. The expression of OPN and OPG induced by water and methanol at 1 ×10~(-3) and 1×10~(-4) dilution was detected by RT-PCR. RESULTS AND CONCLUSION: Immunocytochemistrical results indicated that both water and methanol extracts of eucommia bark simulated OPN and OPG expression, in particular with dilution of 1×10~(-4). The methanol extracts had a stronger effect than water extract, but the expression of OPG did not change obviously. RT-PCR demonstrated that at the 3rd day of inducement, the level of OPN expression induced by water extract was higher than that of methanol extract, and no OPG expression was detected. Osteogenic differentiation of rat MSCs induced by eucommia bark water/methanol extracts relates to stimulating expression of OPN, which has no correlation to OPG. OPN expression induced by water extract is early than that of methanol extract.