骨碎补总黄酮促进骨膜细胞增殖及对兔骨不连的治疗作用

Osteopractic total flavone promotes periosteal cell proliferation and treats the nonunion in rabbits

背景:骨碎补总黄酮是一种传统中药,目前认为其主要具有预防骨质疏松、调节血脂等功效。骨碎补总黄酮对骨膜细胞的相关作用尚无相关研究。目的:探讨骨碎补总黄酮对兔骨膜细胞增殖的影响,进一步研究其对骨膜细胞治疗兔骨不连的促进作用。方法:①选取第3代兔骨膜细胞,分别用10^(-5),10^(-6),10^(-7) g/L骨碎补总黄酮及10^(-8) mol/L雌二醇培养,于第1,2,4,6天检测骨膜细胞增殖情况;②选取第3代兔骨膜细胞,分别用10^(-5)g/L骨碎补总黄酮、10^(-8)mol/L雌二醇、纯净水培养,于第1,5,9,13天检测骨膜细胞中碱性磷酸酶的活性,于第5,10,15,20天用Von Kossa法检测骨膜细胞中钙结节数量;③选取第3代兔骨膜细胞,分别用10^(-5)g/L骨碎补总黄酮、10^(-5)g/L骨碎补总黄酮+1μmol/L雌激素受体拮抗剂ICI182780、PBS培养,于第1,2,4,6天检测骨膜细胞增殖情况;④建立兔桡骨骨不连模型,将10^(-5)g骨碎补总黄酮和1×10~3骨膜细胞注入骨不连处,观察其对骨不连愈合的影响。结果与结论:①各组骨膜细胞吸光度值均随时间增加而增加,以10^(-5)g/L骨碎补总黄酮组和雌二醇组最明显,并于第6天达最大值;②10^(-5)g/L骨碎补总黄酮组和雌二醇组骨膜细胞碱性磷酸酶活性均高于对照组(P <0.05),两组碱性磷酸酶活性比较差异无显著性意义(P> 0.05);③骨碎补总黄酮诱导骨膜细胞的增殖作用被10^(-8) mol/L雌激素受体拮抗剂ICI182780完全阻断;拮抗剂组与对照组相比,骨膜细胞吸光度值差异无显著性意义(P> 0.05);④骨碎补总黄酮和骨膜细胞联合治疗能促进骨愈合;⑤结果表明,骨碎补总黄酮能促进骨膜细胞增殖和成骨分化,其促进细胞增殖的作用与雌激素受体有关;骨碎补总黄酮能在体内和骨膜细胞一起共同治愈骨不连。

BACKGROUND: Osteopractic total flavone is a traditional Chinese medicine, and has the effects of preventing osteoporosis and regulating blood lipids. The effect of osteopractic total flavone on periosteal cells is little known. OBJECTIVE: To investigate the effect of osteopractic total flavone on the proliferation of rabbit periosteal cells, and to further study its therapeutic effect on nonunion in rabbits. METHODS:(1) The third generation of rabbit periosteal cells was cultured with 10-5, 10-6 and 10-7 g/L osteopractic total flavone and 10-8 g/L estradiol, separately. The cell proliferation was detected at 1, 2, 4 and 6 days.(2) The third generation of rabbit periosteal cells was cultured by 10-5 g/L osteopractic total flavone, 10-8 g/L estradiol and purified water. The alkaline phosphatase activity was detected at 1, 5, 9 and 13 days. The count of calcium nodules was measured by Von Kossa assay at 5, 10, 15 and 20 days.(3) The third generation of rabbit periosteal cells was cultured by 10-5 g/L osteopractic total flavone, 10-5 g/L osteopractic total flavone +1 μmol/L ICI182780(estrogen receptor antagonist) and PBS, separately. The cell proliferation was detected at 1, 2, 4 and 6 days.(4) The rabbit model of nonunion was established, and 10-5 g/L osteopractic total flavone and 1× 103 periosteal cells were injected into the nonunion area to observe the effects on the nonunion. RESULTS AND CONCLUSION:(1) The absorbance value of periosteal cells was increased with time, especially in the 10-5 g/L osteopractic total flavone and estradiol groups, and peaked on day 6.(2) The alkaline phosphatase activity in the 10-5 g/L osteopractic total flavone and estradiol groups was higher than that in the control group(P < 0.05), and there was significant difference between 10-5 g/L osteopractic total flavone and estradiol groups(P > 0.05).(3) The cell proliferation induced by osteopractic total flavone was completely inhibited by 10-8 mol/L estrogen receptor antagonist ICI182780. There was no significant difference in the absorbance value between antagonist and control groups(P > 0.05).(4) The combination of osteopractic total flavone and periosteal cells could promote bone healing.(5) These results imply that osteopractic total flavone can promote osteoblast proliferation and differentiation, which is related to estrogen receptors. Osteopractic total flavone combined with periosteal cells can cure nonunion in vivo.