【作者】 张文海； 李秀兰； 张杨； 冯俊奇； 白人骁；

【机构】 天津市天津医院风湿骨病科；

【摘要】 因多种疾病引起的骨缺损在,临床当中十分常见,但治疗困难。近年来组织工程学的创立和迅速发展,为骨组织修复提供了新的思路和方法。骨组织工程研究主要有三个方面:种子细胞,生长因子,支架材料。其中,骨髓间充质干细胞(MSCs)及其诱导因子是目前生命科学研究的重点。众多学者多采用化学药物(如地塞米松,维生素C,β-甘油磷酸钠等)或生长因子(如骨形态发生蛋白2)将骨髓间充质干细胞(MSCs)诱导为成骨细胞,用于修复骨缺损。但上述骨诱导因子仍存在着诱导转化率不稳定、使用安全性等诸多问题。结合中药生肌膏能够治疗感染开放性骨折的临床经验,本试验尝试将生肌液作为MSCs的刺激因子进行研究。目的:考察中药生肌液对体外培养的免骨髓间充质干细胞(MSCs)成骨活性的影响及其作用特点,为骨组织工程研究找寻一种新的作用因素,探索中药在骨组织工程领域的新应用。方法:1取家兔胫骨骨髓,用密度梯度离心-贴壁筛选的方法,获取并培养鉴定MSCs。 2以不含生肌液的基础培养液(含10％胎牛血清的MEM培养基)为空白对照,以含地塞米松10-8M,维生素C0．05 ／ml,β-甘油磷酸钠10mM的基础培养液为标准对照。在基础培养液中加入生肌液,分别制成1:20(生肌液浓度为0．15g／ml)、1:60(生肌液浓度为0．05g／ml)、1:100(生肌液浓度为0．03g／ml)的条件培养液。将不同浓度的生肌液作用于免MSCs,采用空白对照、标准对照,用镜下观察、四环素标记、胶原I免疫组织化学染色和检测细胞成骨标志物碱性磷酸酶 (ALP)、骨钙素(OCN)、乳酸脱氢酶(LDH)的方法,计算出其比值(ALP／LDH和OCN／LDH),考察MSCs 的成骨活性。结果:在条件培养后的2周、3周时间点:生肌液组MSCs细胞四环素标记呈阳性(可见钙结节荧光),胶原I免疫组织化学染色亦呈阳性,空白对照组为阴性;成骨标志物ALP和OCN在生肌液组均有表达,且ALP／LDH和OCN／LDH比值都是以1:20生肌液组最高(P<0．05)。结论:生肌液对MSCs的诱导成骨作用肯定,且与剂量有关,高浓度生肌液的诱导成骨活性最强。更多还原

【Abstract】 It is difficult to cure the bone defect that is common in clinic field caused by many kinds of diseases. In the resent years, the development of bone tissue engineering has given us a new kind of thought and method , that includes three aspects: seed cell, growth factor and scaffolds. Now, the emphasis of life science is mesenchymal stem cell (MSCs)and its inductive factor. Chemical drugs and growth factors are popular for osteogenesis.But their safety problem and unstability of transformation effiency are ever existed. Basing on the clinic experience that Sheng-Ji Ointment can successfully cure infectious open fractures, the experiment tries to make Sheng-Ji liquor as a stimulative of mesenchymal stem cells. Objective:to investigate the effect of Sheng-Ji liquor on osteogenesis of bone mesenchymal stem cells(MSCs)from rabbit tibia,in vitro. Method: 1.Bone marrow is from rabbit tibia. MSCs are isolated by the method of density gradient centrifugation with stick to plastic, then, cultivated and identified.2 The experiment puts Sheng-Ji liquor to MSCs in different concentration. Using a blank control and standard control, it can observe the osteogenesis of MSCs. Three are some ways: cell morphology, acheomycin labeling (for mineralized nodes) ,immunocytochemistry (for Coll) and detect alkaline phosphatase (ALP), osteocalcin(OCN) and lactate dehydrogenase(LDH). Result: At the 2-week and 3-week time, the results show us something followed. Conditioned cultured groups is positive in acheomycin labeling and immunocytochemistry, but blank control is negative. ALP and OCN are positive in conditioned cultured groups. The 1:20 group is higher than other groups in ALP/LDH and OCN/LDH (P<0.05). Conclusion: The Sheng-Ji liquor has definitive inductive osteogenitive effect on MSCs, relatively to dosage.The high concentration is the best for osteogenesis.更多还原