华中科技大学
硕士学位论文
骨形态发生蛋白2活性肽在体内体外诱导成骨的实验研究
姓名：卢宏伟
申请学位级别：硕士
雷锋作文
专业：外科学（骨外科）
指导教师：郭晓东
2010-04
骨形态发生蛋白2活性肽在体内体外
诱导成骨的实验研究
享受孤独华中科技大学同济医学院附属协和医院骨外科
试用期自我鉴定
硕士研究生：卢宏伟
指导教师：郭晓东教授
摘要
【目的】探讨根据骨形态发生蛋白2（BMP-2）自行研制合成的活性多肽在体内和体外诱导成骨的作用。
【方法】①制备骨形态发生蛋-2活性多肽。②制备体内实验所需的水凝胶载体。③将重组人骨形态发生蛋-2（rhBMP-2）及骨形态发生蛋-2活性多肽与水凝胶复合作为实验组，以空白水凝胶作为对照组。④动物试验分6组。A组：单纯水凝胶作为空白对照；B组：5 mg活性多肽与水凝胶复合组；C组：10 mg活性多肽与水凝胶复合组；D 组：20 mg活性多肽与水凝胶复合组；E组：40 mg活性多肽与水凝胶复合组；F组：5 μg重组人骨形态发生蛋-2与水凝胶组复合组作为对照组。⑤将60只大鼠随机按上述方法分组，每组10只。每只大白鼠左侧大腿作1cm的切口，制备股四头肌肌袋模型。将各组实验材料植入股四头肌肌袋中。大鼠培养至第6周作放射学检查（X-ray，CT），并处死作组织学（HE染色、Vonkossa染色）检查。比较各组成骨效果。⑥将重组人骨形态发生蛋-2及不同浓度的活性多肽与鼠成骨前体细胞（MC3T3-E1）复合培养。重组人骨形态发生蛋-2作为对照组，普通培养基作为空白对照。分别在3、5、7天检测碱性磷酸酶（ALP）活性，比较各组差异。
【结果】①第6周时X线和CT扫描见：A、B、D组无明显高密度影；E组可见不甚明显的高密度影；C、F组有明显团块状高密度影。②组织学观察见：6组的水凝胶载体均充分降解，未见残留物。A、B、D组未见成骨细胞、骨小梁及新生骨；E组见部分成骨细胞和软骨陷窝；C、F组见团块状新生骨和新生骨小梁，周围包绕大量成骨细胞，外周可见新生血管。③碱性磷酸酶检测见：重组人骨形态发生蛋白-2与不同剂量活性
多肽所复合的细胞的碱性磷酸酶活性比空白对照组明显增高。重组人骨形态发生蛋白-2组碱性磷酸酶活性最高。活性多肽组随剂量升高碱性磷酸酶活性逐渐增高，并有时间相关性，随培养时间增加，碱性磷酸酶活性逐渐增高，在第5天达到高峰，随后逐渐下降。
【结论】骨形态发生蛋白活性多肽在大鼠体内能够诱导异位成骨，并能诱导鼠成骨前体细胞向成骨细胞分化，效果明显，其作用接近于骨形态发生蛋白-2，在骨修复方面具有良好的应用前景。
【关键词】水凝胶；骨形态发生蛋白活性肽；骨诱导；异位成骨；成骨分化；鼠成骨前体细胞
Osteoinduction of a Bone Morphogenetic Protein-2 Related
Peptide in Vitro and in Vivo
Department of Orthopaedics， Union Hospital，Tongji Medical College，
Huazhong University of Science and Technology
Candidate: Hongwei Lu
Supervisor: Prof. Xiaodong Guo
Abstract
Objective: To investigate the activity of osteoinduction of a synthetic peptide developed according to the bone morphogenetic protein 2 (BMP-2) in vitro and in vivo.
Methods: BMP-2 related peptide and hydrogel vector were prepared. The recombinant human bone morphogenetic protein-2 (rhBMP-2) and BMP-2 related peptide combined with hydrogels respectively were the experimental groups and the blank hydrogel was the blank control group. 60 SD rats were divided into 6 groups randomly with 10 in each group. The blank hydrogel were implanted in group A as blank control group. 5 mg，10 mg，20 mg，40 mg active peptide combined with hydrogel were implanted in group B，C，D，E respectively. Complex of 5 µg rhBMP-2 and hydrogel were implanted in group F as control group.The quadriceps muscle pouch model was prepared of 1cm incision on the left thigh in each rat and implanted with experimental materials resp
ectively. Radiological examinations (X-ray， CT) and HE staining were performed 6 weeks after modeling to compare the osteogenic effect in each group. Culture the rhBMP-2 and different concentrations of active peptides with the rat osteoblast precursor cells (MC3T3-E1)，rhBMP-2 as the control group and normal culture medium as blank control. Detection the activity of alkaline phosphata(ALP) in 3，5，7 days respectively to compare the
differences between each group.
①，B， D showed no significant high density， group E had
接待工作方案Results: Group A
洛川神irregular high density; group C，F showed a significantly higher density of block shadow暖和的近义词是什么
②
in X ray and CT scan. Hydrogel vector in each group were fully degraded histologically. No osteoblasts，trabecula and new trabecular bone could be obrved in group A，B and D; group E showed some of osteoblasts and cartilage lacuna; in group C and F the bulky massive new bone and new trabecula wrapping around by a large number of osteoblasts and peripheral neovasculariza
tion could be obrved.③The activity of cellular ALP in the compound of rhBMP-2 peptides and different dos of BMP-2 related peptide were significantly higher than the blank control group， especially in rhBMP-2 group. ALP activity in peptide group incread with the do gradually went up and showed time dependence that ALP activity gradually incread with increasing time and reached a peak in the 5th day and then decread gradually.
Conclusion: Bone morphogenetic protein peptides can induce ectopic bone formation in rats and can induce rat osteoblast precursor cells differentiating into bone cells significantly as bone morphogenetic protein -2 and has a good prospect in bone regeneration.
Key words: Hydrogel; Bone morphogenetic protein-2 related peptide; Osteoinduction; Ectopic bone formation; Bone differentiation; MC3T3-E1
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