【作者】 徐天睿；

【导师】 白人骁； 李秀兰；

【作者基本信息】 天津医科大学 ， 外科学， 2007， 硕士

【摘要】 临床上各种原因引起的骨缺损很常见，且治疗困难。目前采用的自体骨和异体骨移植均有各自的不足之处：自体骨移植的骨量受到限制，另外也增加了病人的痛苦；而异体骨移植可能出现免疫排斥反应和其它的并发症。1987年美国科学基金会提出组织工程概念，国内外组织工程研究取得令人注目的阶段性研究成果，应用组织工程骨修复骨缺损将成为可能，将组织工程骨用于骨缺损的修复是临床治疗的趋势所在。传统外用中药生肌膏对于感染性开放骨折疗效显著，将其有效成分优化制备成生肌液。本实验旨在研究体内环境中生肌液对骨髓基质细胞(Marrow Stromal Cells，MSCs)增殖和分化功能的影响，为骨组织工程研究活性骨诱导因子提供新思路。目的：1建立和完善MSCs分离和培养技术，最大限度的获得MSCs。2将中西医结合治疗感染性开放骨折的特色中药应用于骨缺损的修复研究，观察生肌液在体内对MSCs增殖和分化的影响，从而进一步加深我们对中药作用的认识，探索中药在骨组织工程领域的新应用。方法：1抽取大耳白兔胫骨骨髓，经密度梯度离心-贴壁筛选，获取MSCs并培养、扩增。2以纤维蛋白胶和脱基质骨(Dematrix Bone，DMB)为MSCs载体，构建组织工程骨，分别为：空白组(DMB-MSCs复合物)；实验组包括骨形态发生蛋白(Bone Morphgenetic Protein，BMP)组(含BMPDMB-MSCs复合物)、生肌液组(生肌液-DMB-MSCs复合物)、BMP+生肌液组(生肌液-含BMPDMB-MSCs复合物)。3制作标准的兔桡骨10mm完全缺损模型，植入已构建的组织工程骨，制作修复模型。4术后观察动物的一般情况，分别于4、8、12、16周摄X线片动态观察新生骨痂生长；4、8、12、16周取材进行组织学染色观察骨缺损修复过程中的细胞学变化；4周进行BrdU免疫组化染色观察标记的MSCs在骨修复中的作用；8周进行Ⅰ型胶原免疫组化染色观察骨缺损修复中Ⅰ型胶原的分泌活性、Von Kossa染色以及四环素荧光标记观察新骨形成状态；16周取材进行标本大体观察和骨密度检测以综合评价骨缺损修复情况。结果：1采用密度梯度离心—贴壁筛选的方法，可获得大量骨髓中的MSCs。MSCs于原代培养13—14天，80％融合，进行传代培养。传代后MSCs为成纤维细胞样形态结构，呈指纹状生长，具有强大的增殖能力。2动态放射学和组织学检查结果提示生肌液-BMP组编织骨生成量最多，成熟也最早；BMP组骨痂生成量和成熟度都强于生肌液组；空白组最差，骨痂生长缓慢，量少且幼稚。3组织学观察发现骨缺损修复兼有膜内成骨和软骨内成骨的方式，且新生编织骨贴支架材料生长；含生肌液组新生血管较其它组更丰富。4术后4周标本BrdU标记可见支架材料中细胞胞核呈深棕色，显示植入的MSCs参与了骨缺损修复。5术后8周标本四环素荧光标记观察四环素荧光带的亮度及带间宽度BMP+生肌液组＞BMP组＞生肌液组＞空白组。6术后8周标本Ⅰ型胶原免疫组化观察显示大量骨基质生成，且各实验组明显多于空白组。7术后8周标本Von Kossa染色显示骨折断端和支架材料旁新生编织骨。结论：1采用密度梯度离心—贴壁筛选法，分离大耳白兔骨髓中的单个核细胞层，可获得具有强大增殖能力、高纯度的MSCs；2在体内环境，中药生肌液具有促进MSCs增殖和分化为成骨细胞的能力。生肌液与BMP复合能极大增强BMP的功能，更加有效的促进成骨；3 DMB具有天然的多孔网状结构，生物相容性和生物降解性好，可以作为骨组织工程的支架材料；与生肌液复合，复合物有良好的骨传导和诱导成骨能力；4使用纤维蛋白胶将MSCs加载于DMB支架上可保证支架内细胞存活；5目前，还不能明确生肌液中哪种(些)有效成分在对MSCs增殖活性和成骨分化起作用，需要我们进一步研究生肌液中单味药和组方药的各自有效成分及诱导MSCs增殖和分化成骨的机理。更多还原

【Abstract】 Bone defect is common in clinic, which caused by many kinds of diseases, and it’shard to treat. The present treatment for the bone defects includes autogenous andallogenous bone transplantation, the former is restricted in amount and may causedistress, the latter can arouse the immunological rejection and other complications. Sincethe American Science Foundation introduced the concept of "Tissue Engineering" in1987, the study on tissue engineering have received noticeable achievement, it is possibleto use the tissue-engineering bone to repair the bone defect and it becomes one trend inclinic. According to Chinese traditional herb ShengJiYe can cure infectious open bonefracture, this experiment study the influence of ShengJiYe on MSCs’ proliferation anddifferentiation in vivo.Aim: 1 To establish and perfect the method to isolate and culture MSCs, and getmore seed cells possibly. 2 Put the characteristic Chinese herb ,which can cure infectiousopen fracture, in MSCs’ proliferation and differentiation in vivo. Explore new applicationof Chinese herb medicine in tissue-engineering bone, and deepen our knowledge onChinese herb medicine’s action.Method: 1 Get bone marrow from rabbits’ tibias, obtain MSCs by density gradientcentrifugation and plastic-adherence, then cultivate and amplify them. 2 Put fibrin sealantasnd dematrix bone (DMB)as carrier of MSCs,construct tissue- engineering bone ofMSCs-DMB composite as blank group,and MSCs-BMP-DMB composite,MSCs-ShengJiYe-DMB, MSCs- ShengJiYe- BMP-DMB as experimental group to repairrabbit 10mm segmental radial defect.3 The efficiency of osteogenesis was evaluation byradiography, histology, immuno- histochemical staining, tetracycline fluorescencetest, Von Kossa staining and BMD analysis.Result: 1 MSCs can be isolated by means of density-gradient-centrifugation andtheirs adherence to plastic. The primary generation can be cultured for 13-14 days. Afterpassaged, the MSCs look fibroblastoid, growing in the form of fingerprint and can proliferate. 2 The radiographic and histologic evaluation show: BMP-ShengJiYe has thestrongest power to form the new bone callus and make them mature; BMP has the secondcapability to form and mature new bone callus, and ShengJiYe has the lesser ability toinduce osteogenesis. The pure scaffold-MSCs form and mature new bone callus slowliest.3 Histologic analysis confirrn the mechanism of osteogenesis are both intramembranousand endochondral ossification, and the new-born woven bone grow along the scaffold. 4BrdU labelled specimen 4 weeks post operation show MSCs take part in repairing bonedefect. 5 Tetracycline fluorescence test reveal the volumn and forlning rate of new bornbone: BMP-ShengJiYe group＞BMP group＞ShengJiYe group＞blank group.6 ColⅠimmunohistochemical staining show much new bone matrix, and experimentalgroup have more than blank group. 7 Von Kossa staining display new born boneexist near the fracture and scaffold.Conclusion: 1 Isolate the layer of mono-nucleus cells in the rabbit bone marrow bymeans of density-gradient-centrifugation and plastic-adherence, and the obtained MSCsare pure and can proliferate. 2 In vivo, ShengJiYe has the ability to make MSCsproliferate and to induce MSCs differentiating to osteoblasts. And ShengJiYe canenhance BMP’s ossification. 3 Dematrix bone have natural porous structure, goodbiocompatibility and biodegradability, can be used to as the scaffold oftissue-engineering bone. DMB-ShengJiYe composite have good osteoconduction andosteoinduction. 4 Fibrin sealant can ensure the viability of the cells in the scaffold ascell-carrier. 5 At present, we can’t confirm the definite components acting on MSCs’proliferation and differentiation, We should further study the single medicine in ShenJiYeand the effective components of the medicine complex, and explore the mechanism ofMSCs’ proliferation and differentiation to osteoblast.更多还原