【作者】 金翔；

【导师】 许建安；

【作者基本信息】 南京中医药大学 ， 中医骨伤科学， 2010， 博士

【摘要】 背景及目的关节软骨缺损修复困难,历来是医学界的难点及研究热点。目前,组织工程技术已经成为其主要的研究方向,并且取得了一定的进展,组织工程技术包括种子细胞、支架材料、生长因子三部分。另外,中医药在该领域中,已经被证实具有良好的临床疗效。我们筛选单味中药—威灵仙作为研究对象,有研究证明威灵仙能够维持和促进软骨细胞合成蛋白多糖与Ⅱ型胶原,并可能通过抑制白细胞介素1水平的增高保护关节软骨。本课题将威灵仙与组织工程技术结合,观察威灵仙促进软骨细胞增殖、干预C/β-GP凝胶复合同种异体软骨细胞体外三维培养以及修复软骨缺损的作用,探讨中医药与现代医学结合的方式与价值。方法1.体外分离培养兔膝关节软骨细胞,甲苯胺蓝染色鉴定后,取处于对数生长期第3代细胞随机分组进行培养,倒置显微镜观察软骨细胞形态变化,MTT法检测不同浓度威灵仙对软骨细胞增殖的影响,RT-PCR法检测TGF-β1mRNA表达。2.按7：1比例制备C/β-GP复合软骨细胞凝胶,随机分为实验组和对照组,分别给予相应处理,MTT法检测不同浓度威灵仙对凝胶中软骨细胞增殖的影响,HE、TB和Ⅱ型胶原免疫组织化学染色观察1、4周凝胶中软骨细胞的组织学变化。3.复制关节软骨缺损模型,24只新西兰白兔随机分为3组,即实验组(C/β-GP+软骨细胞+威灵仙培养基关节腔注射组)、对照组(C/β-GP+软骨细胞+普通培养基关节腔注射组)、模型组(模型组),分别给予相应处理,术后6、12周后分别行大体观察及HE、TB、Ⅱ型胶原免疫组织化学染色,并进行Wakitani评分。结果1.不同浓度威灵仙均能促进软骨细胞增殖,尤以0.5mg/ml增殖效果明显,且增殖高峰出现在威灵仙干预后的第3天。0.05、0.1、0.5、1.0mg/ml威灵仙均能促进软骨细胞TGF-β1 mRNA表达,4组组间差异无显著性意义,但作用的高峰为0.5mg/ml组。2.软骨细胞能良好地在C/β-GP凝胶内伸展、粘附、生长和增殖。各浓度组威灵仙均能促进软骨细胞在凝胶中的增殖,其中0.5mg/ml威灵仙的效果最明显,且能促进软骨细胞Ⅱ型胶原、细胞外基质的分泌。3.实验组、对照组可见缺损关节面被较好地填充,并形成透明软骨样结构,实验组表面平整度及与周围组织整合程度较对照组好,组织学切片上可见类软骨形成并分泌软骨基质和特异性Ⅱ型胶原,模型组未见修复,可见组织增生退变。实验组修复组织与周围组织整合程度、组织学及Ⅱ型胶原分泌量均好于对照组。Wakitani评分：实验、对照两组分值显著低于模型组分值(P<0.01),且实验组分值明显低于对照组分值(P<0.05)。结论1.威灵仙能促进软骨细胞增殖及TGF-β1 mRNA的表达,这种类细胞生长因子作用可能是其促进软骨修复的机制之一；2.C／β-GP凝胶与软骨细胞具有良好的相容性,威灵仙能够促进软骨细胞在C／β-GP凝胶中的增殖、Ⅱ型胶原、蛋白多糖的分泌及成软骨化发展。3.威灵仙能够促进可注射性C／β-GP凝胶复合同种异体软骨细胞修复关节软骨缺损,这为中医药与组织工程技术的结合提供了新思路、新方法。更多还原

【Abstract】 Background and objectiveOnce the articular cartilage defect,the repair is quite difficult,and it has always been the difficulty and research focus of the medical profession. At present,tissue engineering technique has been main research direction and has got advancement.The technique includes of seeds cell, frame and growth factor.Chinese medicine has been manifested satisfactory clinical therapeutic effect on repairing cartilage defect. We filt Weilingxian as study object. Studies have shown that Weilingxian can maintain and promote the synthesis of proteoglycan and collagen II of chondrocyte, and protect articular cartilage by inhibiting the level of interleukin-1 (IL-1) possibly.The topic combine Weilingxian with tissue engineering technique,and observe effect of Weilingxian on developing proliferation of chondrocytes and interfereing in injectable chitosan/β-glycerol PHosPHate(C/β-GP)encapsulating allograft chondrocytes there-dimension cultured in vitro and repairing articular cartilage defects. Our study try to explore methode and value of combineding Chinese medicine with modern medicine.Methods1.chondrocytes were isolated and cultured by the way of enzymatic digestion in vitro,after the PHenotype was identified by toluidine blue staining,the third-passage cells in the logarithmic growth PHase were randomly divided into groups and given the treatment respectively.MorPHology of chondrocytes was observed under inverted PHase contrast microscope,Methyl Thiazolyl Tetrazolium (MTT) assay method was adopted to observe the influence of Weilingxian with different concentrations on the proliferation of chondrocytes, and anti-transcription-polymerase chain-type reaction (RT-PCR) was used to assay the expression changes of transforming growth factor-β1 mRNA.2.Chitosan/β-glyceroPHosPHate gel encapsulating allograft chondrocytes was prepared at ratio of 7:1,then was randomly divided into experimental and control groups divided into groups and given corresponding treatment respectively.MTT assay method was used to detect the influence of Weilingxian with different concentrations on the proliferation of chondrocytes three-dimensional cultured in gel;HE, TB and type II collagen immunohistochemical staining was used to detect histological changes of chondrocytes at 1、4 week.3.After the model of knee articular cartilage defects was established,24 New Zealand rabbits were randomly divided into three groups,that is experimental group (C/β-GP+chondrocytes+Weilingxian medium intra-articular injection group), control group (C/β-GP+chondrocytes+normal medium intra-articular injection group), model group (model group),and given corresponding treatment respectively.The specimens were excised at the 6th and 12th week,examined grossly and analyzed by haematoxylin eosine,toluidine blues staining and typeⅡcollagen immunohistochemistry reaction.Wakitani score method was counted to evaluate the effect of repair.Result1.All groups of Weilingxian can promote proliferation of chondrocytes,effect of 0.5mg/ml group was especially obvious,and the peak of proliferation was at the third day.Group of 0.05、0.1、0.5 and 1.0 mg/ml could promote the expression of TGF-β1 mRNA, and there was no significant difference between the four groups (P>0.05), but the peak occurred at 0.5 mg/ml group.2.Chondrocytes behave good state of stretch、adhesion、proliferation in C/β-GP gel.All groups of Weilingxian can promote chondrocyte proliferation in the gel,and the effect of 0.5mg/ml group was especially obvious.Weilingxian can also enhance the secretion of collagen II and extracellular matrix.3.Defects of articular surface were well filled in experimental group and control group,and hyaline cartilage-like structure were formed,the surface flatness and degree of integration with surrounding tissue of experimental group was better than control group,and model group was not repaired.Formation of cartilage-like and secretion of cartilage matrix and specificity of collagen type II were found in histological slices.Integration of repair tissue with surrounding tissue,histology and amount of type II collagen secretion in experimental group were better than control group. Wakitani scores:scores of experimental group and control group were significantly lower than model group (P<0.01),and scores of experimental group was significantly lower than group control group (P<0.05).Conclusions1.Weilingxian can promote chondrocyte proliferation and TGF-β1mRNA expression,these role like cell growth factor may be one of the possible mechanisms to promote the repair of cartilage defect.2.It has good compatibility of chondrocytes in the co-culture with C/-GP gel,Weilingxian can promote chondrocytes proliferation and secretion of collagenⅡ、proteoglycan cultured in the C/β-GP gel, and also can induce the development to cartilage.3.Weilingxian can promote repair of cartilage defect by the injectable C/β-GP gel composite allogenic chondrocytes,this offer a new idea and method for the combination of Chinese medicine and tissue engineering technologies.更多还原