【作者】 张国荣；

【导师】 欧阳宏伟；

【作者基本信息】 浙江大学 ， 人体解剖与组织胚胎学， 2010， 硕士

【摘要】 背景:随着国民素质的提高以及人们对健康的追求,体育锻炼和竞赛活动明显增加,运动造成的临床损伤也越来越多,其中韧带肌腱损伤占50%以上。由于韧带肌腱再生能力差,所以治疗后常由小直径胶原纤维组成,力学性能和组织结构低下,常导致重复断裂。如何让损伤肌腱再生大直径胶原纤维是目前肌腱修复研究的主要难题。实验发现V型胶原和蛋白聚糖decorin在损伤肌腱中表达异常升高,且V型胶原和蛋白聚糖是调节胶原纤维生长自聚的重要因子。所以本研究将利用基因抑制手段降低肌腱细胞中V型胶原和decorin的表达,建立组织工程肌腱模型,在体外研究胶原纤维生长自聚的生物学信息,在体内评价损伤肌腱的修复效果。本研究分为四个部分:（1）建立组织工程肌腱模型,为体外检测肌腱再生过程中相关细胞外基质的作用和体内修复损伤肌腱提供可行手段;（2）siRNA抑制肌腱细胞中高表达的V型胶原,在体外检测其对胶原纤维形成的影响;（3）反义核酸法抑制蛋白聚糖decorin表达,在体外检测其对胶原纤维形成的影响;（4）构建组织工程肌腱,用慢病毒介导的siRNA抑制decorin的表达,修复损伤肌腱评价其修复效果。本研究探求了V型胶原和蛋白聚糖decorin在肌腱损伤修复过程中对胶原纤维生长自聚的影响,为促进损伤肌腱再生,达到结构和功能的完全恢复提供有用的基础生物学信息。第一部分构建应用于生物学研究的组织工程肌腱目的:本研究建立体外组织工程肌腱模型,为体外分析肌腱再生过程中相关细胞外基质因子的作用和体内修复损伤肌腱提供可行手段。方法:高糖DMEM培养肌腱细胞,培养基中加入10%胎牛血清（FBS）和50μg/mL维生素C,防止细胞老化和促进细胞外基质分泌。三天更换一次培养基,培养两周后细胞分泌的胞外基质逐渐累积和细胞一起在培养皿底部形成连续的细胞片。用1ml枪头尖部沿培养皿的边缘,用向上剪切力慢慢卷起细胞片,形成长条形组织。继续培养一周,使细胞片光滑面与粗糙面充分融合形成组织工程肌腱。组织学检测组织工程肌腱的结构特征,透射电镜检测其胶原纤维的微观结构。结果:此种方法构建的组织工程肌腱表面光滑,结构紧密。组织学结果显示有波浪式的胶原纤维生成,细胞存活良好,分散在成束纤维中间。透射电镜结果显示组织工程肌腱形成了直径大小相对均一的胶原纤维。结论:本研究由培养的肌腱细胞成功构建了组织工程肌腱,该组织工程肌腱中细胞存活良好,细胞外基质分泌丰富。具有波浪形的胶原纤维结构,胶原纤维直径大小均匀,类似于损伤肌腱组织,可以用于体外检测胶原合成效率与胶原纤维直径大小,分析肌腱完全再生过程中相关细胞外基质的作用。也可以用来修复缺损肌腱组织,应用于进一步的体内研究实验。是体内外研究肌腱损伤修复的良好生物学模型。第二部分siRNA抑制V型胶原的表达对胶原纤维形成的影响目的:损伤肌腱组织中V型胶原表达异常升高,修复组织常由小直径胶原纤维组成,力学性能低下,易重复断裂。本实验利用RNAi技术抑制V型胶原的表达,体外构建组织工程肌腱,探讨V型胶原在损伤肌腱中的调节作用。方法:根据V型胶原的分子序列COL5（α1）₂（α2）,分别设计与COL5A1和COL5A2亚基互补的siRNA序列,转染到大鼠肌腱细胞内。分别用real time PCR和免疫荧光法检测V型胶原在基因水平和蛋白水平的表达。确认V型胶原的表达被抑制后,检测细胞增殖和凋亡情况,以及组织工程肌腱的胶原含量和胶原纤维直径大小。分析COL5A1和COL5A2亚基在肌腱胶原纤维形成中的作用。并按不同比例混合培养抑制COL5A1表达的细胞和非抑制细胞,分析COL5A1不同水平的抑制,组织工程肌腱中胶原纤维的形成情况。结果:阴性对照组（FAM绿色荧光标记的乱序siRNA）siRNA的转染效率>70%。Real time PCR与免疫荧光结果显示V型胶原在基因水平和蛋白水平的表达均被明显抑制。COL5A1和COL5A2亚基对其他相关基因表达和胶原纤维形成的影响不同。抑制COL5A1表达后,蛋白聚糖decorin的表达明显降低,胶原纤维形成障碍且直径比对照组更小。抑制COL5A2表达后,Ⅰ型胶原表达降低,形成的胶原纤维无明显变化。在混合培养的实验中,抑制COL5A1表达的细胞和未抑制细胞混合比例为1∶1和1∶0.5时,胶原纤维状态良好且直径增大。结论:RNAi是一种有效的抑制基因表达的手段。在胶原纤维形成过程中,COL5A1亚基比COL5A2亚基的作用显著。在肌腱愈合过程中,V型胶原的水平至关重要,V型胶原过多会导致小直径胶原纤维的形成,过少会导致胶原纤维形成障碍,只有合适的水平才能促进损伤肌腱修复再生。第三部分反义核酸法抑制蛋白聚糖decorin表达对胶原纤维形成的影响目的:损伤肌腱组织中蛋白聚糖decorin表达异常升高,修复组织常由小直径胶原纤维组成,力学性能低下,易重复断裂。本实验利用反义核酸法抑制蛋白聚糖decorin的表达,体外构建组织工程肌腱,分析蛋白聚糖decorin在损伤肌腱中的作用。方法:根据蛋白聚糖decorin的分子序列,设计反义核酸（antisense）、正义核酸（sense）序列,转染兔子肌腱细胞。分别用PCR和免疫荧光法检测蛋白聚糖decorin在基因水平和蛋白水平的表达。确认decorin的表达被抑制后,检测组织工程肌腱的胶原纤维直径大小和相关基因表达,分析蛋白聚糖decorin在肌腱胶原纤维形成中的作用。结果:通过对FAM绿色荧光标记的核酸序列检测,转染效率>70%。PCR与免疫荧光结果显示decorin在基因水平和蛋白水平的表达均被明显抑制。蛋白聚糖decorin的表达被抑制后,组织工程肌腱的胶原纤维直径明显增粗。结论:反义核酸法是一种有效的抑制基因表达的手段。适当抑制蛋白聚糖decorin的表达有利于大直径胶原纤维的形成。第四部分慢病毒介导的siRNA抑制蛋白聚糖decorin表达对损伤肌腱修复的研究目的:损伤肌腱中蛋白聚糖decorin表达异常升高,构建组织工程肌腱,用慢病毒介导的siRNA抑制decorin的表达,修复损伤肌腱,检测肌腱修复效果,分析蛋白聚糖decorin在损伤肌腱中的作用。方法:根据蛋白聚糖decorin的分子序列,设计互补的siRNA序列,包装慢病毒。感染大鼠肌腱细胞。分别用real time PCR和免疫荧光法检测蛋白聚糖decorin在基因水平和蛋白水平的表达。确认decorin的表达被抑制后,检测细胞增殖和凋亡情况及其他相关基因的表达。用感染病毒的细胞构建组织工程肌腱,修复大鼠髌韧带1cm×4cm全层窗口缺损。四周后,检测修复效果。结果:检测慢病毒中GFP绿色荧光蛋白,确认感染效率>70%。Real time PCR与免疫荧光结果显示蛋白聚糖decorin在基因水平与蛋白水平的表达均被明显抑制。decorin被抑制后,细胞增殖能力增强。体内修复组织结果显示,用被抑制decorin的组织工程肌腱修复的肌腱组织胶原纤维直径明显增大,组织学结构良好,力学性能增强。结论:RNAi是一种有效的抑制基因表达的手段,包装成慢病毒后可以持续抑制目的基因的表达,便于体内实验研究。抑制蛋白聚糖decorin的表达有利于肌腱细胞增殖和大直径胶原纤维的形成。更多还原

【Abstract】 Background:With the improvement of people’s pursuit of health, physical exercise and competitions increased significantly. The clinical damage caused by sports is growing more and more, in which tendon and ligament injuries account for over 50%. As the ligament and tendon have poor regenerative capacity, in the healing tendon, a uniform distribution of small diameter collagen fibrils has been found with poorer mechanical properties than native tissue and shows no improvement of these properties. How to promote the injured tendon to regenerate large diameter collagen fibers is currently a big challenge in tendon repair study. Recently, type V collagen and proteoglycans are found to be important regulatory factors for collagen fibrillogenesis. Their expression is abnormally increased in injured tendon, which may cause the formation of samll diameter collagen fibers. Therefore, this study was designed to inhibit the expression of type V collagen and decorin by gene therapy, construct a tissue engineered tendon, and investigate the biology of collagen fibrillogenesis in in vitro and in vivo studies. Our study included four parts. Part I: Construct a tissue engineered tendon for the following studies. Part II: Inhibit the expression of type V collagen by siRNA and detect its regulatory effect on collagen fibrillogenesis; Part III: Inhibit the expression of proteoglycan decorin by antisense and detect its effects on collagen fibrillogenesis in vitro; Part IV:Repair the injured tendon using tissue engineered tendon which was infected with siRNA-inhibited decorin Lentivirus and evulate the efficiency of tendon regeneration in vivo. This study explored the effects of collagen type V and proteoglycan decorin on fibrillogenesis in tendon repair process. This may provide a basis for future development of novel cellular- and molecular biology-based therapeutics for tendon diseases.Part I: Construction of tissue-engineered tendonOBJECTIVE: To conveniently detect the collagen fibrillogenesis in vitro and repairinjured tendon in vivo, a scaffold-free tissue engineered tendon was developed withtendon cell sheets.METHODS: Tendon cells were cultured in high glucose DMEM with 10% (v/v) FBSand 50μg/mL ascorbic acid. Cells proliferated rapidly and formed coherent cellularsheets within 2 weeks. It could be detached from the substratum by applying a smallroll-up force. The cell sheet was cultured for one more week and then a scaffold-freetissue engineered tendon with living cells and collagen matrix was developed. Thetissue engineered tendon was used for histological examination and transmissionelectron microscopy analysis.RESULTHS: The tissue engineered tendon with living cells and collagen matrix had arelatively compact structure. Hematoxylin-eosin and Masson trichrome stainingrevealed a tissue-specific tendon structure: organized bundles of highly crimped fibersand cells oriented in parallel. Transmission electron microscopy analysis showed thatthe in vitro tissue engineered tendon had a more uniform distribution of smallerdiameter collagen fibrils than native tissue.CONCLUSIONS: Tissue engineered tendon is scaffold-free, so there are fewerexogenous factors, It possesses both the simple and controllable properties of cellculture models. It can be easily and efficiently handled with RNAi and antisense techniques which are not convenient for animal models. Moreover, the in vitro tissue engineered tendon has a tendon-specific structure of well-organized collagen fibrils, so the factors regulating collagen content and fibril formation can be investigated conveniently in this model. In addition, the tissue engineered tendon may be an excellent model to investigate the injured tendon because it has a uniform distribution of small collagen fibrils, which is characteristic of injured tendon. Although the tissue engineered tendon lacks a fair bit from the mechanical perspective, it illustrates a novel model and strategy for future tendon biology research.Part II: Regulatory effect of collagen V on the fibrillogenesis in a tissue engineered tendon modelOBJECTIVE: The presence of uniformly small collagen fibrils in repaired tendon is believed to play a major role in suboptimal tendon healing. Collagen V is significantly elevated in healing tendons and related to fibrillogenesis. This study aimed to investigate the effect of particular subunits of collagen V on the fibrillogenesis of tenocytes.METHODS: RNA interference gene therapy was used in this study to knock down the expression of two subunits of collagen type V and a tissue engineered tendon model was used to detect the regulatory effect of collagen V on collagen fibrillogenesis.RESULTS: The results showed that siRNA againstα1 andα2 chains of collagen type V (COL5A1 siRNA, COL5A2 siRNA) had different effects on collagen I and decorin gene expression. Tissue engineered tendon treated with COL5A1 siRNA had smaller collagen fibrils with abnormal morphology, while those formed by coculrure of COL5A1 siRNA treated and cultured tenocytes with a ratio of 1:0.5 or 1:1 had better fibrogenesis and larger collagen fibrils than that of cultured tenocytes.CONCLUSIONS: Our studies demonstrated that tissue engineered tendon is a novel and useful model for collagen fibrillogenesis biological investigation. The type V procollagenα1 andα2 chains have different effects on tendon matrix gene expression. An optimal level of collagen V is vital in regulating collagen fibrillogenesis. This may provide a basis for future development of novel cellular- and molecular biology-based therapeutics for tendon diseases.Part III: Decorin antisense gene therapy improves regeneration of large collagen fibril using tissue engineered tendon modelOBJECTIVE: Injured ligaments heal with scar tissue, which has uniformly smallercollagen fibrils and poor mechanical properties contrast to those of normal ligaments.Small leucine-rich proteoglycan decorin is known to regulate collagen fibrillogenesis.We hypothesized that the down-regulating proteoglycan decorin would improve largecollagen fibril regeneration.METHODS: The sense and antisense oligonucleotides which were against rabbitdecorin were transfected into rabbit tendon cells. Collagen synthesis, fibril organizationand collagen fibril diameter of tissue engineered tendon were analyzed.RESULTS: RT-PCR and immunofluorescence detection indicated that decorin had beenefficiently down regulated by antisense oligonucleotides in mRNA and protein level.TEM examination showed that down regulation of proteoglycan decorin led to anincreased development of larger collagen fibrils.CONCLUSIONS: This study demonstrated that tissue engineering tendon model is anovel and useful model for biological study of tendon. Down regulation of decorin canimprove larger diameter collagen fiber regeneration.Part IV: Decorin RNAi gene therapy improves regeneration of large collagen fiber in rat patellar tendon defect model.OBJECTIVE: Injured tendons heal with uniformly smaller collagen fibrils and poor mechanical properties. We hypothesized that the down-regulating proteoglycan decorin would improve large collagen fibril regeneration in vivo. METHODS: In this study, we constructed lentivirus containing siRNA against decorin. A tissue engineered tendon which was infected with siRNA-lentivirus was used to repaire rat patellar tendon defect. The repair effect was valued by detecting the ability of collagen synthesis, the microstructure of collagen fiber and histological examination.RESULTS: Real time PCR and immunofluorescence indicated that decorin had been efficiently down regulated by lentivirus containing siRNA against decorin in mRNA and protein level. Down regulation of decorin promote cell proliferation and led to an increased development of large collagen fibrils in repaired tendon tissue.CONCLUSIONS: The results demonstrated that RNA interference technology is a powerful tool for silencing genes in mammalian cells and tissue engineered tendon is a novel and useful model for tendon defect repair. Down regulation of proteoglycan decorin can improve large diameter collagen fiber regeneration and increase mechanical property. This may provide a promising approach to improve functional healing of injured tendon.更多还原