【作者】 李鑫；

【导师】 高忠礼； 陈迁；

【作者基本信息】 吉林大学 ， 外科学， 2011， 博士

【摘要】 背景目前,每年有数以万计的人由于各种原因发生骨折,尽管其中大部分病人经过治疗获得痊愈,但是仍有5-10%病人无法完全康复,饱受骨折不愈合或骨折延迟愈合带来的痛苦[1]。骨折愈合障碍会导致病人运动功能缺失,因此深入研究骨折愈合的病理生物学机理,可以为我们开发促进骨折愈合的生物学及临床治疗方法奠定基础。骨折愈合是十分复杂的生物修复过程,包含细胞内外环境的成骨分子信号调控,其分子生物学机制目前仍不明确。这个过程中,在胚胎发育期调控骨骼生长的分子生物学机制在骨折愈合过程中再次起作用[2]。除了分子生物学影响因素,骨折愈合过程还受力学因素影响。如果骨折端获得足够稳定的固定,骨折愈合过程将以直接愈合方式进行;相反,如果骨折端有微动,骨折愈合过程将以间接愈合方式进行。实际上大部分骨折都以间接骨折愈合方式进行。这种骨折愈合方式所经历的是软骨内成骨过程,即未分化的基质细胞先聚集在骨折部位,经过增生和分化过程形成软骨,而后再骨化最终由骨取而代之的过程。多种细胞外基质蛋白在间接骨折愈合过程中发挥作用,Matrilin-1(MATN1)便是其中之一。MATN1是一种软骨特异性细胞外基质蛋白,在软骨内成骨过程中的软骨化骨阶段有重要作用。现有研究证实MATN1与Ⅱ型胶原纤维和蛋白多糖结构关系密切,并且能够在软骨内部形成软骨依赖性和非依赖性纤维网结构[3] [4]。但是,其在动物体内的生物学作用还尚未明确。既然间接骨折愈合与软骨密切相关,且MATN1又与软骨关系密切,由此我们假设MATN1会在骨折愈合过程产生一定作用。方法基因敲除小鼠是一种经过基因工程技术处理过的小鼠,通过对基因的靶向突变使一个或几个基因失去功能。目前,基因敲除小鼠已成为研究基因功能的重要动物模型。本实验使用了MATN1敲除鼠(MATN1-/-)和野生型鼠(Wild Type, WT)研究MATN1在小鼠骨折愈合过程中的作用。我们使用了经典的小鼠胫骨闭合骨折髓内针固定模型,该模型具有制作过程简便,感染率低,对设备要求低的特点。在实验中我们使用了如下方法评价骨折愈合情况:使用X线影像技术观察骨折愈合过程;使用荧光分子断层体内成像技术(FMT)对骨折部位血供情况和成骨情况进行定量分析分析;微CT技术和生物力学扭力测试用于评价骨痂情况;组织学Safranin-O/Fast-green染色能清楚地显示骨痂内软骨细胞情况;实时定量聚合酶链式反应(Real time-PCR)定量分析骨痂内血管生成标记物和成骨标记物目的基因表达水平。其中值得加以简略介绍的是如下三种技术方法:FMT技术平台能够对实验小动物的全身组织,包括深层组织进行无创伤性地分子生物学水平信息分析。目前在美国,该系统平台已成功应用于研究肿瘤,炎症,肺脏疾病,心血管疾病和骨骼疾病。通过使用该平台,研究人员能够实时监控并定量分析生物学标记物和信号通路变化,为进一步深刻理解疾病机制和明确治疗效果提供有力帮助。本实验是FMT系统平台第一次用于研究骨折愈合过程。微CT能够对小鼠细小的骨痂内部成分进行定量分析,精确计算骨痂内骨量(BV)和骨痂整体体积(TV)。生物力学扭力测试目前已被认为是评价骨折愈合情况的金标准。该平台通过测定能够使骨痂断裂的最大扭矩和刚度,来评价骨折愈合的生物学功能情况。结果实验中,我们观察到MATN1-/-小鼠较WT小鼠骨折愈合快。X线影像和组织学染色观察结果显示MATN1-/-小鼠较WT小鼠愈合快,骨痂成骨增高。Safranin-O/Fast-green染色分析,发现MATN1-/-小鼠组较WT小鼠组提前进入骨化阶段。微CT结果显示MATN1-/-小鼠有更大的整体骨痂体积(TV)和骨体积(BV),但是两组的BV/TV值却相当。随后的生物力学扭力测试结果证实了MATN1-/-小鼠的骨痂与WT小鼠相比,能够承受更高的最大扭矩,有更优良的生物力学功能。在FMT系统平台上使用了血管生成荧光探针和成骨荧光探针实时跟踪评估分析两组骨折愈合过程中血管生成情况和成骨情况。我们得到的结果为,MATN1-/-小鼠较WT小鼠有更多的血管和骨生成,且实验结果完全符合已知的骨折愈合过程中血管和新骨生成随时间的变化之间的关系。Real time PCR分析结果显示MATN1-/-小鼠较WT小鼠,在骨折后第14天和第21天,血管发生相关基因,如血管内皮生长因子(VEGF)和成骨相关基因水平,如骨形态发生蛋白(BMP),都分别明显高于后者。除此之外,MATN1-/-小鼠骨痂内的基质细胞衍生因子(SDF-1)基因水平也明显高于WT小鼠。综合上述实验结果,我们第一次证实了MATN1在体内的作用,即MATN1对小鼠骨折愈合过程起负向调节作用。MATN1抑制骨折愈合的分子生物学机制目前尚不清楚。但是,我们认为可能是如下原因造成了这种现象:第一,MATN1抑制新血管生成的作用。骨折部位的血供对骨折愈合至关重要。第二, MATN1可能是通过抑制SDF-1的作用来负向调节骨折愈合的。SDF-1已经被证实有促进成骨的作用,并且能够通过促进造血干细胞、基质干细胞和其他炎症细胞在损伤部位聚集。所以,SDF-1功能受到抑制时,骨折愈合过程势必会受到影响。本实验首次明确了MATN1在体内的生理学作用,MATN1这个软骨特异性基质分子,可能通过抑制血管生成和骨生成,造成骨折愈合延迟。未来仍需要大量研究工作以阐明MATN1在骨折愈合过程中的作用机制。更多还原

【Abstract】 Back ground There are millions of people who get fractured every year for different reasons. Most of them can be recovered, but there is still 5-10% sufferring from un-union or delayed union [1]. The impaired fracture healing can cause disability. Under such an urgent situation like this, we need to dig the mechanism of fracture healing deeply, and try our best to find efficient ways to induce fracture healing in both of biochemical and clinical methods.Fracture healing is a complex event that involves the coordination of variety different processes, including the ex- and in-cellular osteogenic cells signaling pathways. Till now, the mechanism of fracture healing is not totally understood. Molecular mechanisms known to regulate skeletal tissue formation during embryological development are repeated during the fracture healing process [2].Besides the molecular biology factors, the progress of fracture healing is infected by the mechanical factors as well. Direct fracture healing occurs when rigid internal fixation anatomically reduces the fracture fragments. However, the majority of fractures heal by indirect fracture healing, which involves callus formation through endochondral ossification. Matrinlin-1(MATN1) is one of the many kinds of excellular matrix proteins, which are involved in this bone repair presses.MATN1 is a cartilage specific extracellular matrix protein which is critical for the transition from cartilage to bone during endochondral ossification. MATN1 is associated with collagen type II fibrils and proteoglycans, and forms collagen-independent and collagen-dependent filamentous networks in cartilage[3] [4]. But its function in vivo has not been studied yet. Since, MATN1 and fracture healing all have connections with the collagen; our work thereby is to determine whether MATN1 plays a role in fracture healing. Method A knockout mouse is a genetically engineered mouse in which one or more genes have been turned off through a targeted mutation.Nowadays, knockout mice are important animal models for studying the role of genes whose functions have not been identified. In this study, MATN1-/- and WT mice were used to investigate the effect of MATN1 in fracture healing. The classical mouse closed tibial fracture with intramedullary needle fixation model was used, which has some advantages, such as the easy manipulation, low infection rate and less requiry for the equipments. The methods used to value the fracture healing are as follows:the fracture healing process was followed by X-ray radiography, fluorescence molecular tomography (FMT) was used to analysis the vascularity and bone formation,micro-CT was also used to quantitifiy the fracture callus ,biological mechanical torsional test was used to value mechanical quality of the callus, Safranin-O/Fast-green staining histological analysis was included to value the callus as well, real time PCR was used to quantitify the target genes expression in the callus. There are three of them deserved to be introduced briefly. FMT technology provides non-invasive, whole body, deep tissue imaging in small animal models information-rich results. Now across the US, these systems are used for research in oncology as well as inflammatory, pulmonary, cardiovascular and skeletal disease. Biological targets and pathways can be monitored and quantified in real time - giving a deeper understanding of the biology underlying disease mechanisms and therapeutic response. The presented study is the first one in which FMT system is applied to study the fracture healing in the world wide. Micro-CT determined the newly formed bone, and quantified the new bone and unmineralized cavities [total volume (TV) in mm3] and volume of mineralized bone tissue [bone volume (BV) in mm3]. Biological mechanical torsional test is recognized as golden standard to value the fracture healing mechanical function, with determining the peak torque.Results In this study, we observed that MATN1-/- mice healed fast than WT mice. The X-ray images and histology staining results showed that MATN1-/- mice had more bone fromation in callus.From Safranin-O/Fast-green staining results, MATN1-/- mice stepped in mineralization stage ahead of WT mice. According to the real time in vivo imagining (FMT) and micro-CT analysis, MATN1-/- mice showed significant more vascular and bone formation than WT mice. The real time PCR results showed that MATN1 -/- mice expressed significant more angiogenesis genes on days 14, such as vascular endothelial growth factor (VEGF) and significant more osteogenic genes on days 21, such as bone morphogenetic protein (BMP). Besides that, the stromal cell-derived factor 1(SDF-1) is highly expressed in MATN1-/- mice than in WT mice.In summary, all the data we got in this study demonstrate for the first time that MATN1 inhibit bone repair in fracture healing.The molecular mechanism regulating the fracture healing by MATN1 is not clear at present. However, there are some reasons to support this phenomenon. The first possible reason is the inhibition angiogenesis caused by MATN1.The blood supply to the fracture site is vital important to the fracture healing. The second possible reason is that MATN1 may inhale the fracture healing through inhibiting the SDF-1 function.SDF-1 has been determined to have the ability to induce bone formation. So, the fracture healing progress should be infected by the down regulation to SDF-1. For the future, there will be lots of work to reveal the mechanism of matrilin-1’s function in fracture healing.更多还原