研究背景

组织工程化软骨的应力应变能力与正常耳软骨相比仍存在明显差异，是制约组织工程化耳软骨向临床应用转化的主要原因之一。耳软骨的力学性能由细胞外基质组分提供，主要包括胶原纤维、弹性纤维、蛋白聚糖和水。耳软骨是一种弹性软骨，拥有灵活的应力应变能力，以富含弹性纤维区别于透明软骨和纤维软骨。弹性纤维以微纤维（Microfibril）作为支架（微纤维由原纤维蛋白1（Fibrillin1，FBN1）和原纤维蛋白2（Fibrillin2，FBN2）构成），弹性蛋白（Elastin，ELN）为“原料”，在细胞外基质蛋白，如赖氨酸氧化酶（Lysyl Oxidase，LOX）、原纤蛋白4（Fibulin4，FBLN4）、原纤蛋白5（Fibulin5，FBLN5）、潜在转化生长因子β结合蛋白4（Latent Trans growing Factor β Bingding Protein 4，LTBP4）等的辅助下，遵循一定的时间和空间顺序进行组装。据文献报道，其中任一成分缺失或分泌不足，均会导致弹性纤维的积累量不足或形态异常，使弹性纤维含量丰富的组织器官功能异常而引起相应先天或后天性疾病。因此，明确组织工程化软骨中弹性纤维组装相关组分的表达情况，对组织工程化软骨弹性、灵活性等力学性能的提高具有十分重要的意义。当前，对弹性纤维组装相关组分在血管、皮肤等工程化组织再造中的作用已有研究，但在组织工程化弹性软骨中的表达及作用研究还未有报道，有待进一步研究。

研究目的

明确组织工程化软骨中弹性纤维组装关键组分较人耳软骨组织的表达差异，筛选影响弹性蛋白表达的关键组分，探讨其对软骨表型和基质形成的影响，为提高组织工程弹性软骨构建效果提供参数和线索。

研究内容

1.工程化软骨与人耳软骨组织弹性纤维相关组分的mRNA及蛋白水平表达差异                                                                                                                                                         

方法：提取组织工程化软骨及人耳软骨组织RNA，逆转录为cDNA，利用Real-time PCR检测组织工程化软骨及人耳软骨组织中弹性纤维组装相关组分基因（ELN、FBN1、FBN2、LOX、FBLN4、FBLN5及LTBP4）mRNA的表达水平。利用免疫荧光染色检测两组中弹性纤维组装相关组分蛋白的表达差异。

结果：Real-time PCR结果显示ELN、FBN1、LOX在组织工程化软骨中的表达水平显著低于耳软骨组织，FBN2、FBLN4、FBLN5及LTBP4在组织工程化软骨中的表达有下降趋势，但无统计学意义。免疫荧光染色结果显示：（1）ELN、FBN1、LOX、FBLN4、FBLN5及LTBP4的蛋白表达水平低于耳软骨组织，FBN2蛋白表达水平与耳软骨组织相当；（2）ELN、FBN1及LOX蛋白在组织工程化软骨中未能形成耳软骨中的纤维束状形态。

2.弹性纤维相关组分过表达对弹性纤维组装相关基因表达的影响

方法：提取人耳软骨细胞，培养至p2代，分别构建人弹性纤维组装相关组分ELN、FBN1、FBN2、LOX、FBLN4、FBLN5及LTBP4的过表达质粒，在人耳软骨细胞中构建过表达体系，Real-time PCR检测各基因过表达后，弹性蛋白及其他弹性纤维组装相关基因表达水平的变化，明确其中的关键组分。

结果：（1）人耳软骨细胞中过表达ELN后，其他弹性纤维组装组分基因表达水平无明显差异；（2）过表达FBN1后，ELN、FBLN5的表达水平升高；（3）LOX过表达后，弹性纤维主要成分ELN及FBN1的表达水平显著升高；（4）FBLN4过表达后，ELN的表达水平升高；（5）FBLN5过表达后，其他组分的表达水平差异无统计学意义；（6）过表达LTBP4后，FBN1、FBLN5及LOX的表达水平升高。

3.耳软骨细胞中过表达LOX、FBN1对软骨表型及基质形成的影响

方法：提取猪耳软骨细胞，构建LOX及FBN1过表达体系。利用Real-time PCR检测LOX及FBN1过表达对猪耳软骨细胞软骨基质相关基因、肥大成骨分化相关基因表达水平的影响；Transwell检测LOX过表达对猪耳软骨细胞迁移能力的影响；番红O染色检测猪耳软骨细胞蛋白多糖分泌的差异。

结果：在软骨细胞中过表达LOX后，软骨分化相关基因COLⅡ、ACAN、SOX9的表达水平显著升高；软骨肥大成骨分化相关基因MMP13、COLⅩ及RUNX2的表达水平明显下降；弹性纤维组装相关组分基因ELN的表达水平升高；FBLN4、FBLN5的表达水平降低。LOX过表达后，猪耳软骨细胞在24h内的迁移能力增强，蛋白多糖的积累明显增加。FBN1过表达后，软骨分化关键基因SOX9、ACAN及COLⅡ表达水平显著升高。提示我们LOX和FBN1具有维持软骨细胞表型的作用，LOX还具有抑制软骨细胞肥大成骨分化及促进细胞迁移的作用。

研究结论

1. 组织工程化软骨中弹性纤维组装相关组分ELN、FBN1及LOX的基因和蛋白表达显著低于正常耳软骨，而且在组织工程化软骨中未能形成成熟的纤维束状结构，提示这些组分的表达降低可能是组织工程软骨弹性纤维表达不足的主要原因。

2. LOX和FBN1过表达，可以促进软骨表型及相关基质表达，而且LOX可促进细胞迁移，可以作为促进弹性软骨构建的候选基因。 

Research Background

Compared with auricular cartilage, the stress-strain capability of tissue engineered cartilage is still significantly different, which is one of the reasons that restrict the transformation of tissue engineered cartilage into clinical application. The mechanical properties of auricular cartilage are provided by extracellular matrix components, which mainly include collagen fibers, elastic fibers, proteoglycans and water. Auricular cartilage is a kind of elastic cartilage, which has flexible stress-strain capability. It is distinguished from hyaline cartilage and fibrous cartilage for its abundance in elastic fibers. The elastic fiber is mainly consists of microfibril and elastin. And microfibril plays a role as scaffold which is consists of fibrillin 1 (FBN1) and fibrillin 2 (FBN2)).The assemble process also need lysyl oxidase (LOX), fibrillin 4 (FBLN4), fibrillin 5 (FBLN5), latent transforming growth factor β binding protein 4 (LTBP4).The elastic fiber is assembled in a certain time and space order. According to reports in the literature, the lack of any of these components or insufficient secretion will lead to insufficient accumulation of elastic fibers or abnormal morphology, causing abnormal functions of tissues and organs which are rich in elastic fibers and causing corresponding congenital or acquired diseases. Therefore, clarifying the expression of components related to the assembly of elastic fibers in tissue engineered cartilage is of great significance to the improvement of the stress-strain capability of tissue engineered cartilage. At present, the research on the components related to the assembly of elastic fibers has been reported in the engineered reconstruction of blood vessels and skin, but the expression in tissue engineered cartilage is rarely reported and needs further study.

Objectives

Clarifying the differences in the expression of key components of elastic fiber assembly in tissue engineered cartilage and human auricular cartilage，screening the key components that affect the expression of elastin，discussing their effects on cartilage phenotype and matrix formation，and providing parameters and clues for improving the construction of tissue engineered elastic cartilage. 

Research contents

1. Differences in the expression of genes and proteins related to the assembly of elastic fiber in tissue engineered cartilage and human auricular cartilage tissue

Methods: RNA was extracted from tissue engineered cartilage and auricular cartilage tissue, reverse transcribed into cDNA, and Real-time PCR was used to detect mRNA expression levels in genes related to elastic fiber assembly (ELN, FBN1, FBN2, LOX , FBLN4, FBLN5 and LTBP4) in tissue engineered cartilage and normal auricular cartilage tissue. . Immunofluorescence staining was used to detect the differences of protein expression of elastic fiber assembly related components.

Results: Real-time PCR results showed that the expression levels of ELN, FBN1, and LOX in tissue-engineered cartilage were significantly lower than auricular cartilage tissue. The expression of FBN2, FBLN4, FBLN5, and LTBP4 in tissue engineered cartilage showed a downward trend, but no statistical significance. Immunofluorescence staining results showed that: (1) ELN, FBN1, LOX, FBLN4, FBLN5 and LTBP4 protein expression levels were lower than auricular cartilage tissue, FBN2 protein expression levels were comparable to the auricular cartilage tissue; (2) In tissue engineered cartilage: ELN, FBN1 and LOX fails to form the fibrous bundle morphology in auricular cartilage.

2. The effects of the overexpression of elastic fiber related components’ genes on the gene’s expression related to elastic fiber assembly 

Methods: Human chondrocytes were extracted and cultured to generation p2. Overexpression plasmids of human elastic fiber assembly-related components ELN, FBN1, FBN2, LOX, FBLN4, FBLN5 and LTBP4 were constructed, and 293T cells were transfected with lentiviral vectors. The viral supernatant was infected with human auricular chondrocytes of p2 generation, then extracted RNA, reverse transcribed, and used Real-time PCR to detect the effect of every gene overexpression on the gene expression of elastin and other elastic fiber assembly related components, and find the components.

Results: (1)After overexpressed of ELN in human auricular chondrocytes, there was no significant difference in gene expression levels of other elastic fiber assembly components; (2)After overexpressed of FBN1, ELN and FBLN5’s expression level of  have been increased; (3) After overexpressed of LOX, ELN and FBN1’s expression level have been significantly increased, which are the main components of elastic fibers; (4)After overexpressed of FBLN4 ,ELN‘s expression level has been increased; (5) After overexpressed of FBLN5, the expression level other components have no statistically significant difference in expression levels; (6)After overexpressed of LTBP4, FBN1, FBLN5 and LOX’s expression level have been increased.

3. Effects of overexpression of LOX and FBN1 in auricular chondrocytes on the  cartilage phenotype and extracellular matrix expression

Methods: Extract pig auricular chondrocytes from pig auricular, and construct LOX and FBN1overexpressed plasmid, using lentivirus vector transfected 293T cells to collected virus supernatant, and then infected pig auricular chondrocytes. Real-time PCR was used to detect the effects of LOX and FBN1 overexpressed on cartilage-related matrix genes, hypertrophic differentiation related genes’ expression levels of pig auricular chondrocytes; using transwell to detect the effects of LOX overexpression on migration ability of pig auricular chondrocytes; Saffron O staining was used to detect the changes in proteoglycan secretion.

Results: Real-time PCR results showed the differences after LOX overexpressed in chondrocytes, the expression levels of cartilage differentiation related genes: COLⅡ, ACAN, and SOX9 have been significantly increased; cartilage osteogenesis and aging-related genes MMP13, COLⅩ and RUNX2 expression levels decreased significantly; Elastic fiber assembly related component genes ELN’s expression level has been increased, FBLN4, FBLN5’s expression level have been decreased. After LOX overexpression, the migration ability of pig auricular chondrocytes increased within 24 hours, and the accumulation of proteoglycan has been promoted significantly. It is suggested that LOX may have the effect of promoting chondrocyte phenotype, inhibiting chondrocyte hyperostosis and promoting cell migration. After the expression of FBN1, the expression levels of cartilage differentiation key genes: SOX9, ACAN, and COLⅡ has been significantly increased.

Conclusions:

1. The gene and protein’s expression levels of ELN, FBN1 and LOX in tissue-engineered cartilage are significantly lower than those in human auricular cartilage, and failed to form mature fiber bundle-like structures similar to human auricular cartilage, which suggested us that the decreased expression of these components may be the main reason for the insufficient expression of elastic fiber in tissue engineered cartilage.

2. Overexpression of LOX and FBN1 can promote cartilage phenotype and the expression of cartilage related matrix. LOX can promote cell migration and can be used as a candidate gene for promoting the construction of elastic cartilage.