整合素α5β1在膝骨关节炎不同退变程度关节软骨中的差异性表达

发布时间：2018-06-17 19:56

本文选题：整合素 + 骨关节炎　；参考：《泸州医学院》2014年硕士论文

【摘要】：目的：骨关节炎（Osteoarthritis，OA）是一种退行性关节疾病，主要病理表现为关节软骨的退变，好发于中老年，膝关节常受累。随着世界人口老龄化进程的加速，OA的发病率逐年上升，已成为中老年人致残的重要原因之一，给患者带来极大痛苦与经济负担，严重影响患者的生活质量。由于OA的病因与发病机制尚不清楚，目前仍是骨科领域研究的热点与难点。研究发现，整合素（Integrin）是细胞粘附分子家族的一员，存在于细胞表面，是一种跨膜蛋白，在细胞粘附与信号传导中起着重要作用，参与细胞形态、极性、粘附、运动、生长、增殖、分化与凋亡等多种生理功能的调节。软骨细胞可表达多种Integrins，成人软骨细胞中主要表达Integrinα5β1。Integrinα5β1在软骨细胞力学信号转导、细胞增殖与分化等方面具有重要作用。研究发现，Integrinα5β1与软骨细胞的损伤、修复、细胞表型改变等多种软骨病变密切相关。我们推测，Integrinα5β1在不同退变程度软骨中可能存在差异性表达，且与软骨的退变及OA的发展相关。目前，关于Integrinα5β1在不同退变程度软骨中的表达情况及与软骨退变的关系缺乏相关研究。本实验选取膝骨关节炎（Knee Osteoarthritis，KOA）患者股骨远端负重面不同退变程度关节软骨，检测其中Integrinα5β1的表达，探讨Integrinα5β1在KOA不同退变程度软骨中的表达情况及与软骨退变的关系。方法：收集KOA患者全膝关节置换术股骨远端截骨组织20例，经大体观察，依据Outerbridge分级标准选取不同退变程度软骨标本共40例（每例标本沿矢状面等分成三块，一块用10%中性甲醛固定，另两块－80℃冻藏保存），将10%中性甲醛固定的标本脱钙后制成石蜡组织块，3μm切片后，常规HE染色及镜下观察，参照Bobinac分期，将标本分为三组：轻度退变组（0～6分）、中度退变组（7～9分）、重度退变组（10～14分）。采用免疫组化、Real-time PCR及Western blot检测Integrinα5β1在三组不同退变程度软骨中mRNA与蛋白水平的表达。实验数据采用均数±标准差表示，统计分析采用SPSS17.0软件，三组间比较采用单因素方差分析，P＜0.05为差异有统计学意义。结果：本实验20例KOA患者股骨远端截骨顺利，内外髁负重面软骨无人为损伤及污染，共选取软骨标本40例，HE染色均匀、红蓝分明、组织结构清晰、胞浆胞核清晰可辨、镜下观察与判别良好，参照Bobinac分期分成三组，轻度退变组13例、中度退变组15、重度退变组12例。免疫组化染色显示：Integrinα5β1在不同退变程度软骨中均有表达；轻度退变组主要表达在软骨浅表层与移形层；中度退变组除钙化层周围软骨细胞未见明显表达外，其余软骨细胞均有表达，侵及软骨潮线的血管内皮细胞可见表达；重度退变组所有软骨细胞与侵入软骨的血管内皮细胞均有表达。轻度退变组呈低表达，中度退变组表达增强，重度退变组表达明显增强；免疫组化检测结果经Image-Pro Plus6.0软件半定量分析，三组间比较，差异性显著（P＜0.05）。Real-time PCR与Western blot检测显示：Integrinα5β1的mRNA与蛋白在三组不同退变程度软骨中均有表达，两种方法检测结果相同，轻度退变组呈低表达，中度退变组表达增强，，重度退变组表达明显增强；Real-time PCR与Western blot检测结果对应经ABI StepOne PlusReal-time PCR System与Quantity One软件定量分析，同种方法的三组间比较，差异性显著（P＜0.05）。结论：Integrinα5β1在KOA不同退变程度软骨中均有表达，各组间的表达存在差异性；随着软骨退变程度的加重，Integrinα5β1在软骨中的表达由浅表层细胞逐渐扩展到全层软骨细胞，表达量逐渐增强。Integrinα5β1在不同退变程度软骨中的差异性表达与软骨退变及OA的发展具有密切联系。
[Abstract]:Objective: Osteoarthritis (OA) is a degenerative joint disease. The main pathological manifestation is the degeneration of articular cartilage. The knee joint is often affected in the middle and old age. With the acceleration of the aging process of the world population, the incidence of OA has risen year by year. It has become one of the important reasons for the disabled people in the middle and old years, which brings great pain to the patients. And the economic burden seriously affects the quality of life of the patients. Because the etiology and pathogenesis of OA is still unclear, it is still a hot and difficult point in the field of Department of orthopedics. It is found that integrin (Integrin) is a member of the cell adhesion molecule family, which exists on the cell surface, is a kind of transmembrane protein, and plays an important role in cell adhesion and signal transduction. To participate in the regulation of many physiological functions, such as cell morphology, polarity, adhesion, movement, growth, proliferation, differentiation and apoptosis. Chondrocytes can express a variety of Integrins. The main expression of Integrin alpha 5 beta 1.Integrin alpha 5 beta 1 in adult chondrocytes is important in the study of chondrocyte mechanical signal transduction, cell proliferation and differentiation. It is found that Integrin alpha 5 beta 1 is closely related to cartilage damage, repair, cell phenotype change and so on. We speculate that Integrin alpha 5 beta 1 may have differential expression in different degeneration cartilage, and is related to cartilage degeneration and the development of OA. At present, the expression of Integrin alpha 5 beta 1 in cartilage of different degeneration degree The relationship between the condition and the cartilage degeneration is not related. This experiment selected the articular cartilage of the distal femur with different degrees of degeneration in Knee Osteoarthritis (KOA). The expression of Integrin alpha 5 beta 1 was detected, and the expression of Integrin alpha 5 beta 1 in the cartilage of different degree of degeneration of KOA and the relationship with cartilage degeneration were investigated. Methods: 20 cases of distal femur osteotomy in total knee arthroplasty of KOA patients were collected, and 40 cases with different degree of degeneration were selected according to the standard of Outerbridge classification. Three pieces were divided into three blocks along the sagittal plane, one was fixed with 10% neutral formaldehyde, and the other two - 80 centigrade frozen storage was preserved. The target of 10% neutral formaldehyde was fixed. After decalcification, the paraffin tissue was made. After 3 m slices, conventional HE staining and microscopic observation were used. The specimens were divided into three groups: mild degeneration group (0~6), moderate degeneration group (7~9) and severe degeneration group (10~14 points). Immunohistochemistry, Real-time PCR and Western blot were used to detect Integrin alpha 5 beta 1 in three groups of different degrees of degeneration soft The expression of mRNA and protein in bone was expressed. The experimental data were expressed with mean standard deviation, and SPSS17.0 software was used for statistical analysis. The three groups were compared with single factor analysis of variance. The difference was statistically significant in P < 0.05. Results: the distal femur osteotomy was smooth in 20 cases of KOA patients and no one was damaged and polluted by the internal and external condyle bearing face cartilage. 40 cases of cartilage specimens were selected, HE staining was uniform, red and blue, clear tissue structure, clear differentiation of cytoplasm nucleus, good observation and discrimination under the microscope, Bobinac staging was divided into three groups, 13 cases in mild degeneration group, 15 in moderate degeneration group and 12 in severe degeneration group. Immunohistochemical staining showed that Integrin alpha 5 beta 1 was expressed in different degeneration degree cartilage The mild degeneration group was mainly expressed in the superficial cartilaginous surface and the transitional layer, and the other chondrocytes were expressed in the moderate degeneration group except the cartilage cells in the calcification layer, and the vascular endothelial cells that invaded the cartilage tidal line were expressed, and all the cartilage cells in the severe degeneration group were expressed in the vascular endothelial cells that invaded the cartilage. The expression of mild degeneration group was low, the expression of moderate degenerative group was enhanced and the expression of severe degeneration group was obviously enhanced. The results of immunohistochemical detection were semi quantitative analysis by Image-Pro Plus6.0 software. The difference was significant between the three groups (P < 0.05).Real-time PCR and Western blot detection: the mRNA and protein of Integrin alpha 5 beta 1 were in three groups of different degenerative processes. The results of the two methods were the same, the results of the two methods were the same, the mild degeneration group showed low expression, the moderate degeneration group was enhanced, the expression of the severe degeneration group was obviously enhanced; the results of Real-time PCR and Western blot were quantified by ABI StepOne PlusReal-time PCR System and Quantity One software, and compared between the same methods. The difference was significant (P < 0.05). Conclusion: the expression of Integrin alpha 5 beta 1 was expressed in different degree of degeneration of KOA cartilage, and the expression in each group was different. With the aggravation of cartilage degeneration, the expression of Integrin alpha 5 beta 1 in cartilage gradually expanded from superficial layer to whole layer cartilage cell, and the expression gradually increased.Integrin alpha 5 beta 1 in different The differential expression of degeneration in cartilage is closely related to the degeneration of cartilage and the development of OA.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R684.3

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