β-catenin在发育性髋关节脱位动物模型早期软骨退行性变中机制的实验研究

发布时间：2018-06-09 03:10

本文选题：发育性髋关节脱位 + 动物模型　；参考：《复旦大学》2012年博士论文

【摘要】：[背景和目的] 发育性髋关节脱位(Developmental dislocation of the hip, DDH)是小儿矫形外科最常见的疾病之一,骨关节炎(Osteoarthritis, OA)是其最常见的远期并发症。与正常人相比,DDH骨关节炎发病年龄早且病情重,大多数必须进行全髋关节置换术(Total hip arthroplasty,THA),严重影响了生活质量。本课题组前期研究发现发育性髋关节脱位早期可能已经存在关节软骨的退行性改变。目前对于DDH的早期发生关节软骨退行性变及骨关节炎的分子生物学机制尚不明确,近年来有大量研究表明β-catenin及其所在Wnt信号通路在关节软骨的形成和退行性改变中起关键作用,但β-catenin在髋关节脱位关节软骨的早期退变中有无作用的研究尚未见报道。本研究旨在通过检测β-catenin与关节软骨早期退变有关的胶原、基质金属蛋白酶(MMPs)和解聚素等在DDH早期髋臼软骨中的表达的相关性,探讨β-catenin与DDH早期软骨退行性变化的相关性,研究DDH早期软骨退变的机制,为临床DDH的早期预防骨关节炎提供理论依据,为进一步深入研究DDH与骨性关节炎的关系提供新的思路。 [方法] 一、DDH动物模型的制作、病理组织学观察 1.根据襁褓体位好发髋关节脱位,对新生20只Wistar大鼠双下肢双髋双膝医用胶带并拢模拟襁褓体位固定10天,大体观察证实制模成功情况。 2.实验组大鼠固定10天后解除固定继续饲养,对2、4、6、8周DDH模型及对应周期正常发育大鼠各10只髋关节软骨进行大体观察,测量髋臼深度、长度及宽度；股骨头长度及宽度。 3.髋关节软骨标本Safranin O/Fast green染色后光镜下观察关节软骨细胞与细胞外基质的病理组织学改变。二、X型胶原、基质金属蛋白酶-13(MMP-13)、ADAMTS-4、ADAMTS-5、β-catenin在不同时期DDH髋关节软骨中的表达 1.第一部分DDH模型不同年龄和对应期对照鼠各10只髋关节软骨标本。 2.采用免疫组化染色检测X型胶原、MMP-13、β-catenin在不同年龄髋关节软骨中表达的改变。 3.采用qRT-PCR法检测X型胶原、MMP-13、ADAMTS-4、ADAMTS-5、β-catenin 在不同年龄髋关节软骨中表达的改变,SPSS16.0统计和比较组间差异。三、体外软骨细胞培养,激活β-catenin的表达,检测β-catenin对软骨细胞退行性改变的影响 1.8周对照组大鼠髋关节软骨细胞的提取及原代培养,Ⅱ型胶原免疫荧光及甲苯胺蓝染色鉴定,免疫荧光及qRT-PCR检测MMP13及X型胶原的表达,qRT-PCR检测ADAMTS-5的表达,TUNEL法检测软骨细胞凋亡。 2.LiCl处理正常发育8周鼠关节软骨细胞,激活β-catenin的表达,qRT-PCR检测X型胶原、MMP-13和ADAMTS-5的表达；检测软骨细胞凋亡情况。 3. SPSS16.0统计和比较组间差异。 [结果] 一、DDH动物模型的制作及病理组织学观察 1.建模成功率100%(20只新生大鼠固定10天)。 2.髋关节大体观察：实验组关节囊增厚,髋臼浅平,软组织嵌入,股骨头外形扁平,随年龄增长头臼匹配差距增大；测量结果显示2、4周大鼠髋臼及股骨头大小较对照组发育小,但仍呈发育增大趋势,6周后髋臼发育基本停止,髋臼大小开始变小,股骨头发育停止,头臼不对称进一步加重(P0.01)。 3. Safranin0/Fast green染色可见到实验组关节软骨退变征象：Safranin0染色浅层部分染色降低,表明带负电荷蛋白多糖明显降低、4周后关节软骨浅层裂隙形成、浅层中层软骨细胞簇聚及数量减少、关节软骨退变随鼠龄增长而加重。二、X型胶原、基质金属蛋白酶-13(MMP-13)、ADAMTS-4、ADAMTS-5、B-catenin在不同时期DDH髋关节软骨中的表达 1.实验组X型胶原、MMP-13在软骨层软骨细胞中不均匀表达,成簇细胞表达明显,胞浆内明显棕色深染细胞数目百分比较对照组多。4周及以后与对照组比较差异有统计学意义(P0.01),且随鼠龄增长而表达增高。 2.实时荧光定量PCR结果显示实验组X型胶原和MMP-13mRNA在4周及以后表达较对照组增加,且随鼠龄增长表达增加；ADAMTS-5在8周时表达增高,与对照组比较差异有统计学意义(P0.01)。ADAMTS-4与实验组比较无统计学差异。 3. β-catenin:免疫组织化学中可见β-catenin在对照组大鼠2周时表达较多,随鼠龄增长表达逐渐下降,8周时免疫组化基本无表达；实验组β-catenin表达无下降趋势,持续高表达；实时荧光定量PCR mRNA表达呈相似结果。三、体外软骨细胞培养,检测β-catenin对软骨细胞退行性改变的影响 1.软骨细胞培养及鉴定：软骨细胞培养后Ⅱ型胶原免疫荧光及甲苯胺蓝染色鉴定为软骨细胞。 2. LiCl激活β-catenin表达：LiCl处理8周正常软骨细胞后免疫荧光及qRT-PCR均提示β-catenin表达明显增高。 3.激活β-catenin引起软骨退行性改变：LiCl处理软骨细胞免疫荧光提示X型胶原及MMP-13表达增高明显；qRT-PCR提示X型胶原、MMP-13、ADAMTS-5表达增高(P0.01);TUNEL染色提示LiCl处理后软骨细胞凋亡明显增加(P0.01)。 [结论] 1.DDH动物模型早期关节软骨发育落后并在4周出现发育停滞退变、软骨蛋白多糖丢失等关节软骨退变,这种早期发育异常及软骨退变可能是DDH最终发生骨关节炎的病理学基础。 2.与关节软骨退变有关的X型胶原、MMP-13及ADAMTS-5在DDH早期就表达增多,可以作为反映DDH软骨退变的早期敏感生物学指标。 3.β-catenin在正常发育早期有促进关节软骨发育的作用,但成熟后不再表达,而在DDH整个发育过程中,关节软骨出现持续高表达提示其可能是参与了软骨退行性变。 4.关节软骨发育异常导致β-catenin在DDH动物模型早期持续高表达,并可能引起X型胶原、MMP-13及ADAMTS-5等合成增多,软骨细胞凋亡增加,由此导致关节软骨早期退行性改变,这可能是DDH最终导致骨关节炎发生的机制之一
[Abstract]:[background and purpose]
Developmental dislocation of the hip (Developmental dislocation of the hip, DDH) is one of the most common diseases in pediatric orthopedics. Osteoarthritis (Osteoarthritis, OA) is the most common long-term complication. Compared with normal people, DDH osteoarthritis is early and is seriously ill, and most of them must be replaced by total hip arthroplasty (Total hip Arthr). Oplasty, THA), seriously affected the quality of life. Earlier studies in our group have found that there may have been degenerative changes in articular cartilage in the early stage of developmental dislocation of the hip. The molecular biological mechanism of osteoarthritis in the early stages of DDH is not clear. In recent years, a large number of studies have shown that beta -catenin and The Wnt signaling pathway plays a key role in the formation and degenerative changes of articular cartilage, but the study of the role of beta -catenin in the early degeneration of articular cartilage in the hip joint dislocation has not been reported. The aim of this study was to detect collagen, matrix metalloproteinase (MMPs) and depolymerization of beta -catenin and the early degeneration of articular cartilage. The correlation between the expression of element in the early acetabular cartilage of DDH and the correlation between the early chondrodegenerative changes of beta -catenin and DDH and the study of the mechanism of early cartilage degeneration in DDH provide a theoretical basis for the early prevention of osteoarthritis of the clinical DDH, and provide a new idea for further in-depth study of the relationship between DDH and osteoarthritis.
[method]
The production of DDH animal model, histopathological observation
1. according to the dislocation of the hip joint in the infancy, the new 20 Wistar rats with double hips and double knees and double knees and double knees were closed for 10 days, and the success of the model was confirmed by the general observation.
2. the rats in the experimental group were reared for 10 days after fixation. The 2,4,6,8 week DDH model and the 10 hip articular cartilage of the normal developing rats were observed, and the depth, length and width of the acetabulum were measured, and the length and width of the femoral head were measured.
3. histopathological changes of articular cartilage cells and extracellular matrix were observed by Safranin O/Fast green staining.
Two, the expression of type X collagen, matrix metalloproteinase -13 (MMP-13), ADAMTS-4, ADAMTS-5, and beta -catenin at different stages of DDH hip cartilage.
1. part I DDH model of 10 different age and corresponding control rats.
2. immunohistochemical staining was used to detect the expression of type X collagen, MMP-13 and beta -catenin in hip cartilage at different ages.
3. X collagen, MMP-13, ADAMTS-4, ADAMTS-5 and beta -catenin were detected by qRT-PCR.
Changes in cartilage expression in hip articular cartilage of different ages, SPSS16.0 statistics and comparison between groups. Three, in vitro chondrocyte culture, activation of the expression of beta -catenin, and detection of chondrocytes by beta -catenin
Effects of degenerative changes
The chondrocytes of the hip joint in the 1.8 week control group were extracted and cultured, the collagen type II was identified by immunofluorescence and toluidine blue, the expression of MMP13 and X collagen was detected by immunofluorescence and qRT-PCR, the expression of ADAMTS-5 was detected by qRT-PCR, and the apoptosis of cartilage cells was detected by TUNEL.
2.LiCl treated normal development of chondrocytes in 8 weeks and activated the expression of beta -catenin. QRT-PCR was used to detect the expression of X type collagen, MMP-13 and ADAMTS-5, and the apoptosis of cartilage cells was detected.
3. SPSS16.0 statistics and comparison between groups.
[results]
One, DDH animal model making and histopathological observation.
1. the success rate of modeling was 100% (20 newborn rats were fixed for 10 days).
2. hip joint gross observation: the joint capsule in the experimental group was thickened, the acetabulum was shallow, the soft tissue was embedded, the shape of the femoral head was flat, and the gap between the head of the head of the femoral head increased with age. The measurement results showed that the size of the acetabulum and the femoral head in 2,4 week rats was smaller than the control group, but the growth trend was still growing, the development of acetabulum was basically stopped after 6 weeks and the size of acetabulum began to begin. The development of femoral head was stopped and the asymmetry of the head and socket was further aggravated (P0.01).
3. Safranin0/Fast green staining showed the signs of articular cartilage degeneration in the experimental group: the superficial staining decreased in the superficial layer of Safranin0 staining, indicating that the negative charged protein polysaccharide was obviously reduced, the shallow fracture of the articular cartilage was formed after 4 weeks, the cartilage cells in the shallow layer were clustered and the number decreased, and the degeneration of articular cartilage was aggravated with the age of rat.
Two, the expression of type X collagen, matrix metalloproteinase -13 (MMP-13), ADAMTS-4, ADAMTS-5 and B-catenin in DDH hip cartilage at different stages.
1. the type X collagen in the experimental group was unevenly expressed in the chondrocytes of the chondrocytes, and the expression of the cluster cells was obvious. The number of brown deep stained cells in the cytoplasm was more than that of the control group for more than.4 weeks and after the control group, the difference was statistically significant (P0.01), and the expression increased with the growth of the rat age.
2. real time fluorescence quantitative PCR results showed that the expression of type X collagen and MMP-13mRNA in the experimental group was increased in 4 weeks and after the control group, and the expression increased with the age of rat. The expression of ADAMTS-5 increased at 8 weeks. There was a significant difference between the control group and the control group (P0.01).ADAMTS-4 and the experimental group.
3. beta -catenin: immunohistochemical staining showed that the expression of beta -catenin was more in the control group at 2 weeks, and gradually decreased with the growth of rat age. The expression of immuno histochemistry was basically no expression at 8 weeks, and the expression of beta -catenin in the experimental group had no downward trend and continued high expression, and the real-time fluorescence quantitative PCR mRNA table showed similar results.
Three, in vitro chondrocyte culture, to detect the effect of beta -catenin on the degeneration of chondrocytes.
1. chondrocyte culture and identification: chondrocytes cultured after type II collagen immunofluorescence and toluidine blue staining were identified as chondrocytes.
2. LiCl activated the expression of beta -catenin: immunofluorescence and qRT-PCR showed that the expression of beta -catenin increased significantly after LiCl treatment for 8 weeks in normal chondrocytes.
3. activation of beta -catenin induced degeneration of cartilage: LiCl treated chondrocyte immunofluorescence suggested that the expression of type X collagen and MMP-13 increased obviously; qRT-PCR suggested X collagen, MMP-13, ADAMTS-5 expression increased (P0.01); TUNEL staining suggested that apoptotic chondrocyte apoptosis was significantly increased after LiCl treatment (P0.01).
[Conclusion]
The early development of articular cartilage in the 1.DDH animal model is backward and the development of stagnation and degeneration, cartilage protein polysaccharide loss and other articular cartilage degeneration appear at 4 weeks. This early dysplasia and cartilage degeneration may be the pathological basis of osteoarthritis of DDH.
2. the expression of type X collagen, MMP-13 and ADAMTS-5 related to articular cartilage degeneration is increased in the early stage of DDH, which can be used as an early sensitive biological indicator to reflect the degeneration of DDH cartilage.
3. beta -catenin can promote articular cartilage development at the early stage of normal development, but no longer expression after maturation, and the continuous high expression of articular cartilage in the whole development of DDH suggests that it may be involved in degeneration of cartilage.
4. articular cartilage abnormal development leads to the high expression of beta -catenin in early DDH animal model, and may cause X collagen, MMP-13 and ADAMTS-5 synthesis increase, cartilage cell apoptosis increase, resulting in the early degeneration of articular cartilage, which may be one of the mechanisms that DDH eventually leads to the occurrence of osteoarthrosis.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R726.8

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