【作者】 康映泉；

【导师】 马瑞雪；

【作者基本信息】 复旦大学 ， 儿科学， 2011， 硕士

【摘要】 [背景和目的]发育性髋关节发育不良(Developmental dysplasia of the hip, DDH)是小儿常见的四肢畸形之一。随着早期筛查的开展,很多DDH患儿能够得到闭合复位等早期治疗,但其中相当一部分会出现残余髋臼发育不良,以后进展为骨关节炎(Osteoarthritis, OA)。目前对于残余髋臼发育不良治疗时机的选择有不同观点,残余髋臼发育不良在什么时间治疗才能逆转其病理变化尚无定论。已有研究对髋臼发育不良髋臼软骨的病理变化及其可逆性进行观察,但对股骨头软骨的变化及其时序性与可逆性的研究尚未见报告。由于髋关节内、外侧应力不同,本研究拟分别对不同时段髋臼发育不良兔模型股骨头内、外侧软骨的组织形态学变化和I型胶原、基质金属蛋白酶-13(Matrix metalloproteinase-13, MMP-13)表达情况进行观察,以探讨髋臼发育不良与骨关节炎的相关性,并通过对股骨头退行性变可逆性时间节点的观察为临床对残余髋发育不良治疗时机的选择提供理论依据。[方法]选取5周龄新西兰大白兔27只,随机分为A、B、C三组,每组9只。左后肢为实验侧,以石膏管型外固定保持伸膝屈髋位,三组固定时间分别为2周、4周、6周；右后肢为对照侧,不做处理。所有实验兔在固定2周后行骨盆正位片检查,证实造模成功。每组在达到各自固定时间后随机选取5只取材,构成幼兔组a、b、c组；各组另外4只拆除石膏继续喂养至6月龄时取材,构成成兔A、B、C组。股骨头内、外侧软骨应力部位不同,分别进行观察、分析。标本HE常规染色后光镜下进行软骨组织形态学观察,并进行改良Mankin评分；采用免疫组化染色观察Ⅰ型胶原、MMP-13表达情况,通过Image-Pro Plus 6.0软件测量累积光密度值(IOD值)进行定量分析。运用Stata 7软件进行统计分析。采用两样本均数t检验进行实验组与对照组以及同一股骨头内、外侧间的比较。取P<0.05为差异有统计学意义。[结果]1.组织形态学观察与改良Mankin评分：幼兔对照组中a、b、c各组均见软骨表面光滑,潮线清楚；实验组中a组见软骨细胞增多,b组见潮线模糊、多层潮线,c组见软骨表面轻度不规则,部分软骨细胞增生成簇。尽管组织形态学观察有所不同,但幼兔a、b、c各组实验组与对照组之间以及内、外侧之间改良Mankin评分均无统计学差异。成兔A、B组实验组与对照组均见部分软骨表面轻度不规则,潮线中断、模糊较多见。实验组与对照组之间以及内、外侧之间改良Mankin评分均无统计学差异；成兔C组对照组软骨表面轻度不规则,细胞排列规则；实验组软骨表面不规则,可见裂隙,细胞明显增多,增殖成簇,潮线模糊。实验组内、外侧改良Mankin评分均比对照组高(P<0.01),内、外侧之间改良Mankin评分无统计学差异。2.Ⅰ型胶原表达：Ⅰ型胶原表达于软骨细胞胞浆内,在幼兔及成兔各组中均有表达。幼兔各组随固定时间延长表达逐渐增多,幼兔a、b组实验组与对照组比较、外侧与内侧比较均无统计学差异；幼兔c组实验组内、外侧均比对照组表达增多(外侧P=0.0013,内侧P=0.0201),实验组外侧比内侧表达增多(P=0.0185)。成兔A、B组实验组与对照组比较、外侧与内侧比较无统计学差异；成兔C组实验组内、外侧均比对照组表达增多(外侧P=0.0004,内侧P=0.0083),实验组外侧比内侧表达增多(P=0.0043)。3.MMP-13表达：MMP-13表达于软骨细胞胞浆内,在幼兔及成兔各组中均有表达。幼兔各组随固定时间延长表达逐渐增多,幼兔a组实验组与对照组比较、外侧与内侧比较均无统计学差异；幼兔b、c组实验组内、外侧均比对照组表达增多(P<0.01,c组外侧P<0.05),外侧与内侧比较无统计学差异。成兔A组实验组与对照组比较、外侧与内侧比较无统计学差异；成兔B组实验组外侧比对照组表达增多(P=0.0067),内侧与对照组比较无统计学差异；实验组外侧比内侧表达增多(P=0.0488),对照组外侧与内侧比较无统计学差异；成兔C组实验组内、外侧均比对照组表达增多(外侧P=0.0118,内侧P=0.044),内外侧比较无统计学差异。[结论]1.髋臼发育不良股骨头软骨退行性变具有时序性与一定时段的可逆性。2.髋臼发育不良股骨头软骨退行性变时I型胶原、MMP-13表达增多；MMP-13是反应软骨退变更敏感的指标。3.股骨头内、外侧应力的差异在一定时段可能导致软骨退行性变出现差异。更多还原

【Abstract】 [Background and Object]Developmental dysplasia of the hip (DDH) is one of the common deformities of limbs in children. Most of DDH patients were diagnosed earlierly now, because of early screening during infant. Even the hip development improve after close or open reduction, there are still around 70% patients suffering from residual acetabular dysplasia(RAD), which is potential cause of osteoarthritis(OA) when they are adults. Controversy always exists in terms of the most optimalized age to treat RAD. So far it is unknown that whether the pathophysiological changes in RAD cartilage can be reversible or not. Our previous data suggested that the degeneration of acetabular cartilage in acetabular dysplasia (AD) was time-dependent and reversible. But the changes of femoral head cartilage and the sequence and reversibility of the changes in AD are still unknown. So histomorphological changes, type I collagen and matrix metalloproteinase-13(MMP-13) of femoral head cartilages of serial immature rabbit AD models were observed in this study, in order to study the relevance between AD and OA, and to provide theory evidence for choosing the correct age for treating AD in children by observing the time point of reversibility of femoral head cartilage degeneration in AD.[Methods]Twenty seven 5-week-old New Zealand rabbits were randomly divided into three groups, namely A, B, C. And every group owned 9 rabbits. The left hind limb as the experimental side, which was given cast immobilization, maintaining knee extended and hip flexed for 2,4,6 weeks for each group respectively. The right hind limb served as the control side with no treatment. The anteroposterior pelvic radiograph of all rabbits were taken after 2 weeks cast immobilization. The results shows AD models were established. Five rabbits were sacrificed from each group after achieving their own casting time, composing the immature groups a,b and c. And the other four were given cast removal and were sacrificed at 6 months old, composing the mature groups A,B and C. The medial and lateral parts of femoral head cartilages were observed respectively. The morphology of femoral head cartilages was observed using HE staining, and the modified Mankin scores were gotten according to the morphology. Expression of type I collagen and Matrix metalloproteinase-13 (MMP-13) in femoral head cartilages were detected by the Immunohistochemical staining (IHC). Using Image-Pro Prus 6.0 to measure the integrated optical density(IOD). Stata 7 was adopted for statistics. Using two samples t test to compare experimental groups with control groups and medial parts with lateral parts.[Results]1. Histomorphological study and modified Mankin scores:Articular cartilages on control sides had smooth surfaces and tide lines were smooth in immature group a,b and c. On experimental side number of cartilage cells increased in immature group a; Tide line was blurred in immature group b; Cartilage had slightly irregular surface, cartilage cell number increased and a few cell clusters formed in immature group c. No significant difference of the modified Mankin scores in immature group a, b and c was found between experimental side and control side, nor did it between medial part and lateral part. In mature models, articular cartilages had slightly irregular surface, tide lines were blurred and interrupted both on control side and on experimental side in group A and B. No significant difference of the modified Mankin scores in group A and B was found between experimental side and control side, nor did it between medial parts and lateral parts. In mature group C cartilage surfaces were irregular, and there were a few hiatuses in the surfaces on experimental sides, cartilage cell number increased greatly, and the array of cells was disordered; Cell clusters were found frequently on experimental side. Tide lines were blurred. The modified Mankin score in group C experimental side was higher (P< 0.01). But no significant difference was found between medial part and lateral part.2. The expression of type I collagen in femoral head cartilage of the ADType I collagen was expressed in cytochylema in cartilage cells. In immature groups, expression of type I collagen increased gradually after 2,4,6-weeks immobilization. But no significant difference was found between experimental side and control side, nor did it between medial part and lateral part in group a and b. In group c, expression of type I collagen in experimental side is higher both in medial part and in lateral part (lateral part P=0.0013, medial part P=0.0201), and it is highe in lateral part than that in medial part in experimental side (P=0.0185). In mature groups, no significant difference was found between experimental side and control side, nor did it between medial part and lateral part in group A and B. Expression of type I collagen in experimental side is higher both in medial part and in lateral part (lateral part P=0.0004, medial part P=0.0083), and it is higher in lateral part in experimental side (P=0.0043) in group C.3. The expression of MMP-13 in femoral head cartilage of the ADMMP-13 was expressed in cytochylema in cartilage cells. In immature groups, expression of MMP-13 increased gradually after 2,4,6-weeks immobilization. No significant difference was found between experimental side and control side, nor did it between medial part and lateral part in group a. Expression of MMP-13 in experimental side was higher both in medial part and in lateral part (P<0.01, lateral part of group c P<0.05), but there was no significant difference between medial part and lateral part both in group b and in group c. In mature groups, no significant difference was found between experimental side and control side, nor did it between medial part and lateral part in group A. Expression of MMP-13 in experimental side was higher only in lateral part (P=0.0067). In medial part no significant difference was found in group B. Expression of MMP-13 in experimental side was higher both in medial part and in lateral part (lateral part P=0.0118, medial part P=0.044), but there was no significant difference between medial part and lateral part in group C.[Conclusions]1. Degeneration of femoral head cartilage in the acetabular dysplasia is sequential, and it is reversible in some stage.2. The expression of type I collagen and MMP-13 in femoral head cartilage in AD is higher when cartilage degenerated. MMP-13 may be a more sensitive monitor indicating cartilage degeneration.3. The difference of mechanical stress between medial part and lateral part in femoral head may cause difference of cartilage degeneration in some stage.更多还原