Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (46): 8069-8074.doi: 10.3969/j.issn.2095-4344.2013.46.015
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He Ming-jiang, Zhang Hong-mei, Jing Lin, Tao Zhi-kun, Hu Rui
Online:
2013-11-12
Published:
2013-11-30
Contact:
Zhang Hong-mei, Chief physician, Professor, Wangjing Hospital of China Academy of Chinese Medical Sciences, Beijing 100102, China
wangjingzhm@sina.com
About author:
He Ming-jiang★, Master, Physician, Wangjing Hospital of China Academy of Chinese Medical Sciences, Beijing 100102, China
bandian503@126.com
Supported by:
the Natural Science Foundation of Beijing, No. 7102166*
CLC Number:
He Ming-jiang, Zhang Hong-mei, Jing Lin, Tao Zhi-kun, Hu Rui. Optimal animal model of osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(46): 8069-8074.
2.1 非手术方法 2.1.1 关节制动 通过对动物的关节制动所产生的应力改变诱导产生关节软骨退变建立骨关节炎动物模型。Okazaki等[1]研究表明,家兔膝关节伸直位制动可成功建立骨关节炎模型,制动时间越长,关节软骨退变呈逐渐加重。张洪等[2]采用兔膝关节伸直位石膏管形固定法制作骨关节炎动物模型,制动五六周后动物模型细胞凋亡情况呈典型骨关节炎特征。尚平 等[3]研究则进一步阐明过屈位和过伸位固定制作骨关节炎模型的优劣,其认为过屈位法简便、固定牢靠、成功率高。 2.1.2 关节腔注射药物 尿激酶:石辉等[4]将尿激酶注射入成年兔膝关节腔后的第4,8,12周分批取兔滑膜组织进行病理组织学观察,同时取软骨进行病理组织学观察及电镜检查,并与关节内注射生理盐水组对照,结果表明12周所制作的试验兔模型符合骨关节炎的病理组织特征。 木瓜蛋白酶:Havdrup等[5]分别于实验的第1,4,7天将4%的木瓜蛋白酶0.3 mL注入兔髋关节中,成功建立骨关节炎模型。Muehleman等[6]采用木瓜凝乳蛋白酶进行兔膝关节腔注射,2周后即可见膝关节面纤维变性、软骨浅层破坏;4周后可见关节面软骨层广泛破坏、骨面粗糙不平,退变呈进行性发展;6周后关节软骨退变符合骨关节炎改变。 胶原蛋白酶:Kikuchi等[7]通过使用不同剂量的胶原酶进行兔膝关节腔内注射建立骨关节炎模型。在注射后2,4,6周进行组织学观察,6周时即观察到关节软骨及滑膜变性。结果表明,该方法所使用胶原酶剂量比木瓜蛋白酶低,诱导简便可靠。邓宇等[8]发现低剂量Ⅱ型胶原蛋白酶与较高剂量的木瓜蛋白酶比较,前者可诱导出更严重的软骨退变。 碘乙酸盐:Guzman等[9]使用碘乙酸盐注入Wistar大鼠膝关节成功建立骨关节炎模型,在注射后1周及4周后进行组织学观察表明,注射1周后,破骨细胞和成骨细胞数目增加;4周后破骨细胞活性明显增强,骨小梁有局灶性断裂、塌陷和纤维化。Pomonis等[10]研究则认为碘乙酸盐与骨关节炎疼痛有关。 另外,肾上腺皮质激素、透明质酸酶、软骨碎片或微粒、异物等注入动物关节腔内也可导致关节软骨退变,形成骨关节炎模型[11-12]。 2.2 手术方法 2.2.1 关节内手术 前交叉韧带切断法:前交叉韧带切断是近年采用较多的建立骨关节炎模型的方法,因其手术创伤小,对动物生理结构改变较少,能较全面反映骨关节炎软骨退变的病理过程[13]。Stoop等[14]研究发现切断Wistar大鼠的膝关节前交叉韧带后,表层带软骨细胞数量减少、剩余的软骨细胞肿胀,表层带纤维化,且随时间推移,上述表现呈进行性发展,术后10周时变性的Ⅱ型胶原降解产物在纤维化的区域明显增加。由于该法造模时间长,王君等[15]在前交叉韧带切断法基础上固定手术对侧肢体,造成术肢负重增加,可在更短时间内获得理想的骨关节炎模型。 半月板切除法:Moskowitz等[16]采用切除兔膝关节内侧半月板建立骨关节炎模型,造模后6个月可观察到早期骨关节炎的形态学和生化改变。在这一方法基础上,通过去神经支配能加速动物模型的关节退变,弥补单纯半月板切除造模时间长的缺点。李忠等[17]经关节镜切除小型猪双膝外侧半月板,术后12周观察显示,膝关节出现局限性软骨表面纤维化,股骨髁间切迹骨赘形成;组织学检测结果显示,软骨细胞数量和蛋白多糖含量减少,成簇排列细胞数量增加;MRI检查结果显示,软骨厚度变薄和骨软骨信号强度改变。 Hulth法:此方法为经典手术造模方式[18],其主要通过切除前后交叉韧带、内侧副韧带及内侧半月板造成关节失稳,是关节面间磨损增加,促使关节软骨退变。在Hulth法术后3个月,组织学观察可见软骨面出现裂隙、破坏。王云峰等[19]采用切断前交叉韧带、切除内侧半月板而不切断内侧副韧带的方法,在不同的造模时间内成功复制出不同时期骨关节炎。造模2周可肉眼观察到滑膜充血、炎症渗出;6周可出现为关节软骨亮度下降等改变,10周可见软骨明显溃疡、变薄等,个别标本在胫骨平台内缘处出现骨质增生;14周可见软骨缺失、骨质暴露及明显增生的骨赘。由于Hulth法造模时间较长,方锐等[20]以Hulth法造模,并在术后第7天驱赶动物,增加术肢负重,促使软骨退变加剧,发现造模6周可见骨关节炎早期改变,8周为中期,12周为晚期。 关节划痕法:通过关节内手术途径造成关节失稳建立骨关节炎模型,可以较好地观察骨关节炎发展,但由于滑膜炎症的存在,会影响诸如针对软骨保护和修复等治疗措施疗效的观察。而Marijnissen等[21]研究在狗的膝关节股骨髁上刻痕,不损伤软骨下骨的方法建立骨关节炎模型,在40周后观察到关节软骨退变与临床骨关节炎相似,且关节炎症表现轻微。因此认为,通过关节刻痕法可建立观察骨关节炎早期表现及药物疗效的理想模型。 2.2.2 关节外诱导 选择性切断臀肌法:白希壮等[22]通过选择性切断豚鼠臀大、中、小肌远侧端1 cm,使其关节生物力学紊乱,从而获得满意的髋骨关节炎模型。术后12周可观察到软骨细胞呈双柱状及灶巢样增生,潮线有血管通过,并伴有新骨形成,软骨表面剥脱及水平裂隙;术后24周可观察到软骨细胞混杂增生及变性坏死,软骨表面进一步破溃、粗糙,进而软骨下骨裸露呈象牙状。选择性臀肌切断法通过关节外操作,可以避免关节内手术所致关节内滑膜炎症产生对实验的干扰,是观察骨关节炎早期病理改变以及筛选治疗药物的良好模型。 改变关节应力:目前研究认为, 关节活动所形成的正常应力环境对关节软骨意义非凡。应力增加或降低均可导致骨关节炎产生[23]。余存泰等[24]研究表明,应力减小可引起软骨细胞代偿增生,并进一步引起基质破坏,从而导致整个关节软骨退变;应力增加则首先造成基质破坏,同时由于软骨细胞代偿增生,使软骨细胞退变,从而导致整个关节软骨退变。 破坏关节血液循环:在关节退变的影响因素中,关节血液循环异常扮演着一个重要的角色。吴并生等[25]通过双重结扎兔单下肢股静脉,12周建立了符合早期骨关节炎改变的动物模型。刘蜀彬等[26]通过阻断兔股骨干骺端髓内血液供应,16周亦获得符合早期骨关节炎改变的动物模型。戴七一等[27]通过无菌手术结扎并切断新西兰兔臀下静脉、股静脉和大隐静脉,可在术后8周获得早中期骨关节炎动物模型。 卵巢切除:通过切除卵巢所建立骨关节炎模型是研究雌激素及其类似物对关节软骨保护作用的理想模型。Pernille等[28]通过切除SD鼠卵巢制作骨关节炎模型,结果表明代表骨吸收的CTX-Ⅰ(collagen typeⅡdegradation products)及代表软骨变化的CTX-Ⅱ的水平与关节最终的退变呈明显相关性。 2.3 自发动物模型 2.3.1 C57黑鼠 Silberberg于1941年首先发现C57黑鼠具有自发形成骨关节炎的特性,但C57黑鼠骨关节炎模型与人存在一定的差别,主要表现为几乎无关节软骨的微纤维化,关节软骨剥离脱落呈腐蚀状,软骨细胞几无集簇形成,滑膜炎症不明显,骨关节炎进展过程中不伴有GAG、DNA合成增加[29]。而增加运动负荷以后,能增加C57黑鼠骨关节炎自发率,但其病变程度更多表现为早期骨关节炎表现[30]。 2.3.2 Hartley豚鼠 12个月龄繁殖低下的雄性Hartley豚鼠膝关节可出现软骨表层不平整,表层软骨细胞丢失,胶原不同程度的溶解、断裂和排列紊乱,糖胺聚糖分布异常[31]。 C57黑鼠体积小、不易观察、骨关节炎表现与人骨关节炎存在一定区别,而与之相比,Hartley豚鼠发病率与人骨关节炎相似,易于饲养、观察且费用相对较低。 2.3.3 B6C3F1栗鼠 B6C3F1栗鼠因为脚尖行走,踝关节软骨面可出现不规整、龟裂,软骨细胞的数目减少,排列紊乱,软骨边缘骨质增生,与人骨关节炎有类似的病理变化[32]。 2.4 转基因动物模型 随着转基因技术的发展,骨关节炎转基因动物模型为骨关节炎研究提供了新的选 择[33]。Rodriguez等[34]研究研究发现小鼠Col11a1基因的核苷酸缺失会导致胶原纤维的异常增厚和软骨蛋白多糖的增加,实验中发现15个月时Col11a1基因敲除的cho/+小鼠因为Ⅺ型胶原的突变,不能合成正常的胶原纤维,从而导致小鼠出现骨关节炎表现。Hyttinen等[35]研究则发现Col2a1等位基因失活将导致软骨内骨形成障碍,进而导致关节软骨软化。Salminen等[36]研究发现Del1基因突变可增加骨关节炎发病率,Del1转基因小鼠在3个月时膝关节的关节软骨形成表浅原纤维,其可迅速渗透到软骨并引起骨硬化、半月板退变,囊肿形成及软骨下骨裸露。其研究还发现,雄性Del1小鼠骨关节炎表现比雌性严重,并且胫骨和股骨外侧髁的退变都较内侧髁严重。 在Ⅺ型胶原突变诱发的小鼠膝骨关节炎中出现关节软骨细胞盘状结构域受体2的上调和基质金属蛋白酶 13的表达[37],过度诱导表达盘状结构域受体2可引起软骨细胞表面基质金属蛋白酶 13表达的升高,导致软骨细胞的破坏,诱发骨关节炎的发生[38-39]。"
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