卵巢去势对骨折修复中骨组织超微结构和骨形态发生蛋白4基因定位分布的影响(英文)

Experimental study on the bone microstructure and bone morphogenetic protein-4 expression during fracture healing in a rat osteoporotic model prepared by ovariectomy

背景:在卵巢去势后雌激素缺乏状态下,由于骨质疏松时全身及局部骨结构发生骨量减少和骨微细结构以及生长因子基因定位分布变化,无疑会对骨折愈合产生明显的影响。目的:观察雌激素对骨折愈合过程超微结构变化和骨生长因子骨形态发生蛋白4基因表达及定位分布的影响。设计:随机对照观察。单位:云南大学分子生物学实验室。对象:实验于1999-07/2002-07在云南大学分子生物学实验室完成。选用健康2月龄雌性SD大鼠96只,平均质量160～200g。方法:①取96只大鼠腹腔麻醉后,其中48只作双侧卵巢切除术为骨质疏松组,余48只行假手术为对照组。所有大鼠1个月后,在麻醉状态下于双侧胫骨骨干中段行手法折断,任其自行修复,制作成骨折愈合模型。两组动物分别于骨折后1,3,5,7,14,28,56,112d麻醉后处死取骨痂和周围软组织,每个时间点取6只。②大鼠胫骨骨组织超微结构采用电镜下观察。③骨痂组织和软组织骨形态发生蛋白4表达采用原位杂交法观察,杂交液中不加标记探针作为阴性对照。选取两组1,3d时间点原位杂交阳性表达切片各6张,每张在25倍物镜下随机选取3个视野,统计每个视野所有阳性表达颗粒数。主要观察指标:①两组大鼠胫骨骨组织超微结构观察结果。②骨痂和软组织骨形态发生蛋白4表达结果。结果:96只大鼠均进入结果分析。①两组大鼠胫骨骨组织超微结构观察结果:骨折28d以后,对照组骨性骨痂、骨胶原纤维性排列致密,骨陷窝中见骨细胞,体积小、胞质少,但体积小骨小梁周围见破骨细胞。骨质疏松组:纤维性骨痂、骨基质中胶原纤维排列疏松,成骨细胞数量多、体积大,破骨细胞多见。②骨痂和软组织骨形态发生蛋白4表达结果:骨折后1～3d,对照组<骨质疏松组(23.7143±5.0568,21.7143±5.0238比51.2857±8.1387,49.5714±9.0711,P<0.01)。表达局限于骨折周围骨痂形成区的软组织内。结论:卵巢切除后骨质疏松组大鼠骨形态发生蛋白4基因表达增多,并呈现一定的区域性,即局限于骨折周围的软组织内。骨折愈合过程中骨痂基质的形成和质量明显低于对照组。

BACKGROUND： As the result of estrogen shortage due to ovariectomy, osteoporosis occurs in the general and local bones, displaying bone loss and changes in bone microstructure and growth factor mRNA expression, which definitely has an important effect on fracture healing. OBJECTIVE： To probe into the effect of estrogen on bone microstructure and bone morphogenetic protein-4 mRNA expression and location during fracture healing. DESIGN： Randomized controlled study. SETTING：Laboratory of the Department of Molecular Biology of Yunnan University. MATERIALS： This study was conducted at the laboratory of Molecular Biology Department of Yunnan University between July 1999 and July 2002. We recruited 96 healthy female SD rats of 2 months old and with the mean body mass of 160 to 200 g. METHODS：①Of the 96 rats that received intraperitoneal anesthesia, 48 rats were subjected to bilateral ovariectomy （osteoporotic group）, and the other 48 rats received sham operation （control group）. One month later, bilateral tibia fracture at the middle segment was artificially made on all rats under anesthesia, and no treatment was given so as to prepare fracture healing model. Then rats of both groups were put to death for collecting callus and the surrounding parenchyma at postoperative 1, 3, 5, 7, 14, 28, 56 and 112 days, with 6 rats in each time point. ② The tibia bone microstructure was observed under electron microscope. ③ The mRNA expression of bone morphogenetic protein-4 at callus and the surrounding parenchyma was detected by RT-PCR method; no probe in hybrid fluid was used as negative controls. Six in situ hybridization slices with positive expression were selected from both groups at postoperative 1 and 3 days time points, and 3 visual fields were randomly selected from each slice for observing the positive granules under 25 times field lens. MAIN OUTCOME MEASURES： ①The tibia bone microstructure; ② bone morphogenetic protein--4 mRNA expression at callus and the surrounding parenchyma. RESULTS： All the 96 rats entered the final results analysis. ① Observation of the tibia bone microstructure： at 28 days after tibia fracture, osseous callus and ostein fibers were found arranged densely in control group. Osteocytes, small with fewer cytoplasts, were observed in osseous lacuna, but osteoclaats were found surrounding small-sized bone trabecula. In osteoporotic group, fibrous callus and collagenous fibers in bone matrix were arranged loosely, lots of big osteoblasts could be observed with osteocytes easily seen. ② Bone morphogenetic protein-4 mRNA expression at callus and the surrounding parenchyma： it was less expressed in control group than in osteoporotic group at postoperative 1 to 3 days （23.714 3±5.056 8, 21.714 3±5.023 8 vs 51.285 7±8.138 7,49.571 4±9.071 1, P 〈 0.01） and the expression was mainly observed in parenchyma surrounding the fracture where callus was formed. CONCLUSION： Bone morphogenetic protein--4 mRNA expression is increased in osteoporotic group and is mainly observed in parenchyma surrounding the fracture, displaying a manner of regional expression. However, the formation and quality of callus matrix during fracture are obviously poorer than in control group.