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促红细胞生成素预防激素性骨质疏松的实验研究

Experimental study on the protective effect of EPO in glucocorticoid-induced osteoporosis

摘要:

目的:研究促红细胞生成素(erythropoietin,EPO)对于激素性骨质疏松的大鼠模型的预防作用并初步探索其机制。方法:通过动物实验的方法,将SD大鼠分为3组:骨质疏松组,每周2次大鼠后腿肌注甲基泼尼松龙20 mg/kg,连续用药6周;EPO组,每日腹腔内加用500 U/kg的rHuEPO;生理盐水组同样方法注射生理盐水,12周后取大鼠股骨颈标本。通过HE染色观察组织学大体标本的变化证实其抗骨质疏松作用和观察组织学结构和细胞形态变化,通过免疫组化CD31染色检测其微血管变化,Western Bolt验证伴随的VEGF的变化和PCNA检测其细胞增殖的改变,利用ELISA检测血清中骨代谢标记物OPN、PINP、CTX-1的变化并进行统计学分析。结果:组织学:骨质疏松组可见骨小梁明显稀疏、变窄、断裂、形态不规则,并伴有较多破骨细胞出现,部分可见骨细胞胞核皱缩、溶解、消失;EPO组较骨质疏松组病变有明显改善,部分结构已经接近对照组,骨小梁分割程度较高且可见较多成骨细胞。免疫组化高倍镜视野结果:EPO组CD31阳性细胞16.60±4.88,骨质疏松组12.96±4.54,生理盐水组25.84±7.97,EPO组较骨质疏松组CD31高表达,差异有统计学意义( P<0.05)。Western Bolt:生理盐水组VEGF/β-actin灰度比值0.570±0.022,骨质疏松组0.446±0.083,EPO组0.584±0.009;生理盐水组PCNA/β-actin比值0.541±0.158,骨质疏松组0.187±0.099,EPO组0.733±0.257;EPO组VEGF和PCNA表达均高于骨质疏松组,而EPO组与生理盐水组相比差异无统计学意义( P>0.05)。ELISA血清学检测,生理盐水组血清OPN水平为(78.34±17.28) pg/ml,骨质疏松组(368.48±97.23) pg/ml,EPO组(217.62±39.11) pg/ml,差异有统计学意义( P<0.05);生理盐水组血清P1NP为(1 507.00±58.49) ng/ml,骨质疏松组(1 196.00±91.32) ng/ml,EPO组(1 621.00±65.57) ng/ml,差异无统计学意义( P>0.05);生理盐水组CTX-1水平为(27.10±4.78) ng/ml,骨质疏松组(39.46±9.23) ng/ml,EPO组(31.17±4.11) ng/ml,差异有统计学意义( P<0.05)。 结论:同时性注射EPO可以一定程度上预防激素性骨质疏松,促血管作用和促进成骨以及抑制破骨是其可能的机制。

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abstracts:

Objective:To study the mechanism of protective effect of erythropoietin (EPO) on glucocorticoid-induced osteoporosis in rats through promoting vascular action mediated by VEGF, promoting osteogenesis, and inhibiting bone resorption.Methods:An animal experiment of 24 SD rats in total were divided into three groups: the osteoporosis group (20 mg/kg BW of methylprednisolone biwim for 6 weeks); the EPO group (MP 20 mg/kg biw+EPO 500 u/kg qdim); the nature salt group (0.9%NS). After 12 weeks, rats were harvested and received examination of histology (HE staining) for demonstration of protective effect, immunohistochemistry with CD31 stainingfor microvascular changes ,changes of VEGF and PCNA expressions using Western Boltfor microvascular and cell proliferation, and ELISA to detectOPN, PINP, CTX-1 in serumasbone turnover marker.Results:Hematoxylin and eosin staining in the model group showed that the bony trabeculae had become obviously narrow and sparse with discontinuity of the integrity. The integrity of the trabeculae was better in the EPO group. Immunohistochemical results: the EPO group CD31+ cell 16.60±4.88, the osteoporosis group 12.96±4.54, the NS group 25.84±7.97. CD31 expression was higher in the EPO group than the osteoporosis group. Western Bolt: in the NS groupthe ratio of VEGF/β-actin greyscalewas 0.570±0.022, with the osteoporosis group 0.446±0.083 and the EPO group 0.584±0.009; The ratio of PCNA/β-actingreyscale was 0.541±0.158 in the NS group, withthe osteoporosis group 0.187±0.099, the EPOgroup 0.733±0.257. VEGF and PCNA expression in the EPO group were higher than those in the osteoporosis group. ELISA: OPN results: the NS group 78.34±17.28 pg/ml, the osteoporosis group 368.48±97.23 pg/ml, the EPO group 217.62±39.11 pg/ml; P1NP results: the NS group 1 507.00±58.49 ng/ml, the osteoporosis group 1 196.00±91.32 ng/ml, the EPO group 1 621.00±65.57 ng/ml; CTX-1 results: the NS group 27.10±4.78 ng/ml, the osteoporosis group 39.46±9.23 ng/ml, the EPO group 31.17±4.11 ng/ml. The level of OPN and CTX-1 in the EPO group were lower than that in the osteoporosis group, and P1NP was higher than that in the osteoporosis group.Conclusion:EPO generates certain protective effect on bone of rats withglucocorticoid-induced osteoporosis. Its potential mechanism is to promote vascular action, promote osteogenesis, and inhibit bone resorption.

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作者: 颜勇卿 [1] 徐人杰 [2] 魏鹏 [3]
作者单位: 中国科学院大学宁波华美医院(原宁波市第二医院)骨科中心,中国科学院大学宁波生命与健康产业研究院 315010 [1] 苏州市立医院,南京医科大学附属苏州医院骨科 215000 [2] 宁波市第一医院手足及显微创伤外科 315000 [3]
期刊: 《中华骨科杂志》2020年40卷21期 1469-1477页 ISTICPKUCSCDCA
栏目名称: 基础研究
DOI: 10.3760/cma.j.cn121113-20190519-00215
发布时间: 2020-11-16
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