雷公藤红素防治人工关节磨损颗粒诱导骨溶解的实验研究

发布时间：2019-05-09 12:39

【摘要】：目的:通过体内外实验观察不同剂量和细胞培养浓度雷公藤红素对磨损颗粒诱导的小鼠颅盖骨骨溶解及破骨细胞分化的影响,为将来雷公藤红素应用于防治人工关节磨损颗粒诱导的骨溶解提供理论依据。方法:体外实验中,先通过CCK-8法确定雷公藤红素对小鼠BMMs来源的单核-巨噬细胞的安全浓度范围;然后,在无毒浓度范围内,观察给予不同浓度雷公藤红素刺激后,对M-CSF和RANKL诱导小鼠BMMs来源的单核-巨噬细胞分化为破骨细胞的影响。体内实验中,将20只6周龄雄性C57BL/6J小鼠随机分为4组,空白对照组(Sham组)、单纯磨损颗粒组(Vehicle组)、磨损颗粒+低剂量雷公藤红素组(Low组1 mg/kg)和磨损颗粒+高剂量雷公藤红素组(High组3mg/kg)。利用磨损颗粒诱导小鼠颅骨骨溶解模型,分别按3mg/kg(高剂量组)、1mg/kg(低剂量组)雷公藤红素的剂量腹腔注射,隔日给药,空白对照组和单纯颗粒组小鼠注射同等体积的生理盐水。建模2周后采用摘除眼球法取血后处死小鼠,完整取下小鼠颅盖骨标本。小鼠血清通过ELISA试剂盒检测与骨吸收代谢特征性指标TRAP5b和CTX-Ⅰ的表达水平;小鼠颅盖骨先进行Micro-CT扫描,统计分析各项骨参数,再通过制作病理切片HE染色观察颅骨组织形态并进行定量分析。结果:不同剂量、浓度雷公藤红素处理组小鼠存活及细胞增殖均未受到影响。体外实验发现,在安全浓度范围内,雷公藤红素可以显著抑制小鼠BMMs来源的单核-巨噬细胞向破骨细胞分化。体内实验中,磨损颗粒能够有效诱导小鼠颅盖骨溶解,在给予雷公藤红素治疗后,颅盖骨骨溶解被明显抑制了,而且这种抑制效应表现出剂量依赖性。小鼠颅盖骨经Micro-CT扫描后,统计分析感兴趣区域(ROI)的各项骨参数,骨矿物质密度(BMD)、骨体积分数(BVF)和骨孔隙率的结果进一步证实了上述结论。小鼠颅骨组织切片HE染色发现,在雷公藤红素治疗组内,炎症细胞浸润、骨质破坏明显减少。小鼠血清TRAP5b和CTX-Ⅰ检测发现,药物组水平低于单纯磨损颗粒组。结论:安全浓度范围内雷公藤红素在体外能有效抑制破骨细胞形成;人工关节磨损颗粒能有效诱导小鼠颅盖骨骨溶解;雷公藤红素在安全剂量范围内能有效防治磨损颗粒诱导的小鼠颅盖骨骨溶解。
[Abstract]:Objective: to observe the effects of triptoline on osteolysis and osteoclast differentiation induced by wear particles in vitro and in vivo. It provides a theoretical basis for the application of tripterine in the prevention and treatment of osteolysis induced by wear particles of artificial joint in the future. Methods: in vitro, the safe concentration range of triptoline on monocyte-macrophage derived from mouse BMMs was determined by CCK- 8 method. Then, in the range of non-toxic concentration, the effects of tripterine on the differentiation of BMMs-derived monocytes into osteoclasts induced by M-CSF and RANKL in mice were observed. In vivo, 20 6-week-old male C57BL/6J mice were randomly divided into 4 groups: blank control group (Sham group), simple wear granule group (Vehicle group). Wear particles low dose triptolide group (Low group 1 mg/kg) and wear particles high dose triptolide group (High group 3mg/kg). The osteolysis model of mouse skull induced by wear particles was induced by intraperitoneal injection of triptoline in 3mg/kg (high dose group) and 1mg/kg (low dose group), then administered every other day. The mice in the blank control group and the simple granule group were injected with the same volume of saline. After 2 weeks of modeling, the mice were killed after extirpation of eyeball, and the skull specimens of the mice were taken off completely. The expression of TRAP5b and CTX- 鈪，

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