發(fā)布時間：2019-05-09 12:39

【摘要】：目的:通過體內(nèi)外實驗觀察不同劑量和細(xì)胞培養(yǎng)濃度雷公藤紅素對磨損顆粒誘導(dǎo)的小鼠顱蓋骨骨溶解及破骨細(xì)胞分化的影響,為將來雷公藤紅素應(yīng)用于防治人工關(guān)節(jié)磨損顆粒誘導(dǎo)的骨溶解提供理論依據(jù)。方法:體外實驗中,先通過CCK-8法確定雷公藤紅素對小鼠BMMs來源的單核-巨噬細(xì)胞的安全濃度范圍;然后,在無毒濃度范圍內(nèi),觀察給予不同濃度雷公藤紅素刺激后,對M-CSF和RANKL誘導(dǎo)小鼠BMMs來源的單核-巨噬細(xì)胞分化為破骨細(xì)胞的影響。體內(nèi)實驗中,將20只6周齡雄性C57BL/6J小鼠隨機分為4組,空白對照組(Sham組)、單純磨損顆粒組(Vehicle組)、磨損顆粒+低劑量雷公藤紅素組(Low組1 mg/kg)和磨損顆粒+高劑量雷公藤紅素組(High組3mg/kg)。利用磨損顆粒誘導(dǎo)小鼠顱骨骨溶解模型,分別按3mg/kg(高劑量組)、1mg/kg(低劑量組)雷公藤紅素的劑量腹腔注射,隔日給藥,空白對照組和單純顆粒組小鼠注射同等體積的生理鹽水。建模2周后采用摘除眼球法取血后處死小鼠,完整取下小鼠顱蓋骨標(biāo)本。小鼠血清通過ELISA試劑盒檢測與骨吸收代謝特征性指標(biāo)TRAP5b和CTX-Ⅰ的表達(dá)水平;小鼠顱蓋骨先進行Micro-CT掃描,統(tǒng)計分析各項骨參數(shù),再通過制作病理切片HE染色觀察顱骨組織形態(tài)并進行定量分析。結(jié)果:不同劑量、濃度雷公藤紅素處理組小鼠存活及細(xì)胞增殖均未受到影響。體外實驗發(fā)現(xiàn),在安全濃度范圍內(nèi),雷公藤紅素可以顯著抑制小鼠BMMs來源的單核-巨噬細(xì)胞向破骨細(xì)胞分化。體內(nèi)實驗中,磨損顆粒能夠有效誘導(dǎo)小鼠顱蓋骨溶解,在給予雷公藤紅素治療后,顱蓋骨骨溶解被明顯抑制了,而且這種抑制效應(yīng)表現(xiàn)出劑量依賴性。小鼠顱蓋骨經(jīng)Micro-CT掃描后,統(tǒng)計分析感興趣區(qū)域(ROI)的各項骨參數(shù),骨礦物質(zhì)密度(BMD)、骨體積分?jǐn)?shù)(BVF)和骨孔隙率的結(jié)果進一步證實了上述結(jié)論。小鼠顱骨組織切片HE染色發(fā)現(xiàn),在雷公藤紅素治療組內(nèi),炎癥細(xì)胞浸潤、骨質(zhì)破壞明顯減少。小鼠血清TRAP5b和CTX-Ⅰ檢測發(fā)現(xiàn),藥物組水平低于單純磨損顆粒組。結(jié)論:安全濃度范圍內(nèi)雷公藤紅素在體外能有效抑制破骨細(xì)胞形成;人工關(guān)節(jié)磨損顆粒能有效誘導(dǎo)小鼠顱蓋骨骨溶解;雷公藤紅素在安全劑量范圍內(nèi)能有效防治磨損顆粒誘導(dǎo)的小鼠顱蓋骨骨溶解。
[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，

本文編號：2472772