瘦素促进骨髓间充质干细胞成骨分化并增加内植物-骨界面骨生成

Leptin promotes bone marrow derived stem cells osteogenic differentiation and increases bone formation in plant-bone interface models

目的探讨瘦素促进骨髓间充质干细胞(BMSCs)成骨分化和内植物-骨界面骨生成的效果。方法从Sprague-Dawley大鼠骨髓腔获得BMSCs,经过流式细胞仪鉴定后,使用含不同质量浓度瘦素(0、10、20ng/mL,以0为对照组)的成骨诱导液培养21d,采用Western印迹法检测BMSCs碱性磷酸酶(ALP)和成骨特异性转录因子2(RUNX-2)表达,采用细胞计数试剂盒(CCK)8检测试剂并在酶标仪下读取光密度值(D值)反映BMSCs的增殖能力。在内植物-骨界面骨生成模型的新西兰大白兔皮下分别注射瘦素(实验组,术后即刻和术后第3天各注射瘦素20μg/kg)和0.9%氯化钠溶液(对照组,术后即刻和术后第3天各注射0.9%氯化钠溶液0.1mL),在术后4周处死实验兔,应用微型CT扫描并三维重建检测内植物表面之外厚度为0.3mm的环形感兴趣区域,计算骨体积分数(BVF)反映内植物-骨界面骨生成效果,并对模型兔的相关脏器进行病理学检查。结果与对照组相比,BMSCs以含10、20ng/mL瘦素的成骨细胞诱导液培养21d表达ALP和RUNX-2均显著增多(P值均<0.05);CCK-8增殖检测发现,10和20ng/mL瘦素处理组的D值均显著高于对照组(P值均<0.05)。内植物-骨界面骨生成模型的检测发现,实验组的BVF值显著高于对照组(P<0.05),光学显微镜下兔模型的心、肝、肾、脾组织细胞均未见变性、坏死。结论瘦素可以促进BMSCs成骨分化并且增加内植物-骨界面骨生成,且生理剂量瘦素对机体重要器官并未造成损害。

Objective To explore the effect of leptin on osteogenic differentiation of bone marrow derived stem cells （BMSCs） and bone formation in implant-bone interface model. Methods After obtained from Sprague- Dawley （SD） rats and identified by flow cytometry, BMSCs were cultured in osteogenic liquid with different concentrations of leptin （0, 10, 20 ng/mL） for 21 d. The expression of alkaline phosphatase .（ALP） and runtrelated transcription factor 2 （RUNX-2） in BMSCs were detected by Western blot. The value of optical density （D） which represents the capacity of BMSC proliferation was obtained from the microplate reader using Cell Counting Kit-8 （CCK-8）. Different doses of leptin （control group= 0 μg/kg, experimental group= 20 μg/kg, respectively） were injected subcutaneously in New Zealand rabbit model of implant-bone interface bone formation. The rabbits were sacrificed on day 28 after operation. The circle shaped region of interest outside of the implant-bone interface within 0.3 mm in thickness were scanned by micro computed tomography （Micro-CT） and then underwent three- dimensional （3D） reconstruction to calculate the bone volume fraction （BVF） which reflects the effect of bone formation in implant-bone interface. Meanwhile, pathological examination was carried out in the rabbit models. Results Compared with the control, ALP and RUNX-2 expressions were significantly increased in the leptin- cultured BMSCs of two experimental groups （all P〈0.05）. The D values in the two experimental groups were significantly higher than that in the control group （both P 〈 0.05）. BVF in the experimental groups was also significantly higher than that in the control group （P〈0.05）. No cell degeneration or necrosis was found in the heart, liver, kidney or spleen of the model under the optical microscope. Conclusion Leptin can promote BMSC osteogenic differentiation and increase bone formation in implant-bone interface model. Besides, physiological doses of leptin will not damage important organs.