体外破骨细胞骨吸收模型的建立及骨吸收上清液对前成骨细胞功能的影响及其机制研究

发布时间：2018-07-15 11:48

【摘要】：目的：研究破骨细胞骨吸收活动中的分泌产物对成骨细胞增殖、分化及成骨功能的影响,以了解活化的破骨细胞在体外对成骨细胞的影响；并确定其发生作用的信号通路,以期为治疗牙槽骨吸收提供新的治疗靶点。方法：诱导小鼠RAW264.7细胞为破骨细胞,用抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase,TRAP)染色和破骨细胞特异性基因检测鉴定。破骨细胞与牛骨磨片共培养,甲苯胺蓝染色。收集共培养上清液作用于小鼠MC3T3-E1细胞,用甲基噻唑基四唑法(methyl thiazolyl tetrazolium, MTT)法、酶联免疫吸附法、茜素红染色及反转录聚合酶链反应检测细胞的增殖、骨钙素水平、矿化及成骨特征基因的表达；共培养上清液及P13K特异性抑制剂作用于小鼠MC3T3-E1细胞后,蛋白免疫印迹实验(western-bolt)检测相关信号蛋白的表达。结果：TRAP染色、破骨细胞特异性基因检测及甲苯胺蓝染色,表明RAW264.7细胞可分化为具骨吸收能力的破骨细胞。破骨细胞骨吸收上清液作用,MC3T3-E1细胞增殖受抑制(OD值在0.062±0.004和0.405士0.033之间,P0.05)。骨钙素水平增高(第10日上清组的骨钙素浓度为2.965±0.047μg/L),钙化结节增多,碱性磷酸酶(alkaline phosphatase, ALP)和Runt相关转录因子2(runt related transcription factor2, Runx2)的转录增强。破骨细胞骨吸收上清作用后MC3T3-E1细胞中P13K的调节亚基P-P85及P13K信号通路下游分子P-AKT表达显著增加(P0.05)。特异性抑制剂LY294002阻断后,P-P85及P-AKT均出现了剂量依赖性的降低,且都低于上清处理组(P0.05)。结论：RAW264.7破骨细胞骨吸收上清液具有抑制成骨样细胞增殖,促进分化和钙化成骨的作用。破骨细胞骨吸收上清促进小鼠MC3T3-E1细胞的分化作用是通过PI3K信号通路产生的。
[Abstract]:Aim: to study the effects of the secretory products of osteoclast bone resorption on the proliferation, differentiation and osteogenic function of osteoblasts in order to understand the effect of activated osteoclasts on osteoblasts in vitro, and to determine the signaling pathway of their action. In order to provide a new therapeutic target for alveolar bone resorption. Methods: mouse RAW264.7 cells were induced to be osteoclasts and identified by tartrate resistant acid phosphatase-trap staining and osteoclast specific gene detection. Osteoclasts were co-cultured with bovine bone mill and stained with toluidine blue. Mouse MC3T3-E1 cells were treated with co-cultured supernatants. The proliferation and osteocalcin levels of MC3T3-E1 cells were detected by (methyl thiazolyl tetrazolium, assay, enzyme-linked immunosorbent assay (Elisa), alizarin red staining and reverse transcriptase polymerase chain reaction (RT-PCR). After co-culture supernatant and P13K specific inhibitor acted on mouse MC3T3-E1 cells, Western blot assay (western-bolt) was used to detect the expression of related signal proteins. Results the results showed that RAW264.7 cells could differentiate into osteoclasts with bone resorption ability. The results showed that RAW264.7 cells could be differentiated into osteoclasts with bone resorption ability by the detection of specific genes of osteoclasts and toluidine blue staining. Bone resorption supernatant of osteoclasts inhibited the proliferation of MC3T3-E1 cells (OD values were 0.062 卤0.004 and 0.405 卤0.033 respectively, P0.05). The level of osteocalcin increased (the concentration of osteocalcin was 2.965 卤0.047 渭 g / L), the number of calcified nodules increased, and the transcription of alkaline phosphatase (alkaline phosphatase,) and Runt related transcription factor 2 (runt related transcription factor-2 (Runx2) were increased in the 10th day supernatant group. The expression of P13K regulatory subunit P-P85 and the downstream molecule P-AKT of P13K signaling pathway were significantly increased in MC3T3-E1 cells treated with osteoclast bone resorption supernatant (P0.05). Both P-P85 and P-AKT decreased in a dose-dependent manner after blocking LY294002 and were lower than those in supernatant treatment group (P0.05). Conclusion the supernatant of bone resorption of osteoclasts from the osteoclasts can inhibit the proliferation of osteoblasts and promote the differentiation and calcification of osteoblasts. The bone resorption supernatant of osteoclasts promotes the differentiation of mouse MC3T3-E1 cells through PI3K signaling pathway.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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