Transwell小室内建立小鼠成骨-破骨细胞共培养体系  

Establish mouse osteoblast-osteoclast cell co-culture system in a Transwell chamber

作　　者：莫国业 张顺聪[2] 李永贤[1] 郭惠智 郭丹青[2] 李大星[1] 唐永超[2] 莫凌 罗培杰 马延怀 MO Guo-ye , ZHA NG Shun-cong , LI Yong-xian , GUO Hui-zhi , GUO Dan-qing ,LI Da-xing , TA NG Yong-chao ,MO Ling, LUO Pei-jie , MA Yan-huai.(Department of Orthopaedics ,the Third Affiliated Hospital of Guangzhou University of China Medicine, Guangzhou 510100, Guangdong , Chin)

机构地区：[1]广州中医药大学,广东广州510405 [2]广州中医药大学第一附属医院脊柱骨科,广东广州510405 [3]广州中医药大学第三附属医院骨科,广东广州510100

出　　处：《中国骨伤》2018年第3期241-247,共7页China Journal of Orthopaedics and Traumatology

基　　金：广东省自然基金项目(编号:2016A030313641);广州市科技计划项目(编号:2017010460);广东省中医药局科研项目(编号:20172043);广东省科技厅项目(编号:2016A020215137)~~

摘　　要：目的 :Transwell小室内建立体外小鼠成骨-破骨细胞共培养体系,并检测体系对成骨及破骨细胞活性的影响。方法:体外培育小鼠成骨细胞MC3T3-E1和小鼠单核巨噬细胞RAW264.7,RANKL诱导小鼠单核巨噬细胞RAW264.7分化为成熟破骨细胞后,于Transwell小室内建立成骨-破骨细胞共培养体系。通过CCK-8实验、茜素红染色、TRAP染色检测细胞的成骨、破骨活性。采用PCR、Western Blot方法检测成骨细胞MC3T3-E1中OPG、ALP、RANKL、TGF-b1的基因表达以及RANKL的蛋白表达,检测破骨细胞RANK、NF-κB的基因表达和蛋白表达。结果 :小鼠成骨细胞MC3T3-E1和小鼠破骨细胞可在Transwell小室内建立共培养体系;共培养体系影响小鼠成骨细胞与破骨细胞的分化活性,镜下可见成骨细胞分化增多,破骨细胞分化稍减少。共培养体系中成骨细胞基因OPG(0.65±0.08)、ALP(0.16±0.01)较单独培养OPG(1.00±0.08)、ALP(1.01±0.16)表达下降,而TGF-b1(4.42±0.21)、RANKL(4.12±1.04)较单独培养组TGF-b1(1.00±0.10)、RANKL(1.00±0.09)表达上升;破骨细胞相关RANK(0.63±0.06)、NF-κB(0.64±0.08)基因表达较单独培养组的RANK(1.00±0.08)、NF-κB(1.00±0.09)下降,差异均有统计学意义。同时共培养组的OPG(0.43±0.05)、NF-κB(0.59±0.05)的蛋白表达较单独培养组的OPG(0.84±0.06)、NF-κb(1.13±0.03)减少;共培养组RANKL(0.54±0.03)的蛋白表达则较单独培养组的RANKL(0.31±0.03)增加,差异有统计学意义,均与基因表达变化趋势一致。结论:小鼠成骨细胞MC3T3-E1和小鼠破骨细胞可在Transwell小室内建立共培养体系,共培养体系中成骨细胞活性高于破骨细胞活性。Objective： To establish osteoblast -osteoclast cell co-culture system in a Transwell chamber,and detect cell viability of osteoblasts and osteoclasts in system. Methods： Osteoblast MC3T3-E1 and mouse monocytes RAW264.7 were cultivated in vitro. RANKL-induced mouse RAW264.7 monocytes differentiated into mature osteoclasts,osteoblast-osteoclast cell co-culture system was established in Transwell chamber. Cell activity of osteoblasts and osteoclasts were detected by CCK-8 experimenting,Alizarin Red staining,TRAP staining. The expression of OPG,ALP,RANKL,TGF-b1 gene and RANKL protein in osteoblast MC3T3-E1 were detected by PCR,Western-Blot methods. Also,the expression of RANK,NF-κB in gene and protein level in osteoclast were measured through the same method respectively. Results： The co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell were established in Transwell chamber. Co-culture system affected cell division activities of osteoblasts and osteoclasts. Differentiation of osteoblasts were increased,while differentiation of osteoclast division were slight decreased under microscope observation. OPG （0.65±0.08） and ALP （0.16±0.01） gene expression of co-culture system were less than single culture OPG（1.00±0.08） and ALP （1.01±0.16）; TGF-b1（4.42±0.21） and RANKL（4.12±1.04） of osteoblasts in co-culture system were higher than TGF-b1（1.00±0.10） and RANKL（1.00±0.09） under single culture. However,gene expression of RANK（0.63±0.06） and NF-κB（0.64±0.08） in co-culture system were decreased than RANK（1.00±0.08） and NF-κB（1.00±0.09）,in single culture,and had significant differences. Similarly,protein expression of OPG（0.43±0.05） and NF-κB（0.59±0.05） of co-culture system were less than OPG（0.84±0.06） and NF-κB（1.13±0.03） of single culture. While RANKL protein expression （0.54±0.03）of co-culture system was more than single culture RANKL（0.31±0.03）,and had statistically differences,which was in agreement of the trend of gen

关 键 词：成骨细胞 破骨细胞 基因表达 

分 类 号：R68[医药卫生—骨科学]