Transwell小室内建立小鼠成骨-破骨细胞共培养体系
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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 MC3 T3-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 MC3 T3-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 MC3 T3-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 gene expression change. Conclusion:Co culture system of mouse MC3 T3-E1 cells and RAW264.7 cell was viable in Transwell chamber,and the activity of osteoblasts is higher than osteoclasts in co culture system.
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 MC3 T3-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 MC3 T3-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 MC3 T3-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 gene expression change. Conclusion:Co culture system of mouse MC3 T3-E1 cells and RAW264.7 cell was viable in Transwell chamber,and the activity of osteoblasts is higher than osteoclasts in co culture system.
引文
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[2]Tamura T,Udagawa N,Takahashi N,et al.Soluble interleukin-6receptor triggers osteoclast formation by interleukin 6[J].Proc Natl Acad Sci U S A,1993,90(24):11924-11928.
[3]Baek SW,Kim C,Chang H.The relationship between the spinopelvic balance and the incidence of adjacent vertebral fractures fol鄄lowing percutaneous vertebroplasty[J].Osteoporos Int,2015,26(5):1507-1513.
[4]Morita Y,Yamamoto S,Ju Y.Development of a new co culture sys鄄tem,the"separable close co culture system,"to enhance stem cell to chondrocyte differentiation[J].Biotechnol Lett,2015,37(9):1911-1918.
[5]Dougall WC,Glaccum M,Charrier K,et al.RANK is essential for osteoclast and lymph node development[J].Genes Dev,1999,13(18):2412-2424.
[6]Zhang YM,Zhang ZM,Guan QL,et al.Co culture with lung cancer A549 cells promotes the proliferation and migration of mesenchymal stem cells derived from bone marrow[J].Exp Ther Med,2017,14(4):2983-2991.
[7]Pandurangan M,Hwang I.Application of cell co culture system to study fat and muscle cells[J].Appl Microbiol Biotechnol,2014,98(17):7359-7364.
[8]Boyce BF,Rosenberg E,de Papp AE,et al.The osteoclast,bone re鄄modelling and treatment of metabolic bone disease[J].Eur J Clin In鄄vest,2012,42(12):1332-1341.
[9]Indran IR,Liang RL,Min TE,Yong EL.Preclinical studies and clin鄄ical evaluation of compounds from the genus Epimedium for osteo鄄porosis and bone health[J].Pharmacol Ther,2016,162:188-205.
[10]Tatsumi S,Ishii K,Amizuka N,et al.Targeted ablation of osteo鄄cytes induces osteoporosis with defective mechanotransduction[J].Cell Metab,2007,5(6):464-475.
[11]郭海玲,赵咏芳,王翔,等.淫羊藿苷对人成骨细胞增殖及OPG蛋白表达的实验研究[J].中国骨伤,2011,24(7):585-588.GUO HL,ZHAO YF,WANG X,et al.Experimental study on the mechanism of icariin improving human osteoblasts proliferation and the expression of OPG protein[J].Zhongguo Gu Shang/China J Orthop Trauma,2011,24(7):585-588.Chinese with abstract in English.