| Transwell小室内建立小鼠成骨-破骨细胞共培养体系 |
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Received:October 25, 2017
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| 作者 | Author | 单位 | Unit | E-Mail |
| 莫国业 |
MO Guo-ye |
广州中医药大学, 广东 广州 510405 |
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| 张顺聪 |
ZHANG Shun-cong |
广州中医药大学第一附属医院脊柱骨科, 广东 广州 510405 |
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| 李永贤 |
LI Yong-xian |
广州中医药大学, 广东 广州 510405 |
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| 郭惠智 |
GUO Hui-zhi |
广州中医药大学, 广东 广州 510405 |
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| 郭丹青 |
GUO Dan-qing |
广州中医药大学第一附属医院脊柱骨科, 广东 广州 510405 |
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| 李大星 |
LI Da-xing |
广州中医药大学, 广东 广州 510405 |
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| 唐永超 |
TANG Yong-chao |
广州中医药大学第一附属医院脊柱骨科, 广东 广州 510405 |
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| 莫凌 |
MO Ling |
广州中医药大学第三附属医院骨科, 广东 广州 510100 |
Department of Orthopaedics, the Third Affiliated Hospital of Guangzhou University of China Medicine, Guangzhou 510100, Guangdong, China |
spine2016@sina.com |
| 罗培杰 |
LUO Pei-jie |
广州中医药大学, 广东 广州 510405 |
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| 马延怀 |
MA Yan-huai |
广州中医药大学, 广东 广州 510405 |
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| 期刊信息：《中国骨伤》2018年31卷，第3期，第241-247页 |
| DOI：10.3969/j.issn.1003-0034.2018.03.010 |
| 基金项目: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） |
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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。 |
| [关键词]:成骨细胞 破骨细胞 基因表达 |
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| Establish mouse osteoblast -osteoclast cell co-culture system in a Transwell chamber |
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Abstract:
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 gene expression change.
Conclusion: Co-culture system of mouse MC3T3-E1 cells and RAW264.7 cell was viable in Transwell chamber,and the activity of osteoblasts is higher than osteoclasts in co-culture system. |
| KEYWORDS:Osteoblast Osteoclast Gene expression |
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| 中文格式: | 莫国业,张顺聪,李永贤,郭惠智,郭丹青,李大星,唐永超,莫凌,罗培杰,马延怀.Transwell小室内建立小鼠成骨-破骨细胞共培养体系[J].中国骨伤,2018,31(3):241~247 |
| 英文格式: | MO Guo-ye,ZHANG Shun-cong,LI Yong-xian,GUO Hui-zhi,GUO Dan-qing,LI Da-xing,TANG Yong-chao,MO Ling,LUO Pei-jie,MA Yan-huai.Establish mouse osteoblast -osteoclast cell co-culture system in a Transwell chamber[J].zhongguo gu shang / China J Orthop Trauma ,2018,31(3):241~247 |
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