Establish mouse osteoblast -osteoclast cell co-culture system in a Transwell chamber / 中国骨伤
China Journal of Orthopaedics and Traumatology
;
(12): 241-247, 2018.
Article
in Chinese
| WPRIM
| ID: wpr-690006
ABSTRACT
<p><b>OBJECTIVE</b>To establish osteoblast-osteoclast cell co-culture system in a Transwell chamber, and detect cell viability of osteoblasts and osteoclasts in system.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Osteoblasts
/
Osteoclasts
/
Cell Differentiation
/
NF-kappa B
/
3T3 Cells
/
Coculture Techniques
/
Cell Biology
/
Transforming Growth Factor beta1
/
RANK Ligand
/
Receptor Activator of Nuclear Factor-kappa B
Limits:
Animals
Language:
Chinese
Journal:
China Journal of Orthopaedics and Traumatology
Year:
2018
Type:
Article
Similar
MEDLINE
...
LILACS
LIS