[Recombinant human tumor necrosis factor-alpha promotes human adipose-derived stromal cells transforming into osteoblast in vitro].
OBJECTIVE
To investigate the effect of recombinant human tumor necrosis factor alpha (rhTNF-α) on the osteogenesis potential of the osteo-induced  human adipose-derived stromal cells (hASCs) in vitro.


METHODS
hASCs at passage 4 were divided into four groups according to culturing conditions:  basal medium [BM, DMEM + 10% FBS + antibiotics], BM with 10 μg/L rhTNF-α, osteogenic medium (OM, BM + dexamethasone + L-ascorbate + β-glycerophosphate) and OM with 10 μg/L rhTNF-α. On days 3, 7, 14 and 21, alkaline phosphatase (ALP) activities were examined. On days 14 and 21, the  staining and quantitation of calcium deposition were performed. For the cells under osteogenic induction, osteoblast-related genes, such as core-binding factor α1 (Cbfa1), Osterix (Osx) and osteocalcin (OC) were analyzed with reverse transcription PCR on days 3, 7, 14, and 21, and real time PCR was  performed to confirm the effect of rhTNF-α on genes expression on day 3 .


RESULTS
rhTNF-α promoted ALP activities of induced hASCs on day 14  (3.527 ± 0.415 vs. 2.345 ± 0.354,P<0.01) and on day 21 (3.106 ± 0.105 vs. 2.442 ± 0.163,P<0.01) and promoted calcium deposition of induced hASCs on  day 14 (2.896 ± 0.173 vs. 0.679 ± 0.173,P<0.01) and on day 21 (2.231 ± 0.233 vs. 1.729 ± 0.229, P<0.01). RT-PCR and Real-time PCR assays showed that  rhTNF-α augmented the expression of Cbfa1, Osx and OC of these cells.


CONCLUSIONS
The findings indicate that 10 μg/L rhTNF-α can promote the  osteogenic potential of osteogenetically induced hASCs in vitro.
Department of Stomatology, Beijing Shijitan Hospital, Capital Medical University, Beijing, China.
Ri-yue
RY
Yu
Bai-jin
BJ
Zeng
Yun-song
YS
Liu
Yong-sheng
YS
Zhou
D017667:Adipocytes / Q000166:cytology*; D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D010006:Osteoblasts / Q000166:cytology*; D010012:Osteogenesis / Q000187:drug effects; D011994:Recombinant Proteins / Q000494:pharmacology*; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000494:pharmacology*
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D000469:Alkaline Phosphatase