[Regulation of differentiation and proliferation of epiphysis stem cells by Notch1 signaling system].
OBJECTIVE
To investigate the regulation of differentiation and proliferation of epiphysis stem cells by Notch1 signaling system.


METHODS
Costocostal cartilage was taken from a SD rat. Epiphysis stem cells were isolated and cultured. Recombinant human nuclear factor-kappaB (rhNF-kappaB), an activator of the Notch signaling system, and gamma-secretase inhibitor (MW167), an inhibitor of the Notch signaling system, were added into the culture medium respectively. The cells cultured in the medium added with phosphate-buffered saline were used as control group. Then the cultured cells were collected. The expression of the homologous Notch receptors and homologous Notch ligands was detected by RT-PCR. Immunohistochemistry was used to detect the levels of collagen II, collagen X, and proliferating cell nuclear antigen (PCNA). MTT method was used to calculate the growth curve. The cell phase was examined by flow cytometry. The level of alkaline phosphatase (AP) was measured. Western blotting was used to detect the protein expression of collagen II, collagen X, and stathmin, a signaling protein of proliferation.


RESULTS
Only 2 the expression of the receptor Notch1 and the ligand Jagged1 was found. The expression of PCNA was stronger in the rhNF-kappaB group than in the other 2 groups. rhNF-kappaB remarkably promoted the expression of collagen II and inhibited the expression of collagen X and MW167 remarkably promoted the expression of collagen X and did not remarkably influence the expression of collagen II. MTT method showed that rhNF-kappaB significantly promote the proliferation of the cells (P = 0.027), and MW167 did not significantly promote the cell proliferation (P > 0.05). The percentage of cells at S phase of the rhNF-kappaB group was 26.54%, significantly higher than those of the MW167 group and control group (8.22% and 6.15%). AP was significantly expressed in the MW167 group, and less expressed in the other groups. Western blotting showed a significantly increased expression of collagen X protein and decreased expression of collagen II protein and stathmin.


CONCLUSIONS
When the Notch signaling system is activated the epiphysis stem cells proliferate, and when the Notch signaling system is suppressed the epiphysis stem cells differentiate.
Zhang
Yi-bei
YB
Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huanzhong University of Science and Technology, Wuhan 430030, China.
Chen
An-min
AM
Guo
Feng-jing
FJ
Huang
Shi-long
SL
Xiong
Wen-hua
WH
Li
Qi
Q
D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D024043:Collagen Type II / Q000378:metabolism; D024121:Collagen Type X / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D007150:Immunohistochemistry; D010455:Peptides / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D051881:Receptor, Notch1 / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects
D024043:Collagen Type II; D024121:Collagen Type X; C432344:MW167; C497761:NOTCH1 protein, human; D010455:Peptides; D018809:Proliferating Cell Nuclear Antigen; D051881:Receptor, Notch1