摘要
目的研究激活的Notch1信号系统对体外培养的骨骺干细胞增殖与分化的调控作用。方法向体外培养的骨骺干细胞中分别加入Notch1激活剂rhNF-κB和抑制剂γ-secretaseinhibitorⅡ(MW167),空白对照加PBS缓冲液。用RT-PCR、免疫组化、MTT、流式细胞仪、碱性磷酸酶染色和Western印迹方法检测。结果骨骺干细胞中有Notch1和Jagged1表达;与对照组相比,rhNF-κB诱导组表达PCNA、胶原Ⅱ蛋白明显增多,促细胞增殖作用明显(P=0.027),并且进入分裂期(S期)的细胞增多(26.54%),而MW167诱导组碱性磷酸酶、CollagenⅩ蛋白表达明显增多,胶原Ⅱ蛋白及分化标志蛋白stathmin表达明显减少。结论当Notch信号系统被激活时,骨骺干细胞进行增殖;当Notch信号系统被抑制时,骨骺干细胞进行分化。
Objective To investigate the regulation of differentiation and proliferation of epiphysis stem cells by Notchl signaling system. Methods Costocostal cartilage was taken from a SD rat. Epiphysis stem cells were isolated and cultured. Recombinant human nuclear factor-KB （rhNF-KB）, an activator of the Notch signaling system, and 3,-secretase inhibitor （MWI67）, an inhibitor of the Notch signaling system, were added into the culture medium respectively. The cells cultured in the medium added with phosphatebuffered saline were used as control group. Then the cultured cells were collected. The expression of the homologous Notch receptors and homologous Notch ]igands was detected by RT-PCR Immunohistochemistry was used to detect the levels of collagen 11 , 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 11 , collagen X, and stathmin, a signaling protein of proliferation. Results Only 2 the expression of the receptor Notchl and the ligand Jaggedl was found. The expression of PCNA was stronger in the rhNF-kB group than in the other 2 groups, rhNF-kB remarkably promoted the expression of collagen 11 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 11. MTT method showed that rhNF-kB significantly promote the proliferation of the ceils （ 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-kB 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 11 protein and stathmin. Conclusion 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.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2005年第48期3430-3434,共5页
National Medical Journal of China
基金
国家自然科学基金资助项目(30371439)
