摘要
目的探讨低强度脉冲超声(LIPUS)对人退变髓核细胞合成细胞外基质的影响及机制。方法取人腰椎退变髓核组织,进行髓核细胞的分离、培养、鉴定,取P3代细胞转至藻酸钙凝珠培养。实验分组如下:LIPUS组:超声刺激1周,20 min/d;对照组:不予超声刺激同条件培养1周;LY294002组:超声刺激并加入LY294002共培养1周。应用ELISA法检测上清中髓核细胞分泌的aggrecan和Ⅱ型胶原(Col2α1);RT-PCR法检测细胞中aggrecan和Col2α1在mRNA水平的表达差异;Western blot法检测细胞中aggrecan、Col2α1、Akt及p-Akt的表达差异。结果予以LIPUS刺激后,上清中的aggrecan和Col2α1浓度较对照组升高(P<0.05),细胞内aggrecan和Col2α1在mRNA及蛋白水平的表达与对照组比较均有增高(P<0.05),Akt的表达无明显变化(P>0.05),p-Akt的表达增高(P<0.05),加入特异性抑制剂LY294002后,aggrecan和Col2α1的合成明显降低(P<0.05)。结论低强度脉冲超声能通过PI3K/Akt信号通路促进藻酸钙凝珠立体培养的人退变髓核细胞合成细胞外基质。
Objective To investigate the effect of low-intensity pulsed ultrasound （LIPUS） on the extracellular matrix synthesis of human degenerative nucleus pulposus cells and explore the underlying mechanism. Methods The nucleus pulposus cells acquired from human degenerative lumbar intervertebral discs were cultured by monolayer culture and identified. After three passages, the cells were cultured in calcium alginate beads. Experimental groups were stimulated by LIPUS （LI- PUS group） for 1 week （20 min/d） or treated with LY294002 simultaneously （LY294002 group）, and control group was trea- ted in the same way without LIPUS stimulation. The concentrations of aggrecan and Col2αl in culture supematant were de- tected by ELISA. The expression levels of aggrecan and Col2α1 mRNA were dertermined by RT-PCR. The expression levels of aggrecan, Col2αl, Akt and p-Akt proteins were examined by Western blotting. Results The concentrations of aggrecan and Col2αl in the LIPUS group group was significantly higher than those in the control group （ P 〈 0.05）. The expressions of aggrecan and Col2αl at both mRNA and protein levels and p-Akt protein significantly increased in the IPUS group as com- pared with the control group （ P 〈 0.05）, but there was no significant difference in the expression of Akt protein between the two groups （ P 〉 0.05）. The levels of aggrecan and Col2αl proteins were significantly lower in the LY294002 group than in the LIPUS group （ P 〈 0.05）. Conclusion The LIPUS can stimulate the extracellular matrix synthesis of degenerative human nucleus pulposus cells cultured in calcium alginate beads via activating PI3K/Akt pathway.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第1期34-38,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81171751)
关键词
低强度脉冲超声
人退变髓核细胞
PI3K/AKT通路
细胞外基质
low-intensity pulsed ultrasound
human degenerative nucleus pulposus cell
PI3K/Akt pathway
extracellular matrix
