摘要
目的探讨脂多糖(LPS)对骨髓间充质干细胞(MSC)Toll 样受体4(TLR-4)基因的表达及其功能的影响。方法采用贴壁培养和密度梯度离心法从大鼠骨髓分离 MSC,通过细胞形态、成骨分化潜能及流式细胞术检测表型以鉴定其纯度;半定量 RT-PCR 和流式细胞术分别检测 MSC 在不同浓度(1、10、100 μg/ml)LPS 存在条件下培养24 h 的 TLR-4 mRNA 相对表达量和共刺激分子(CD80、CD86、MHC-Ⅱ)的表达水平;ELISA 法定量检测 TNF-α的分泌水平。结果骨髓 MSC 低表达 TLR-4mRNA(相对表达量0.61±0.10),同时表达 CD80[(9.56±0.69)%]、CD86[(22.03±2.03)%]、MHC-Ⅱ[(2.51±0.97)%],少量分泌 TNF-α[(4.97±2.98)pg/m1]。MSC 经 LPS 处理后,其 TLR-4mRNA、共刺激分子表达和 TNF-α分泌水平均升高,其中10 μg/ml LPS 培养组升高显著,TLR-4 mRNA相对表达水平为1.55±0.02;CD80、CD86、MHC-Ⅱ阳性细胞率分别为(41.70±2.92)%、(59.72±2.00)%、(24.56±2.19)%;TNF-α分泌水平为(213.12±69.08)pg/ml,与对照组比较,P 值均<0.01。100 μg/ml LPS 处理组与10 μg/ml LPS 处理组相比,各项检测指标均降低,但 MHC-Ⅱ和 TNF-α的降低水平无统计学意义(P>0.05)。结论骨髓 MSC 低表达 TLR-4,体外 LPS 可促进骨髓 MSCTLR-4的表达,且与浓度相关。伴随 TLR-4表达水平的升高,CD80、CD86、MHC-Ⅱ表达水平及 TNF-α水平同时升高。
Objective To investigate the effect of lipopolysaccharide （LPS） on the expression and activity of Toll-like receptor 4（TLR-4） in mesenchymal stem cells（MSC）. Methods MSCs were harvested from adult rats bone marrow cells by density gradient centrifugation and adhesive culture. The purity of MSC were identified with the cell morphology and osteogenic capacity. The phenotypes were assayed by flow cytometry. Cultured MSCs were treated by LPS with various concentration （ 1 μg/ml, 10 μg/ml or 100 μg/ml） for 24 hours. The relative expression levels of TLR-4 mRNA were detected by semiquantitative RT-PCR, and costimulatory molecules （CD80, CD86 and MHC-Ⅱ ） expressed on MSC were analyzed by flow cytometry. The levels of TNF-α in supernatants were determined by double-antibody sandwich ELISA. Results The expression levels of CD80, CD86, MHC-Ⅱ, TLR-4 mRNA and TNF-α in MSC were （9. 56± 0. 69 ）%, （22.03 ±2.03）%, （2.51 ±0.97）%, relative magnitude （0.61 ±0. 10）, （4.97 ±2.98）pg/ml, respectively. After incubation with LPS, MSC expressed higher levels of TLR-4 mRNA and costimulatory molecules, and the levels of TNF-α were higher than that in untreated group. Among the various concentration of LPS, 10 μg/ml emerged as the most effective in increasing the levels of TLR-4 mRNA （ relative magnitude 1.55 ± 0.02）, costimulatory molecules [ CD80 （41.70 ± 2.92 ） % , CD86 （ 59.72 ± 2.00） %, MHC- Ⅱ （ 24.56 ± 2.19） % ] and TNF-α[ （213.12 ± 69.08 ） pg/ml] （ P 〈 0.01 ）. The levels of TLR-4 mRNA, costimulatory molecules and TNF-α began to decrease when MSC exposed to 100 μg/ml LPS（P 〈 0.05）. Except for the levels of TNF-α [ （ 158.05 ± 28.05 ） pg/ml ] and MHC- Ⅱ[ （5.62 ± 2.31 ） % ] （ P 〉 0.05 ）, the levels of CD80, CD86, MHC- Ⅱ and TLR-4 mRNA were significantly lower than the 10 μg/ml treatment group （P 〈 0.01 ）. Conclusion MSCs are able to express TLR-4 mRNA. LPS could activate the expression of TLR-4 in MSC obviously, bu
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2007年第12期828-831,共4页
Chinese Journal of Hematology
关键词
脂多糖类
间充质干细胞
受体
Toll样
基因表达
Lipopolysaccharides
Mesenchymal stem cells
Receptor,Toll-like
Gene expression
