[脂多糖对间充质干细胞中Toll样受体4表达及活性的影响]

[The effect of lipopolysaccharide on the expression and activity of Toll-like receptor 4 in mesenchymal stem cells].

作者信息

Shi Liang, Liu Xing-mei, Hu Xiao-yan, Wang Ji-shi, Fang Qin

机构信息

Department of Hematology, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2007 Dec;28(12):828-31.

PMID:18476595

Abstract

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 microg/ml, 10 microg/ml or 100 microg/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-alpha in supernatants were determined by double-antibody sandwich ELISA.

RESULTS

The expression levels of CD80, CD86, MHC-II, TLR-4 mRNA and TNF-alpha 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-alpha were higher than that in untreated group. Among the various concentration of LPS, 10 microg/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-alpha [(213.12 +/- 69.08) pg/ml] (P < 0.01). The levels of TLR-4 mRNA, costimulatory molecules and TNF-alpha began to decrease when MSC exposed to 100 microg/ml LPS (P < 0.05). Except for the levels of TNF-alpha [(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 microg/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, but the activity is dependent on the specific concentration.

摘要

目的

探讨脂多糖（LPS）对间充质干细胞（MSC）中Toll样受体4（TLR-4）表达及活性的影响。

方法

采用密度梯度离心和贴壁培养法从成年大鼠骨髓细胞中分离培养MSC。通过细胞形态和骨生成能力鉴定MSC的纯度，用流式细胞术检测其表型。将培养的MSC用不同浓度（1μg/ml、10μg/ml或100μg/ml）的LPS处理24小时。采用半定量RT-PCR检测TLR-4 mRNA的相对表达水平，用流式细胞术分析MSC上共刺激分子（CD80、CD86和MHC-II）的表达情况。用双抗体夹心ELISA法测定上清液中TNF-α的水平。

结果

MSC中CD80、CD86、MHC-II、TLR-4 mRNA和TNF-α的表达水平分别为（9.56±0.69）%、（22.03±2.03）%、（2.51±0.97）%、相对量（0.61±0.10）、（4.97±2.98）pg/ml。与LPS孵育后，MSC中TLR-4 mRNA和共刺激分子的表达水平升高，TNF-α水平高于未处理组。在不同浓度的LPS中，10μg/ml在增加TLR-4 mRNA水平（相对量1.55±0.02）、共刺激分子[CD80（41.70±2.92）%、CD86（59.72±2.00）%、MHC-II（24.56±2.19）%]和TNF-α水平[（213.12±69.08）pg/ml]方面最为有效（P<0.01）。当MSC暴露于100μg/ml LPS时，TLR-4 mRNA、共刺激分子和TNF-α水平开始下降（P<0.05）。除TNF-α水平[（158.05±28.05）pg/ml]和MHC-II水平[（5.62±2.31）%]（P>0.05）外，CD80、CD86、MHC-II和TLR-4 mRNA水平均显著低于10μg/ml处理组（P<0.01）。