LPS和NNK对大鼠骨髓间充质干细胞增殖及NF-κB、P-P38、CUGBP1蛋白表达的影响

EFFECTS OF LPS AND NNK ON PROLIFERATION OF BONE MARROW MESENCHYMAL STEM CELLS AND THE EXPRESSION LEVELS OF NF-κB,P-P38,AND CUGBP1 IN RATS

目的观察炎性刺激因子脂多糖(LPS)、4-(甲基亚硝氨基)-1-(3-吡啶基)-1-丁酮(NNK)及LPS联合NNK对体外培养大鼠骨髓间充质干细胞(BMSCs)增殖及核因子κB(NF-κB)、P-P38、CUGBP1蛋白表达的影响。方法应用全骨髓贴壁法体外培养纯化BMSCs。采用MTT法检测不同浓度的炎性因子对BMSCs增殖的影响,选择最佳刺激浓度。应用免疫细胞化学法检测炎性刺激对BMSCs中NF-κB、CUGBP1蛋白表达的影响,采用Western blot法检测炎性刺激对BMSCs中NF-κB、P-P38、CUGBP1蛋白表达的影响。结果不同浓度的炎性因子均能促进大鼠BMSCs增殖,其中以12.5 mg/L LPS、10 mg/L NNK、12.5 mg/L LPS联合10 mg/L NNK对大鼠BMSCs增殖的促进作用最为明显。免疫细胞化学法检测显示,炎性刺激后BMSCs中NF-κB、CUGBP1蛋白的表达量显著增加,差异有显著性(F=165.1~481.8,P<0.01)。Western blot法检测显示,炎性刺激后NF-κB、P-P38、CUGBP1蛋白的表达量增加,差异有统计学意义(F=38.1~1 531.0,P<0.01)。结论炎性因子可能通过激活P38MAPK和NF-κB信号途径促进大鼠BMSCs的增殖。长时间的炎性因子刺激,促进了NF-κB、P-P38、CUGBP1蛋白在细胞核中的表达。

Objective To observe the effects of lipopolysaccharide （LPS）, 4-（methylnitrosamino）-l-（3-pyridyl）-l-buta- none （NNK）, and a combination of LPS and NNK on the proliferation of bone marrow mesenchymal stem cells （BMSCs） and ex- pression levels of NF-κB,P-P38 and CUGBP1 in rats. Methods Whole bone marrow adherent culture was used to culture and purify BMSCs. The effect of different concentrations of inflammatory factors on the proliferation of BMSCs was evaluated by MTT assay, based on which the stimulating concentration was optimized. The expression levels of NF-κB, P P38, and CUGBP1 in BMSCs were measured by immunocytochemistry and Western blot. Results Different concentrations of inflammatory factors could all promote the proliferation of rat BMSCs, and 12.5 mg/L LPS, 10 mg/L NNK, and 12.5 mg/L PLS in combination with 10 mg/L NNK had the most significant promotional effects on the proliferation of rat BMSCs. Immunoeytochemistry revealed that after inflammatory stimulation, NF-κB and CUGBP1 protein levels in BMSCs were significantly increased （F = 165.1--481.g, P〈0.01）. Western blot showed that the expression levels of NF-κB, P-P38, and CUGBP1 in BMSCs were increased after inflammatory stimulation （F=38.1--1 531.0,P〈0.01）. Conclusion LPS, NNK, and LPS in combination with NNK may promote the pro- liferation of BMSCs by activating NF-κB and P38MAPK pathways. Long term inflammatory factor stimulation promotes the ex pression of NF-κB, P-P38, and CUGBP1 in cell nucleus.