牙龈卟啉单胞菌脂多糖对大鼠骨髓间充质干细胞生物学行为影响的研究

Biological behavior of rat bone marrow mesenchymal stem cells affected by Porphyromonas gingivalis lipopolysaccharide

目的研究不同浓度牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis lipopolysaccharide,P.g LPS)对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)增殖、炎症因子分泌能力和成骨分化的影响。方法使用不同质量浓度P.g LPS(1.0、2.5、5.0、10.0μg/mL)刺激大鼠BMMSCs,记为P.g LPS组;同时设立对照组,即不使用P.g LPS培养的大鼠BMMSCs。利用CCK-8法检测细胞增殖活性;实时荧光定量PCR(qRT-PCR)和酶联免疫吸附实验(ELISA)分别检测细胞内和上清液中肿瘤坏死因子(tumor necrosis factor-α,TNF-α)和白细胞介素10(interleukin-10,IL-10)的表达;qRT-PCR和碱性磷酸酶(alkaline phosphatase,ALP)染色分别检测成骨诱导早期的成骨分化情况。结果P.g LPS刺激1、3、5 d后,各组细胞增殖活性差异不明显(均P>0.05);P.g LPS刺激3 d后,各质量浓度P.g LPS组相较于对照组中TNF-α及IL-10的mRNA表达水平均升高;成骨诱导4、7 d后,各质量浓度P.g LPS组相较于对照组中成骨分化相关因子ALP、Runt相关转录因子2(Runt-related transcription factor 2,Runx 2)和Ⅰ型胶原(collagen typeⅠ,COL-Ⅰ)的表达水平均受到抑制(均P<0.05);成骨诱导7 d后,5.0、10.0μg/mL P.g LPS组ALP活性明显低于对照组,1.0、2.5μg/mL P.g LPS组ALP活性相比于对照组差异不明显。结论P.g LPS对BMMSCs的增殖无明显影响,但可增加BMMSCs中炎症因子的表达水平,并影响其成骨分化。

Objective To detect the effects of different concentrations of Porphyromonas gingivalis lipopolysaccharide(P.g LPS) on the proliferation,inflammation factors and osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMMSCs).Methods Bone marrow mesenchymal stem cells were stimulated with different concentrations of P.g LPS(1.0,2.5,5.0,10.0 μg/mL),which were used as the P.g LPS groups,the rat BMMSCs without P.g LPS stimulation were included as the control group.The cell proliferation of BMMSCs was analyzed by CCK-8 kit assay,the expression levels of tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)were analyzed by ELISA and qRT-PCR.Osteogenic differentiation at the early stage of osteogenesis induction was detected by qRT-PCR and alkaline phosphatase(ALP)staining.Results On day 1,3,5 of stimulation with P.g LPS,no significant difference was shown in cell proliferation of BMMSCs in all the P.g LPS groups(P> 0.05).TNF-α and IL-10 expression in all the P.g LPS groups was markedly increased under the treatment of 3 days’ P.g LPS compared with control group.The expression of osteogenic differentiation-related genes ALP,Runt-related transcription factor 2(Runx 2)and collagen type Ⅰ(COL-I)were inhibited(P <0.05) in BMMSCs treated with P.g LPS for4 and 7 days.At 7 days after osteogenesis induction,the ALP activity in the 5.0 μg/mL and 10.0 μg/mL P.g LPS groups was significantly decreased,while in 1.0 μg/mL and 2.5 μg/mL P.g LPS groups ALP activity did not change significantly,compared with the control group.Conclusion P.g LPS has no significant effect on the proliferation of BMMSCs,but can increase the expression of inflammatory factors and affect osteogenic differentiation.