Lipopolysaccharide can influence and regulate proliferation and migration of dental pulp stem cells

doi:10.3969/j.issn.2095-4344.1801

Abstract

Abstract:

BACKGROUND: Numerous studies have demonstrated that human dental pulp stem cells have highly self-renewing and multi-directional differentiation potentials.
OBJECTIVE: To study the effect of lipopolysaccharide on proliferation and migration of activated human dental pulp stem cells, and to explore the effect of lipopolysaccharide on Toll-like receptor 4 (TLR4), mineralization-related genes and mitogen-activated protein kinase (MAPK) signaling pathway.
METHODS: The microenvironment of bacteria invading the pulp in human deep-carious teeth was simulated by lipopolysaccharide. Human dental pulp stem cells were isolated and cultured by tissue explant enzymatic digestion. (1) Human dental pulp stem cells were stimulated by 0, 0.1, 1, 10 mg/L lipopolysaccharide for 1, 3, 5, and 7 days, and the cell proliferation ability was then detected by MTT. (2) Immunofluorescence was used to observe the expression of TLR4 in the cells cultured normally (control group) or intervened with 1 mg/L lipopolysaccharide. RT-qPCR was used to observe the expression of mineralization-related genes alkaline phosphatase, dentin sialophosphoprotein and osteopontin mRNAs in the cells. (3) Transwell test was used to detect the migration ability of human dental pulp stem cells after 24-hour intervention with 0.1, 1, and 10 mg/L lipopolysaccharide. (4) Western blot assay was used to detect the activated levels of p-p38 and p-ERK1/2 in the MAPK signaling pathway after 0, 15, 30, 60 and 120 minutes of 1 mg/L lipopolysaccharide stimulation. The study protocol was approved by the ethic committee of Cadre Sanatorium of Hainan with an approval No. 20180410.
RESULTS AND CONCLUSION: Compared with the blank control group, lipopolysaccharide stimulation decreased the cell proliferation ability, and increased the TLR4 expression as well as the mRNA expression of alkaline phosphatase, dentin sialophosphoprotein and osteopontin in the human dental pulp stem cells. The number of migrated cells was also increased after lipopolysaccharide stimulation. Moreover, the expression of p-p38 and p-ERK1/2 proteins yielded an increase as the lipopolysaccharide continued to work. To conclude, lipopolysaccharide has different effects on the proliferation and migration of human dental pulp stem cells. TLR4 and MAPK signaling pathway may be involved in the directional differentiation of human dental pulp stem cells regulated by lipopolysaccharide.

Key words: lipopolysaccharide, Human dental pulp stem cells, Toll-like receptor 4, p38, ERK1/2, dentin sialophosphoprotein mRNA, osteopontin mRNA, alkaline phosphatase mRNA

CLC Number:

Feng Ruxue, Cai Rengang, Xie Dandan. Lipopolysaccharide can influence and regulate proliferation and migration of dental pulp stem cells [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(29): 4688-4693.

Figures/Tables 2

2.1 牙髓干细胞原代培养结果倒置显微镜下可见，消化后牙髓组织块变得松散，接种3-7 d时，原代细胞从组织块附近游离出，见图1A。当细胞浓度达80%时，按1∶2传代培养，第3代后，细胞呈反射状或涡旋状排列，且形态稳定，见图1B。 2.2 牙髓干细胞的鉴定结果人牙髓干细胞表面标记物阴性表达CD34(0.1%)、CD45(0.31%)；阳性表达CD44(98.3%)、CD90(99.24%)和CD29(99.16%)，符合间充质干细胞表面标记物特性，见图2。 2.3 脂多糖抑制牙髓干细胞的增殖脂多糖刺激牙髓干细胞增殖检测结果显示，与对照组(0 mg/L)相比，脂多糖组细胞增殖能力均出现下降，且在第5，7天时差异有显著性意义(P < 0.05)，见表1。 2.4 脂多糖刺激牙髓干细胞中TLR4的表达细胞免疫荧光结果显示，与对照组相比，牙髓干细胞经脂多糖刺激后绿色荧光明显增强，说明TLR4是脂多糖的关键受体，在其刺激下被激活显色。而脂多糖阴性对照组仅见蓝染细胞核，见图3。 2.5 脂多糖刺激后牙髓干细胞中矿化相关基因的表达变化实时定量PCR结果显示，与对照组相比，7 d和14 d时脂多糖组牙本质涎磷蛋白mRNA表达水平明显增加(P < 0.05)，在21 d时脂多糖组牙本质涎磷蛋白mRNA表达水平比对照组明显下降(P < 0.05)；脂多糖组碱性磷酸酶mRNA表达水平在14 d和21 d时均比对照组增加(P < 0.05)；脂多糖组骨桥素mRNA表达水平在各时间点均明显高于对照组(P < 0.05)，见图4。 2.6 脂多糖促进牙髓干细胞的迁移 Transwell实验结果表明，0.1，1，10 mg/L脂多糖组迁移细胞数量分别为(16.004±1.046)，(20.100±0.350)，(25.330±1.24)/400倍视野均高于对照组[0 mg/L，(11.675±1.109)/400倍视野] (P < 0.05)，见图5。说明脂多糖可以促进牙髓干细胞的迁移。 2.7 脂多糖促进牙髓干细胞中MAPK信号通路的蛋白活化 Western blot检测结果显示，脂多糖能激活p38和ERK1/2，且随着时间增加，p-p38和p-ERK1/2蛋白相对表达水平逐渐增加，这说明脂多糖能使p38和ERK1/2蛋白活化，且p38和ERK1/2蛋白可能参与脂多糖调控的牙髓干细胞定向分化过程，见图6。"

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