Liver X receptor reverses the inhibitory effects of propofol on bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.17.001

Abstract

Abstract:

BACKGROUND: Studies have shown that propofol enables a reduction in the number of adult rat mesenchymal stem cells, while the cell differentiation is also significantly inhibited.
OBJECTIVE: To explore whether liver X receptors (LXRs) can reverse the inhibitory effects of propofol on bone marrow mesenchymal stem cells.
METHODS: Fifteen healthy C57/BL6 mice were randomized into three groups, 5 of which served as blank control group (intraperitoneally treated with normal saline), 5 as propofol treatment group (intraperitoneally treated with 60 mg/kg propofol), and 5 as propofol + LXRs agonist treatment group (intraperitoneally injected with 10 μL/g LXRs at the 1st day, and then injected with 60 mg/kg propofol at the 2nd day). The mice in the three groups were killed at 1-3 hours after treatment to isolate and culture bone marrow mesenchymal stem cells. Cell counting kit-8 and clone formation assay were used to evaluate the abilities of cell proliferation and self-renewal; induced differentiation experiments in vitro were used to evaluate the differentiation ability of cells into adipocytes, osteoblasts and chondrocytes; real-time quantitative PCR was used to detect the expression of differentiation related molecules and Notch signal.
RESULTS AND CONCLUSION: In the propofol-treated mice, cell viability and clone forming ability as well as adipogenic, osteogenic and chondrogenic differentiation of cells decreased significantly compared with the blank control group (P < 0.05), while LXR agonists could reverse these effects significantly (P < 0.05). Notch signal expressions showed no difference among three groups prior to induced differentiation. The expression levels differentiation related molecules downregulated significantly after propofol treatment (P < 0.05), but upregulated significantly after treatment with LXR agonists (P < 0.05). Notch signaling inhibitor treatment could significantly inhibit the multi-directional differentiation of bone marrow mesenchymal stem cells in the three groups. All these findings indicate that activated LXRs can reverse the inhibitory effects of propofol on bone marrow mesenchymal stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Propofol, Mesenchymal Stem Cells, Tissue Engineering

CLC Number:

R394.2

Zhang Yong-qiang, Liu Jun, Chen Sheng-yang, Liu Guo-ze, Tian Jian-min, Yue Xiu-qin. Liver X receptor reverses the inhibitory effects of propofol on bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(17): 2625-2630.

Figures/Tables 2

2.1 小鼠骨髓间充质干细胞的分离与鉴定原代培养首次换液后，贴壁细胞多为单个长梭形细胞，偶尔可见几个细胞的克隆，细胞在传代3次后，形态均一，成长梭形，融合时呈漩涡壮生长(图1A)。成脂诱导分化14 d后，油红O染色，细胞内已形成的脂滴染为红色(图1B)。成骨诱导分化20 d后，茜素红染色，细胞内已形成的骨性结节染为红色(图1C)。成软骨诱导分化20 d后，阿尔辛蓝染色，细胞分泌的多聚糖染为蓝色(图1D)。以上结果显示，已成功建立小鼠骨髓来源的体外间充质干细胞培养体系。 2.2 LXR激活修复丙泊酚对骨髓间充质干细胞增殖及自我更新的抑制作用相比于空白对照组，对照组1，3，5 d后骨髓间充质干细胞活力显著降低(P < 0.01)；相比对照组，实验组骨髓间充质干细胞活力明显提升(P < 0.01)，见图2A。相比空白对照组，对照组骨髓间充质干细胞克隆形成能力显著降低；相比于对照组，实验组骨髓间充质干细胞克隆形成能力明显提升，见图2B。以上研究显示，LXR激活可修复丙泊酚对骨髓间充质干细胞增殖及自我更新的抑制作用。 2.3 LXR激活修复丙泊酚对骨髓间充质干细胞分化的抑制作用油红O染色、茜素红及阿新蓝染色均显示，LXR激活可修复丙泊酚对骨髓间充质干细胞分化的抑制作用，见图3A。同时，实时定量PCR检测成骨分化相关分子(Osterox，Runx2)、成脂分化相关分子(Ap2，PPAP-γ)及成软骨分化相关分子(aggrecan，ALP)的表达，结果显示：与空白对照组相比，对照组所有分子表达水平均下调(P < 0.05)；与对照组相比，实验组所有分化相关分子表达均升高(P < 0.05)，见图3B。以上研究表明，LXRs激活可修复丙泊酚对骨髓间充质干细胞分化的抑制作用。 2.4 LXR通过Notch信号激活修复丙泊酚对骨髓间充质干细胞抑制作用结合前期研究，Notch信号在干细胞干性调控与分化过程中扮演的重要作用，以及LXRs通过Notch信号促进海马神经元细胞分化与发育的实验现象，推测是否LXRs与Notch信号间的关系对干细胞分化具有普遍性。同时也为进一步探究LXRs激活修复丙泊酚对骨髓间充质干细胞抑制作用的具体机制，分别检测3组间充质干细胞在成脂、成骨、成软骨分化前后Notch信号的表达情况。结果显示，诱导分化前，3组细胞内Notch信号的表达无明显差异，见图4A；诱导分化后，相比于对照组，实验组内细胞Notch信号在成脂分化后表达上调[(4.8±0.2)倍，P < 0.01]，在成骨分化后表达上调[(1.2±0.1)倍，P < 0.01]，在成软骨分化后表达上调[(2.6±0.9)倍，P < 0.01]，见图4B。随后给予3组细胞Notch信号抑制剂DAPT处理，结果显示与未经Notch信号抑制剂处理前比较，3组细胞的成脂、成骨、成软骨分化均受抑制，见图4C。以上研究表明，LXRs通过Notch信号激活可修复丙泊酚对骨髓间充质干细胞的抑制作用。"

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