Sinomenine effects on differentiation and maturation of rat bone marrow-derived dendritic cells

doi:10.3969/j.issn.2095-4344.2017.21.019

Abstract

Abstract:

BACKGROUND: It may be an important approach to avoiding organ transplant rejection by utilizing immature dendritic cells to induce donor-specific immunologic tolerance.

OBJECTIVE: To study the effect of sinomenine on the differentiation and maturation of rat bone marrow-derived dendritic cells in vitro.

METHODS: Bone marrow-derived dendritic cells were isolated from the rat femur and tibia, and immature dendritic cells were induced by granulocyte-macrophage colony stimulating factor and interleukin-4. On day 7, lipopolysaccharide was added and the cells were cultured to generate mature dendritic cells. Cells were divided into control group and low-, middle- and high-dose sinomenine treatment groups (SNL, SNM, SNH groups). Forty hours later, dendritic cells were harvested, and cell morphology was observed by inverted phase contrast microscope. The expression of CD80 and RT1B was detected by flow cytometry. ELISA was used to detect the expression of interleukin-12. The mixed lymphocyte reaction was used to detect the ability of dendritic cells to stimulate the activation of allogeneic T lymphocytes.
RESULTS AND CONCLUSION: (1) Under the inverted microscope, the morphology of mature dendritic cells was observed in the control group; in the SNL group most dendritic cells were visible; in the SNM group, there were partially suspended cells with poor maturation; and in the SNH group, most of the cells were not mature. (2) The expression of CD80 in the control group was significantly lower than that in the SNL, SNM and SNH groups (P < 0.05), and the expression of RT1B was significantly reduced in the SNM and SNH groups than the control group. (3) Compared with the control group, the level of IL-12p70 in the cell supernatant was significantly decreased in the SNM and SNH groups (P < 0.01). (4) The ability of dendritic cells to stimulate T lymphocyte proliferation in the SNM and SNH groups was significantly decreased compared with the control group (P < 0.05). To conclude, sinomenine can inhibit the maturation of dendritic 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: Dendritic Cells, Immune Tolerance, Sinomenium, Tissue Engineering

CLC Number:

R394.2

Huang Jiang-bo, Luo Zhi-gang, Gao Hong-qiang, Liu Li, He Qun-jun, Li Jian-jun, Yan Cai-hong, Long Xiang-yang. Sinomenine effects on differentiation and maturation of rat bone marrow-derived dendritic cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3394-3399.

Figures/Tables 1

2.1 一般情况及纯化结果培养至第7天收集所有悬浮细胞计数后发现每只大鼠平均能收集(2.0-3.0)×107个未成熟树突状细胞，完全可以满足实验要求。流式细胞仪检测原代培养未成熟树突状细胞的OX62阳性表达率达到82.8%，经免疫磁珠提纯后OX62阳性表达率达到91.9%，纯度明显提高。 2.2 青藤碱对各组树突状细胞形态的影响大鼠骨髓原代细胞经粒-巨噬细胞集落刺激因子、白细胞介素4诱导培养到第3天时可见细胞形态呈小圆形，少部分细胞呈附壁生长，连续诱导至7 d时的未成熟树突状细胞，在光镜下可观察到呈葡萄串样的细胞集落或悬浮生长，且细胞集落轻轻吹打或摇晃培养板即可脱落，细胞表面颗粒感明显，并开始出现较明显的毛刺样突起。用脂多糖+青藤碱刺激48 h后，光镜下观察各组细胞形态见图1。 2.3 青藤碱对各组树突状细胞表面分子表达的影响与对照组相比，青藤碱低、中、高剂量组CD80表达显著降低(P < 0.05)，差异有显著性意义；与对照组相比，青藤碱中、高剂量组RT1B表达显著降低(P < 0.05)，差异有显著性意义，而低剂量组差异无显著性意义(P > 0.05，见表1)。 2.4 青藤碱对各组树突状细胞分泌白细胞介素12的影响青藤碱干预后，4组树突状细胞培养上清液中IL-12p70水平差异有显著性意义(F=69.21，P < 0.001)；与对照组相比，青藤碱低剂量干预后的树突状细胞培养上清中IL-12p70水平差异无显著性意义(P > 0.05)；青藤碱中、高剂量干预后的树突状细胞培养上清中IL-12p70水平有所降低(P < 0.05)，差异有显著性意义，见表2。 2.5 青藤碱对各组树突状细胞刺激T淋巴细胞增殖能力的影响青藤碱干预后，4组差异均有显著性意义；与对照组相比，在不同的混合比例下，青藤碱中、高剂量组吸光度值均有显著降低(P < 0.01)，差异有显著性意义，而低剂量组吸光度值的改变差异无显著性意义(P > 0.05，见表3)。"

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