GW4869 inhibits the release of exosomes from bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.0407

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) and BMSCs-derived exosomes have similar functions, but the regulatory mechanism underlying the release of exosomes is still unclear.
OBJECTIVE: To investigate the role of GW4869, an inhibition of neutral sphingomyelinase 2, in the release of exosomes in BMSCs and the influence of GW4869 on BMSCs proliferation.
METHODS: Rat BMSCs were divided into three groups: normal control group, 24-hour GW4869 treatment group and withdrawal of GW4869 for 24 hours group (24-hour GW4869 treatment followed by 24-hour successive culture with drug withdrawal). Cultured cells were collected to extract exosomes by ultracentrifugation. Western blot was used to detect exosome-associated proteins CD63 and tumor susceptibility gene 101 (TSG101). The concentration and size distribution of exosomes were measured using nanoparticle tracking analysis. BCA was used to test the level of total proteins in exosomes. Live cell imaging system was used to observe the influence of GW4869 on BMSCs proliferation.
RESULTS AND CONCLUSION: (1) Western blot results showed that exosomes expressed marker proteins such as CD63, TSG101. (2) Findings from the nanoparticle tracking analysis confirmed that the size of released exosomes was about 114 nm. (3) Significantly reduced release of exosomes was found in the two treatment groups compared with the normal control group (P < 0.01), but there was no significant difference between 24-hour GW4869 treatment group and withdrawal of GW4869 for 24 hours group (P > 0.05). (4) No significant difference in the proliferation of BMSCs was found among the three groups (P > 0.05). To conclude, 24-hour W4869 can inhibit the release of exosomes by BMSCs and this inhibitory effect is still sustained within 24 hours after drug withdrawal. However, GW4869 has no influence on the proliferation of BMSCs.

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: Bone Marrow, Mesenchymal Stem Cells, Exosomes, Tissue Engineering

CLC Number:

R394.2

Sun Shu-li, Xiao Pei-xin, Ding Hui, Wang Jing, Liu Zi-quan, Liu Jin-yang, Wang Xue, Shi Sha, Lv Qi, Fan Hao-jun. GW4869 inhibits the release of exosomes from bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(1): 26-31.

Figures/Tables 1

2.1 骨髓间充质干细胞来源外泌体阳性表达外泌体表面标志蛋白CD63、TSG101 外泌体分离及鉴定目前尚缺乏标准化流程，对前期文献总结发现大多数研究者会采用Western blot、电镜、NTA及BCA等多种方法中的2个或更多相结合的方法鉴定[11]。CD63属于外泌体表面四跨膜蛋白超家族蛋白，TSG101为物质选择和内部出芽过程所必须的异质蛋白复合物之一，二者均为目前鉴定外泌体最常用的蛋白分子。Western blot 结果显示：超速离心提取物阳性表达CD63和TSG101，骨髓间充质干细胞裂解物为阳性对照(图1)。 2.2 外泌体的粒径分布及浓度 Nanosight检测外泌体的粒径分布范围以及微粒浓度结果见图2。悬液中的微粒多峰分布，微粒大小为(120.9±42.9) nm，大部分粒子都处于外泌体直径的范围(30-150 nm)，含量最多的粒子直径约为114 nm，浓度为1.8×1011微粒/L。Western blot结果示超速离心获得的沉淀阳性表达外泌体表面标志蛋白CD63和TSG101，纳米示综分析结果证实大部分微粒子都处于外泌体直径的范围(30-150 nm)，因此基本可以认为超速离心获得的沉淀为骨髓间充质干细胞释放的外泌体。 2.3 GW4869对骨髓间充质干细胞外泌体释放的影响用BCA法测定各处理组骨髓间充质干细胞条件培养基提取外泌体蛋白水平，见图3。GW4869处理组和GW4869撤药24 h组外泌体蛋白水平均显著低于正常对照组(F=108.7，P < 0.01)，而GW4869预处理组和GW4869撤药24 h组差异无显著性意义(t=2.75，P > 0.05)。 2.4 GW4869对骨髓间充质干细胞增殖的影响 JULI-stage活细胞成像系统对各处理组细胞每隔2 h采集一次图像，连续116 h的数据分析结果见图4。与正常对照组相比，GW4869处理组和溶剂二甲基亚砜对细胞增殖的影响差异无显著性意义(P > 0.05)。"

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