Department of Burns and Cutaneous Surgery, Xijing Hospital, Air Force Medical University, No.169 Changle West Road, Xi'an, 710032, Shaanxi, China.
Department of Burns and Cutaneous Surgery, Xijing Hospital, Air Force Medical University, No.169 Changle West Road, Xi'an, 710032, Shaanxi, China.
Arch Biochem Biophys. 2020 Mar 15;681:108259. doi: 10.1016/j.abb.2020.108259. Epub 2020 Jan 9.
Wound healing remains a challenge in burns and trauma fields. Adipose derived stem cells exosomes (AD-exos) had been confirmed to have a positive effect on the wound healing and the migration and proliferation of keratinocyte. However, the mechanism of the AD-exos is still unclear. The objective of this article is to observe the function of the miR-21 expressed in the adipose AD-exos and the effect on migration and proliferation of the HaCaT cells.
The full layer dermal wound of BALb/c mouse was used to observe the vitro effect of the AD-exos and detect the expression of miR-21.The co-culture systems were established by transwell plates for observing the migration, proliferation, apoptosis rate, detecting the RNA, and protein expression in different treated groups. MiR-21 plasmid was used to over-express miR-21 by transfection of HaCaT cells. GW4869 was used to inhibit the secreting of exosomes from ADSCs.
The results showed that both ADSCs and the AD-exos could improve the wound healing process of BALb/c mouse full layer skin wound at a similar level, especially at the 7th day post surgery when compared to the control group (p < 0.01) and the highly expressed miR-21 was detected (p < 0.01 compared with control group and p < 0.001 compared to other microRNAs) in the treated groups at the same time point. AD-exos could obviously enhance the migration and proliferation of the HaCaT cells (p < 0.01), and fell back to the same level when the exosomes inhibitor--GW4869 was added compared with control group (p > 0.05). Over-expressed miR-21 could also significantly improve the migration and proliferation of HaCaT cells. But both AD-exos and miR-21 had no significantly effect on the apoptosis rate of HaCaT cells (p > 0.05 compared with each other). Over-expression of miR-21 plasmid could decrease the TGF-βI protein level (p < 0.001 vs. control group) in HaCaT cells while TGF-βI protein level increased again when antagomiR-21 was added in (p < 0.01 vs. empty plasmid group, p < 0.001 vs. miR-21 plasmid group). MiR-21 expression of HaCaT cells could be increased by the transfect ion of miR-21 plasmid (p < 0.001 vs. empty plasmid group) and decreased by antagomiR-21 (p < 0.01 vs. empty plasmid group, p < 0.001 vs. miR-21 plasmid group). MiR-21 appeared to have influence on MMP-9 and TIMP-2 (p < 0.001 compared to control group and p < 0.001 compared to TGF-βI group) but not MMP-2 and TIMP-1 (p > 0.05 compared to control group and TGF-βI group). These processes might act through PI3K/AKT pathway.
This research provide the experimental evidence that the miR-21 is highly expressed in AD-exos and can significantly accelerate the wound healing process and enhance the migration and proliferation of the HaCaT cells. Over-expressed miR-21 can inhibit the TGF-βI expression and excess TGF-βI can also have negative feedback influence on miR-21. We have found a reliable evidence that these two factors can act on HaCaT cells by influencing MMP-2 and TIMP-1 protein expression through the PI3K/AKT signal pathway. These results may provide a potential perspectives on improving the wound healing.
伤口愈合仍然是烧伤和创伤领域的一个挑战。脂肪来源的干细胞外泌体（AD-exos）已被证实对伤口愈合以及角质形成细胞的迁移和增殖具有积极作用。然而，AD-exos 的机制仍不清楚。本文的目的是观察脂肪 AD-exos 中表达的 miR-21 的功能及其对 HaCaT 细胞迁移和增殖的影响。
使用 BALb/c 小鼠全层皮肤创面观察 AD-exos 的体外作用，并检测 miR-21 的表达。通过 Transwell 板建立共培养系统，观察不同处理组的迁移、增殖、凋亡率、RNA 和蛋白表达。使用 miR-21 质粒通过转染 HaCaT 细胞过表达 miR-21。使用 GW4869 抑制 ADSC 分泌外泌体。
结果表明，ADSCs 和 AD-exos 均可在类似水平上改善 BALb/c 小鼠全层皮肤创面的伤口愈合过程，尤其是在术后第 7 天（与对照组相比，p < 0.01），同时在同一时间点检测到处理组中高度表达的 miR-21（与对照组相比，p < 0.01；与其他 microRNAs 相比，p < 0.001）。AD-exos 可明显增强 HaCaT 细胞的迁移和增殖（p < 0.01），当添加外泌体抑制剂 GW4869 时，与对照组相比，结果又回到相同水平（p > 0.05）。过表达 miR-21 也可以显著改善 HaCaT 细胞的迁移和增殖。但 AD-exos 和 miR-21 对 HaCaT 细胞的凋亡率均无显著影响（与对照组相比，p > 0.05）。过表达 miR-21 质粒可降低 HaCaT 细胞中的 TGF-βI 蛋白水平（与对照组相比，p < 0.001），而当添加 antagomiR-21 时，TGF-βI 蛋白水平再次升高（与空质粒组相比，p < 0.01；与 miR-21 质粒组相比，p < 0.001）。miR-21 质粒转染可增加 HaCaT 细胞的 miR-21 表达（与空质粒组相比，p < 0.001），而 antagomiR-21 则降低其表达（与空质粒组相比，p < 0.01；与 miR-21 质粒组相比，p < 0.001）。miR-21 似乎对 MMP-9 和 TIMP-2 有影响（与对照组相比，p < 0.001；与 TGF-βI 组相比，p < 0.001），但对 MMP-2 和 TIMP-1 没有影响（与对照组和 TGF-βI 组相比，p > 0.05）。这些过程可能通过 PI3K/AKT 通路发挥作用。
本研究提供了实验证据，证明 miR-21 在 AD-exos 中高度表达，可显著加速伤口愈合过程，增强 HaCaT 细胞的迁移和增殖。过表达的 miR-21 可抑制 TGF-βI 的表达，过量的 TGF-βI 也可对 miR-21 产生负反馈影响。我们已经发现了可靠的证据，这两个因素可以通过影响 MMP-2 和 TIMP-1 蛋白表达，通过 PI3K/AKT 信号通路作用于 HaCaT 细胞。这些结果可能为改善伤口愈合提供潜在的研究视角。
