人类脂肪来源的干细胞释放的小型细胞外囊泡（sEV），即外泌体，作为将干细胞再生能力传递给受伤细胞以促进创伤愈合的一种手段，其功能日益被认识到。外泌体富含鞯脂和长非编码RNA（lncRNA），例如转移相关的肺腺癌转录本1（MALAT1）。我们在MALAT1中鉴定了潜在的鞯脂应答顺式元件（CRCE）。我们假设CRCE能够响应细胞内的鞯脂水平，以调控外泌体的MALAT1包装。许多细胞内的MALAT1水平超过了编码蛋白基因的水平，而且其在外泌体中的表达同样很高。鞯脂还调节外泌体的合成，但尚未证实通过鞯脂磷脂酶和鞯脂合酶途径对外泌体载体的内容进行调节。我们在收集了培养基后，将ADSC与鞯脂磷脂酶抑制剂GW4869和鞯脂合成的刺激剂C2-和C6-短链鞯脂一起处理，然后从条件培养基中分离出外泌体，并使用外泌体处理人皮肤成纤维细胞（HDF）培养物进行细胞迁移刮痕试验和线粒体应激测试以评估氧耗率（OCR）。通过处理ADSC与GW4869抑制剂处理后，鞯脂磷脂酶的抑制降低了小型EV中MALAT1的水平。使用C2-和C6-鞯脂刺激剂刺激鞯脂合成可以增加细胞和EVs中的MALAT1水平。在将EV施加到HDF中之后，我们评估了sEV MALAT1在HDF中的功能作用。对照组sEV增加了HDF的迁移，并显著增加了ATP产生、基础和最大呼吸的OCR。来自GW4869处理的ADSC的sEV抑制了细胞迁移和最大呼吸。然而，分别从C2-和C6-处理的细胞中获得的sEV在这两个功能上都有所增加，但与对照EV相比，仅在最大呼吸方面显著增加。sEV通常是外泌体，但在ADSC经过GW4869和C6-鞯脂胺处理后，它们变大并被视为微囊泡。鞯脂合成调节了MALAT1外泌体的含量，而鞯脂磷脂酶抑制剂阻断了MALAT1从ADSC外泌体中分泌出来。我们的研究结果与MALAT1增加细胞迁移和线粒体MALAT1改变细胞中最大呼吸有关的报道一致。由于MALAT1对外泌体功能至关重要，因此增加外泌体中的MALAT1应该对创伤愈合有益，正如这些实验所示。视频摘要。© 2023年，《BioMed Central有限公司》，施普林格自然出版集团的一部分。

The function of exosomes, small extracellular vesicles (sEV) secreted from human adipose-derived stem cells (ADSC), is becoming increasingly recognized as a means of transferring the regenerative power of stem cells to injured cells in wound healing. Exosomes are rich in ceramides and long noncoding RNA (lncRNA) like metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). We identified putative ceramide responsive cis-elements (CRCE) in MALAT1. We hypothesized that CRCE respond to cellular ceramide levels to regulate sEV MALAT1 packaging. MALAT1 levels by many cells exceed those of protein coding genes and it's expression is equally high in exosomes. Ceramide also regulates exosome synthesis, however, the contents of exosome cargo via sphingomyelinase and ceramide synthase pathways has not been demonstrated.ADSC were treated with an inhibitor of sphingomyelinase, GW4869, and stimulators of ceramide synthesis, C2- and C6-short chain ceramides, prior to collection of conditioned media (CM). sEV were isolated from CM, and then used to treat human dermal fibroblast (HDF) cultures in cell migration scratch assays, and mitochondrial stress tests to evaluate oxygen consumption rates (OCR).Inhibition of sphingomyelinase by treatment of ADSC with GW4869 lowered levels of MALAT1 in small EVs. Stimulation of ceramide synthesis using C2- and C6- ceramides increased cellular, EVs levels of MALAT1. The functional role of sEV MALAT1 was evaluated in HDF by applying EVs to HDF. Control sEV increased migration of HDF, and significantly increased ATP production, basal and maximal respiration OCR. sEV from GW4869-treated ADSC inhibited cell migration and maximal respiration. However, sEV from C2- and C6-treated cells, respectively, increased both functions but not significantly above control EV except for maximal respiration. sEV were exosomes except when ADSC were treated with GW4869 and C6-ceramide, then they were larger and considered microvesicles.Ceramide synthesis regulates MALAT1 EV content. Sphingomyelinase inhibition blocked MALAT1 from being secreted from ADSC EVs. Our report is consistent with those of MALAT1 increasing cell migration and mitochondrial MALAT1 altering maximal respiration in cells. Since MALAT1 is important for exosome function, it stands that increased exosomal MALAT1 should be beneficial for wound healing as shown with these assays. Video Abstract.© 2023. BioMed Central Ltd., part of Springer Nature.