研究背景

在干细胞治疗领域，寻找理想的细胞来源仍是一大挑战。目前较多应用及研究的干细胞包括骨髓间充质干细胞（Bone marrow mesenchymal stem cells, BMSCs）、诱导多能干细胞（Induced Pluripotent Stem Cells, iPSCs）、胚胎干细胞（Embryonic Stem Cells, ESCs）等。然而，这些细胞来源稀缺、采取手段创伤大，且存在伦理和安全性方面的限制，限制了其临床应用。

脂肪组织含量丰富，易于获取，将脂肪组织来源的干细胞应用于再生医学，为修复重建领域提供了新思路。传统理论认为，含有单房脂滴的成熟脂肪细胞处于终末分化状态，然而，最新研究证据表明，成熟脂肪细胞能够通过去分化过程获得成纤维细胞样形态特征，这类细胞被定义为脂肪源性成纤维细胞或去分化脂肪细胞（Dedifferentiated Fat Cells, DFATs），具有多向分化潜能。和成熟脂肪细胞相比，DFATs下调了脂周蛋白的表达，上调了与细胞增殖、细胞重编程相关的基因。与ADSCs 相比，DFATs 细胞的均一性更好，在30代以内细胞表型可保持稳定，且可通过分泌TSG-6、PGE2等因子抑制过度炎症反应。因此，DFATs在修复重建与再生领域具有巨大的应用潜力。目前研究热点聚焦在ADSCs和ADSCs外泌体的基础研究，尚缺乏探讨DFATs及其外泌体在修复再生与软组织重建领域中的应用。

研究目的

1. 体外比较ADSCs和DFATs的三系分化能力和旁分泌功能，分离并鉴定DFATs外泌体。

2. 探究DFATs利用其旁分泌功能在脂肪移植及脂肪再生方面中的应用潜力。

3. 评估DFATs来源外泌体在糖尿病创面修复中的疗效，并探索其分子机制。

研究方法

第一部分：去分化脂肪细胞及其外泌体的提取和鉴定

选取健康成年女性为脂肪供体，采用低负压吸脂、清洗、棉垫法纯化脂肪的方法获取脂肪。体外用I型胶原酶消化脂肪组织，获得基质血管成分（Stromal Vascular Fraction，SVF）和成熟脂肪细胞，培养成熟脂肪细胞脱去脂滴，去分化为DFATs。流式分析法对ADSCs、DFATs表面标记物及多向分化能力进行比较和鉴定，随后收集条件培养基，测定DFATs旁分泌功能（VEGF、HGF、TNFα、IL-1β），超速离心法从DFATs上清液提取DFATs外泌体，并做外泌体鉴定。

第二部分：去分化脂肪细胞联合脱细胞真皮促进移植脂肪存活及其机制

体外检测ADM颗粒的微观结构及生物相容性。在体内实验中，提前将DFATs接种于ADM进行预培养，使其粘附紧密，然后按1：2的比例混合ADM和脂肪颗粒，移植至裸鼠背部皮下区域。移植后4-、8-、12-周取材，测量移植物体积，同时进行组织学评价，qRT-PCR检测移植物血管生成水平和成脂水平，ELISA检测组织样本炎症水平。

第三部分：去分化脂肪细胞来源外泌体促进糖尿病创面愈合的研究

用GelMA水凝胶作为DFATs外泌体的载药平台，浓度梯度实验筛选出生物相容性最佳的GelMA浓度。体外实验中，DFATs-Exos处理内皮细胞和人皮肤成纤维细胞，研究DFATs-Exos对血管生成和成纤维细胞迁移的影响。体内实验中，将链脲佐菌素注射至Balb/C小鼠腹腔内，构建糖尿病小鼠模型。用GelMA水凝胶缓慢释放DFATs-Exos，将其注射于糖尿病小鼠背部创面上，术后观察创面愈合情况，在术后第7天、14天取材进行HE染色、马松染色、免疫组化染色，进一步探究DFATs外泌体促进创面愈合的相关分子机制。

研究结果

1.体外成功分离提取ADSCs和DFATs，呈干细胞样梭形结构。流式细胞分析显示，90%以上DFATs表面高表达干细胞表面标记物 CD90、CD105 和 CD73。DFATs成脂分化能力强于ADSCs，两者成软骨分化能力、成骨分化能力无统计学差异，DFATs可抑制脂肪细胞分泌炎症相关因子。TEM、NTA、WB结果表明成功分离了DFATs外泌体。

2. ADM的组织相容性良好，作为支架可增强DFATs 成脂相关基因的表达，以及其旁分泌功能。体内研究表明，ADM结合DFATs辅助移植的脂肪组织结构完整，纤维化水平低，炎症因子IL-1β和TNFα水平低。

3. 体外成纤维细胞高糖模型中，DFATs外泌体可促进成纤维细胞增殖，并通过激活Wnt/β-catenin信号通路缓解高糖环境对细胞增殖和迁移的抑制作用。体内实验表明，DFATs-Exos/GelMA复合物可加速创面闭合，促进血管生成，提高创面胶原含量。

研究结论

1. DFATs和ADSCs有相同的组织来源，但DFATs有更好的均一性，更高的成脂分化效率。DFATs和ADSCs分泌的细胞因子均可减少脂肪细胞炎症反应。

2. 接种于脱细胞真皮培养后，DFATs获得更强的旁分泌功能。DFATs用于辅助脂肪移植可促进移植物新生血管形成，降低炎症水平，提高脂肪存活率。

3. 将DFATs-Exos负载于GelMA水凝胶用于创面，可通过激活Wnt/β-catenin信号通路增强血管生成、提高成纤维细胞活性，促进M2型巨噬细胞极化，加速糖尿病伤口愈合。

Background

In the field of stem cell therapy, identifying an ideal source of stem cells remains a major challenge in current scientific research. Commonly studied and applied stem cells include bone marrow mesenchymal stem cells (BMSCs), induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs). However, the limited availability of these stem cells, the invasive extraction methods, as well as ethical and safety concerns, have hindered their clinical application.

Adipose tissue, being abundant, offers a promising new avenue for regenerative medicine. Adipose tissue contains adipose-derived stem cells (ADSCs), which possess paracrine functions and can promote fat regeneration after transplantation through adipogenic differentiation. Studies have shown that individual mature adipocytes can be induced to dedifferentiate into multipotent stem cells lacking lipid droplets, known as dedifferentiated fat cells (DFATs). Compared to mature adipocytes, DFATs downregulate the expression of adipocyte-specific proteins and upregulate genes associated with cellular reprogramming.

Compared to ADSCs, DFATs exhibit better homogeneity, slightly higher telomerase activity at passages 3–5, and maintain stable cellular phenotypes within 30 passages. Like ADSCs and BMSCs, DFATs express low levels of MHC II molecules, and they can suppress excessive inflammatory responses by secreting factors such as TSG-6 and PGE2. Therefore, DFATs have significant application potential in the fields of tissue repair, reconstruction, and regeneration.

Currently, research hotspots mainly focus on ADSCs and their exosomes, while the application of DFATs and their exosomes in tissue repair and soft tissue reconstruction remains underexplored.

Objectives

1. To compare the multilineage differentiation potential and paracrine activity of ADSCs and DFATs in vitro, and to isolate and characterize DFAT-derived exosomes (DFATs-Exos).

2. To evaluate the application of DFATs paracrine functions in fat grafting and adipose tissue regeneration.

3. To investigate the potential of DFATs-Exos in promoting diabetic wound healing and to explore the underlying molecular mechanisms.

Methods

Part 1: Isolation and Characterization of DFATs and DFATs Exosomes
   Adipose tissue was obtained from healthy adult female donors, using low-pressure aspiration and cotton-pad purification techniques to preserve tissue structure and viability. After enzymatic digestion, the stromal vascular fraction (SVF) and mature adipocytes were isolated. DFATs were generated from mature adipocytes via the “ceiling culture” method. Flow cytometry was used to compare surface markers and differentiation potential of ADSCs and DFATs. Subsequently, the conditioned medium was collected to assess the paracrine function of DFATs by measuring secreted factors (VEGF, HGF, TNFα, and IL-1β). Exosomes were extracted through ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blot.

Part 2: DFATs Combined with Acellular Dermal Matrix to Enhance Fat Graft Survival
   ADM microparticles were evaluated for microstructure and biocompatibility in vitro. For in vivo studies, DFATs were seeded onto ADM for stable adhesion, then mixed with lipoaspirate at a 1:2 ratio and implanted into the back of immudefficiency nude mice. At 4-, 8-, and 12- weeks post-implantation, graft volume and histological features were assessed. qRT-PCR was used to evaluate angiogenesis and adipogenesis-related gene expression, while ELISA measured inflammation-related cytokines.

Part 3: Study on DFATs-Exos for Diabetic Wound Healing

Using GelMA hydrogel as a drug delivery platform for DFAT-derived exosomes (DFATs-Exos), a concentration gradient experiment was conducted to identify the GelMA concentration with the best biocompatibility. In vitro experiments involved treating endothelial cells and human dermal fibroblasts with DFATs-Exos to investigate their effects on angiogenesis and fibroblast migration. In vivo, streptozotocin was injected intraperitoneally into Balb/C mice to establish a diabetic mouse model. GelMA hydrogel was used for the sustained release of DFATs-Exos, which were then injected into the dorsal wounds of diabetic mice. Wound healing was observed post-surgery, and on days 7 and 14, tissue samples were collected for H&E staining, Masson's trichrome staining, and immunohistochemical analysis to further explore the molecular mechanisms by which DFATs-Exos promote wound healing.

Results

1. ADSCs and DFATs were successfully isolated and extracted in vitro, both exhibiting a spindle-shaped morphology characteristic of stem cells. Flow cytometry analysis showed that over 90% of DFATs highly expressed stem cell surface markers CD90, CD105, and CD73. DFATs demonstrated a stronger adipogenic differentiation potential compared to ADSCs, while no statistically significant differences were observed between the two in chondrogenic or osteogenic differentiation capabilities. Additionally, DFATs were found to suppress the secretion of inflammation-related cytokines by adipocytes. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot (WB) results confirmed the successful isolation of DFAT-derived exosomes.

2. ADM exhibits good biocompatibility and, when used as a scaffold, enhances the expression of adipogenesis-related genes in DFATs as well as their paracrine functions, such as VEGF secretion. In vivo studies have shown that adipose tissue transplanted with ADM and DFATs retains structural integrity, with low levels of fibrosis and reduced expression of inflammatory factors IL-1β and TNFα.

3. In a high-glucose fibroblast model in vitro, DFATs-Exos promote fibroblast proliferation and alleviate the inhibitory effects of the high-glucose environment on cell proliferation and migration by activating the Wnt/β-catenin signaling pathway. In vivo experiments demonstrated that the DFATs-Exos/GelMA composite accelerates wound closure, promotes angiogenesis, and increases collagen content in the wound area.

Conclusions

1. DFATs and ADSCs share a similar tissue origin, but DFATs exhibit superior homogeneity and uniformity, along with a higher efficiency in adipogenic differentiation. Both DFATs and ADSCs secrete factors that help reduce inflammatory responses in adipocytes.

2. When seeded onto ADM, DFATs acquire enhanced paracrine functions. Their application in fat grafting supports neovascularization, reduces inflammation, and improves graft retention and survival.

3. DFATs-Exos loaded into GelMA hydrogel can be applied to wounds, where they enhance angiogenesis, increase fibroblast activity, promote M2 macrophage polarization, and accelerate diabetic wound healing by activating the Wnt/β-catenin signaling pathway.