背景：局限性硬皮病（Localized scleroderma, LoS）是一种以皮肤纤维化和皮下脂肪组织萎缩为特征的自身免疫性疾病。自体脂肪移植（Autologous fat grafting, AFG）与脂肪来源间充质干细胞（Adipose-derived stem cells, ASCs）是极具潜力的治疗方法。然而，与来自健康供者的ASCs（Healthy-ASCs）相比，来自硬皮病患者的ASCs（LoS-ASCs）在多项特征方面具有改变。

目的：本研究将探讨：1）LoS的黑色素、红斑及超声特征；2）Healthy-ASCs与LoS-ASCs的基本特征与治疗纤维化能力的比较；3）单细胞转录组测序比较LoS患者与健康对照的基质血管组分（Stromal vascular fraction, SVF）；4）通过药效学研究明确ASCs治疗硬皮病的效果和机制。

方法：1）对LoS患者病灶的黑色素与红斑指数、LoSCAT评分与超声特征进行了临床测量。2）通过CCK8实验、细胞因子芯片、实时荧光定量PCR实验等方法比较了来自LoS患者和健康供者的ASCs的差异，并通过Transwell共培养、转录组测序探究两种来源的ASCs治疗纤维化的潜在差异机制。3）建立博来霉素诱导的小鼠皮肤纤维化模型，在体内比较了两种来源的ASCs治疗皮肤纤维化的能力。4）通过对LoS患者和健康供者的基质血管组分（SVF）进行单细胞转录组测序，进一步分析LoS患者非病变部位脂肪组织内的固有异常。5）通过流式细胞分选，验证CD55^high ASCs（间质祖细胞）与CD55^low ASCs（脂肪前体细胞）在动物模型体内治疗纤维化的效果差异。6）通过系统的药效学体外和体内研究，探究健康供者来源的ASCs治疗皮肤纤维化的能力和机制。

结果：临床分析显示LoS患者病灶皮肤黑色素沉积，AFG后黑色素指数显著降低。超声分析发现LoS患者肌肉收缩能力减弱。体外培养的健康供者来源ASCs比LoS患者的ASCs具有更强的增殖能力，能够分泌更高水平的生长因子和细胞因子，包括VEGFA、PDGFB、IL-10等。小鼠体内实验显示健康供者来源ASCs能够更有效地减少真皮厚度和胶原沉积，并增加微血管密度和M2型巨噬细胞的比例。单细胞测序发现LoS患者非病变部位CD55^high ASCs比例下降，ASCs存在固有异常，包括纤维生成能力增强、抗炎能力降低和氧化应激水平升高。小鼠模型体内实验表明，CD55^high ASCs比CD55^low ASCs具有更好的治疗皮肤纤维化的效果。系统药效学实验表明，健康供者来源ASCs能够通过分泌HGF、VEGFA₁₆₅亚型发挥抗纤维化和促血管生成作用，调节脾脏淋巴细胞亚群比例，并能在小鼠模型体内存活至少28天。

结论：LoS病灶存在黑色素沉积和肌肉收缩能力受损。LoS患者非病变部位SVF存在固有异常，间质祖细胞比例降低，治疗皮肤纤维化能力较弱。系统药效学实验验证了健康供者来源ASCs治疗皮肤纤维化的能力。综上所述，从健康供体获得的同种异体ASCs在治疗皮肤纤维化方面更加有效，未来或许可以作为LoS治疗的新方案。

Background: Localized scleroderma (LoS) is an autoimmune disease characterized by skin fibrosis and subcutaneous adipose tissue atrophy. Autologous fat grafting (AFG) and adipose-derived stem cells (ASCs) are promising therapeutic approaches. However, ASCs from localized scleroderma patients (LoS-ASCs) exhibit changes in several characteristics compared to ASCs from healthy donors (healthy-ASCs).

Objective: This study will investigate: 1) the melanin, erythema and ultrasonic characteristics of LoS; 2) comparison of the basic characteristics and treatment ability of Healthy-ASCs and LoS-ASCs; 3) the stromal vascular fraction (SVF) of LoS patients was compared with that of healthy controls by single-cell transcriptome sequencing. 4) clarify the effect and mechanism of ASCs in the treatment of scleroderma through pharmacodynamic studies.

Methods: 1) The melanin and erythema indexes, LoSCAT score and ultrasound characteristics of tissue defects in LoS patients were investigated. 2) The differences in LoS-ASCs and healthy-ASCs were compared by CCK8 assay, cytokine array, and real-time fluorescence quantitative PCR. Transwell co-culture and transcriptome sequencing were used to explore the potential different mechanisms of LoS-ASCs and healthy-ASCs in the treatment of fibrosis. 3) Bleomycin-induced mice models of skin fibrosis were established, and the ability of LoS-ASCs and healthy-ASCs to treat skin fibrosis was compared in vivo. 4) Next, we analyzed the inherent abnormalities in adipose tissue from non-lesional sites in LoS patients by single-cell transcriptome sequencing of stromal vascular fraction (SVF) from LoS patients and healthy donors. 5) Flow cytometry was used to verify the difference between CD55^high ASCs (interstitial progenitors) and CD55^low ASCs (committed preadipocytes) in the treatment of fibrosis in animal models. 6) The ability and mechanisms of healthy donor-derived ASCs to treat skin fibrosis were validated through systematic in vitro and in vivo pharmacodynamic studies.

Results: The skin lesions in LoS patients were hyperpigmented, and the melanin index decreased significantly after AFG treatment. Ultrasound analysis revealed weakened muscle contraction ability in LoS lesions. Healthy-ASCs exhibited a stronger proliferation capacity than LoS-ASCs, and were able to secrete higher levels of growth factors and cytokines, including VEGFA, PDGFB, IL-10, etc. In vivo experiments in mice models found that healthy-ASCs could more effectively reduce dermal thickness and collagen deposition, at the same time increase the microvascular density and the proportion of M2 macrophages. Single-cell RNA sequencing revealed a decreased in the proportion of CD55^high ASCs in LoS patients and inherent abnormalities in LoS-ASCs, including enhanced fibrogenesis, reduced anti-inflammatory capacity, and elevated levels of oxidative stress. In vivo experiments in bleomycin-induced mice models demonstrated that CD55^high ASCs had better therapeutic effect on skin fibrosis than CD55^low ASCs. Systematic pharmacodynamic experiments proved that healthy-ASCs can exert anti-fibrotic and pro-angiogenic effects by secreting HGF and VEGF₁₆₅, regulate the proportion of splenic lymphocyte subgroups, and survive for at least 28 days in animal models to exert their therapeutic effect.

Conclusion: LoS lesions can lead to hyperpigmentation and impaired muscle contraction ability. LoS-ASCs had weaker effect in treating skin fibrosis compared to  healthy-ASCs. LoS patients have inherent abnormalities in the SVF from non-lesional sites, and the proportion of interstitial progenitor cells with high expression of CD55 was significantly reduced. Systematic pharmacodynamic study demonstrated the ability of healthy-ASCs to treat skin fibrosis both in vitro and in vivo. In summary, allogeneic ASCs obtained from healthy donors were more effective in the treatment of skin fibrosis and may be used for LoS treatment in the future.