应用仿生三维共培养体系构建人毛囊诱导微环境模型的实验研究

For those cases of hair loss or alopecia, apparently, hair-follicle (HF) reconstruction is the best option for the treatment. Since HF morphogenesis and development mainly depends on well-orchestrated interactions between epithelial and mesenchymal components (epithelial-mesenchymal interactions, EMIs). As a highly specialized mesenchymal component, dermal papilla cells (DPCs) have an indispensable role that enables HF morphogenesis and regulates hair cycling, while follicle stem cells (FSCs) that originated from epithelium of HF can be induced by DPCs to differentiate into hair structure (hair shaft). Although cultured DPCs has been demonstrated to be able to induce competent follicular stem cells from epithelium into HF structures. However, human DPCs grown in two-dimensional (2D) condition tend to lose such capability during passaging in vitro. Thus, due to great difference from in vivo condition, regeneration of human HFs has not yet been realized in vitro.. In vivo, DPCs are physiologically embedded in extracellular matrix (ECM), forming into a three-dimensional (3D) multi-cellular spheroid tissue. Studies have shown that a formation of HF-like structure can be achieved when follicle cells are grown under 3D culture. Since it was proved that a biomimetic cell-cell interaction microenvironment can be established by utilizing 3D culture, we have previously developed an in vitro 3D model for both controllable and scalable production of hair-inductive human DPCs-microtissues based on an advanced version of microarray hanging-drop culture, which was verified that the formation of 3D microtissues not only restored the hair-inductive properties of DPCs, but also endowed a biomimetic niche between cultured cells.. Many distinct signaling such as PDGFs, FGFs, VEGFs, and TGFs have been proved involved in normal HF morphogenesis and development. As an autologous extract prepared from concentrated plasma, platelet-rich plasma (PRP) that containing various growth factors (eg. PDGF, VEGF, FGF, TGF-β, EGF, etc.) has been used as an inactivated liquid form in surgical procedures and clinical treatments. PRP can also be activated to form hydrogel that containing bioactive molecules for local tissue engineering and regeneration. Hence, studies have reported the successful application of PRP-hydrogel in 3D cell culture. Besides, in our latest work, we have preliminarily demonstrated a considerably beneficial effect of PRP on the proliferation and hair-inductivity of DPCs in vitro.. Based on the aformentioned, we hypothesis that "HF morphogenesis and development can be induced by 3D co-culture system that recapitulating the interacting cell-cell and cell-ECM niche of EMIs." Thus, the aim of this study is to establish an inductive microenvironment model for human HF morphogenesis and development by utilizing 3D biomimetic co-culture system consisted of human DPCs-microtissues, FSCs, and PRP hydrogel. It will not only provide a valuable platform for exploring the biological and pathological mechanism regarding HF, but also confers a potential therapeutic strategy for treating alopecia diseases by culturing functional HF organoids.

毛囊重建是治疗秃发性疾病的最佳选择，虽然目前尚未能在体外构建出人毛囊，但实验显示三维培养有助于细胞形成类毛囊结构。研究表明：调控毛囊形态发生及发育的上皮-间质相互作用(EMIs)是由间质来源的毛乳头细胞(DPCs)和上皮来源的毛囊干细胞(FSCs)所主导。我们在前期研究中基于微阵列三维悬滴平台成功构建出仿生人DPCs微组织，并证实其能恢复和增强DPCs的毛囊诱导能力。预实验则提示：含多种生长因子的PRP有利于DPCs诱导能力的维持和增强。因此，本研究拟采用人DPCs微组织、人FSCs和PRP凝胶构建仿生三维共培养体系，并对培养体系进行筛选优化、体外和体内的生物学验证，以证实"基于共培养体系模拟EMIs中细胞-细胞、细胞-细胞外基质相互作用的微环境因素，能诱导毛囊类器官形成"的假说。该研究不仅能为毛囊重建、秃发致病机制和治疗药物筛选提供实验模型，也为应用毛囊类器官治疗秃发奠定理论和实践基础。

毛囊重建是治疗秃发性疾病的最佳选择，虽然目前尚未能在体外构建出人毛囊，但实验显示三维培养有助于细胞形成类毛囊结构。研究表明：调控毛囊形态发生及发育的上皮-间质相互作用(EMIs)是由间质来源的毛乳头细胞(DPCs)和上皮来源的毛囊干细胞所主导。我们在前期研究中基于微阵列三维悬滴平台成功构建出仿生人DPCs微组织，并证实其能恢复和增强DPCs的毛囊诱导能力。因此，本研究中我们基于DPCs的仿生生物学模型，采用高通量测序探究了3D-DPCs仿生微组织中包括转录组、micro-RNAs、lnc-RNAs等微环境因素的差异表达情况；并通过体外和体内细胞生物学实验证实了3D-DPCs微环境中LncRNA-PCAT1通过介导miR-329/Wnt10b信号轴促进毛囊诱导形成的作用，证实了3D-DPCs中LncRNA-XIST通过miR-424/Shh/hedgehog通路轴诱导了毛囊再生，从而阐明了3D微环境对DPCs诱导毛囊再生的影响及其分子机制。此外，我们基于在3D-DPCs中差异表达的乳酸脱氢酶(LDH)基因，初步探明了低氧条件可促进DPCs细胞的增殖，且在低氧条件下LDH对维持细胞的毛囊诱导活性起着重要作用。该研究不仅能为毛囊再生重建、脱发疾病发病机制和治疗药物筛选提供实验模型，也为将来功能化毛囊种子细胞应用于临床治疗奠定了理论和实践基础。