Correlating functional heterogeneity of human dermal fibroblasts with differences in gene expression

Recent work has identified markers of fibroblast heterogeneity in human dermis. Transforming growth factor-β1 (TGF-β1) promotes fibroblast-to-myofibroblast differentiation, characterised by the expression of α-smooth muscle actin (α-SMA). Human dermal fibroblasts (hDF), treated with TGF-β1, were assayed for differentiation, proliferation and cell shape using the Operetta imaging system. One donor hDF, derived from female 64-year-old breast skin, expressed decreased levels of α-SMA protein. The gene expression profile of this donor hDF was determined using the Agilent microarray system. Four gene candidates (Asporin, ASPN; C-X-C motif chemokine ligand 1, CXCL1; Insulin-like growth factor 1, IGF1; and Wnt family member 4, WNT4) were chosen based on expression values and validated by TaqMan qPCR. Successful knockdown of IGF1 and WNT4 was achieved using MISSION shRNA-based lentiviral treatment. Fibroblast IGF1 knockdown (shIGF1) increased α-SMA mRNA and protein expression; no effect was seen with fibroblast WNT4 knockdown (shWNT4). Here I have characterised hDF phenotype and gene expression with regard to population heterogeneity. This work highlights the role of IGF-1 signalling on α-SMA expression and dermal fibroblast fate. Targeting IGF-1 signalling could provide therapeutic benefit for skin disorders involving aberrant wound healing and excessive fibrosis. Enzyme-derived, human dermal fibroblasts from four adult donors. Treated with/without TGF-β1 for 12h/24h and used for Agilent one colour microarray.

已有近期研究鉴定出人类真皮成纤维细胞异质性的标志物。转化生长因子-β1（Transforming growth factor-β1, TGF-β1）可促进成纤维细胞向肌成纤维细胞分化，该过程以α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）的表达为特征。本研究使用Operetta成像系统，对经TGF-β1处理的人类真皮成纤维细胞（human dermal fibroblasts, hDF）的分化、增殖及细胞形态进行了检测。其中一株源自64岁女性乳房皮肤的供体hDF，其α-SMA蛋白的表达水平出现下调。本研究采用安捷伦（Agilent）微阵列平台对该供体hDF的基因表达谱进行了测定。基于基因表达量筛选出4个候选基因：饰胶蛋白（Asporin, ASPN）、C-X-C基序趋化因子配体1（C-X-C motif chemokine ligand 1, CXCL1）、胰岛素样生长因子1（Insulin-like growth factor 1, IGF1）以及Wnt家族成员4（Wnt family member 4, WNT4），并通过TaqMan定量PCR（TaqMan qPCR）对其进行了验证。本研究采用基于MISSION短发夹RNA（shRNA）的慢病毒载体，成功实现了IGF1与WNT4的基因敲低。对成纤维细胞进行IGF1敲低（shIGF1）后，α-SMA的mRNA及蛋白表达水平均有所升高；而WNT4敲低（shWNT4）则未观察到明显影响。本研究围绕群体异质性对hDF的表型与基因表达特征进行了系统表征，阐明了IGF-1信号通路在α-SMA表达及真皮成纤维细胞命运调控中的作用。靶向IGF-1信号通路或可为异常伤口愈合与过度纤维化相关的皮肤疾病提供治疗策略。本研究使用酶解法分离自4名成年供体的人类真皮成纤维细胞，将其分别经TGF-β1处理或不予处理，处理时长为12小时与24小时，随后采用安捷伦单通道微阵列进行基因表达检测。