The role of basement membrane type VII collagen in aberrant differentiation and cell fate determination in ageing skin

VII型胶原（CollVII）是人上皮细胞中的基底膜（BM）蛋白，是参与表皮-真皮粘附的锚定原纤维（AF）结构的主要成分。在老化的皮肤中，长期暴露在紫外线下会导致异常分化、干细胞标记物表达减少和BM损伤（1,2）。已知老年个体的角质形成细胞和成纤维细胞分泌的CollVII较少，分泌的CollVII对细胞因子调节的反应较弱(3)。我们最近的研究表明，在鳞状细胞癌细胞中敲除CollVII会导致三维培养中上皮明显的无序分化，而重组CollVII可以逆转(4)。已知外用维甲酸（维生素A）可能通过抑制胶原酶表达使人心房纤颤密度加倍(5)。我们将探索在正常人类衰老过程中ColVII的减少会导致异常分化和细胞命运决定的假设，而增加内源性维生素A可能会逆转这一过程。原代角质形成细胞将来源于年轻人（n=5< 30岁）和老年人（n=5 ~ 65岁）的多余皮肤，并获得适当的伦理批准。细胞和培养基中的CollVII将通过免疫印迹法进行定量。器官型3-D表皮培养（OTC）将在dispasase处理（去除CollVII）去表皮真皮（DED）上产生。将对增殖、滤泡间干细胞、凋亡和早期和晚期分化标志物进行免疫染色。将在年轻细胞和老年细胞中进行靶向siRNA完全消耗CollVII，以观察老年细胞是否更容易发生异常分化。重组CollVII也将被注射到旧细胞OTCs的DED中，看看这是否会逆转这一过程。视黄酸代谢结合剂（RAMBAs），如利亚唑和塔拉唑，阻断内源性维生素A的分解代谢，从而增加内源性细胞维生素A的分泌。年轻和年老的原代角质形成细胞将与增加剂量的RAMBAs一起培养，以观察这是否会增加年老和年轻角质形成细胞的CollVII分泌差异。视黄酸将被用作额外的对照。将对DED上的角质形成细胞进行类似的实验，以观察是否存在基底膜上CollVII的沉积增加以及免疫染色评估的分化改变。由于表观遗传机制可能在类视黄酮诱导的分化中发挥作用，年轻细胞和老年细胞的三次生物复制将暴露于塔拉唑中，并使用Illumina Infinium HumanMethylation 27头芯片阵列评估全基因组甲基化。在与芯片杂交之前，DNA将被亚硫酸修饰。同时，将进行全基因组表达阵列。数据将使用Illumina软件和Ingenuity Pathway分析进行分析，专门筛选与胶原、核激素受体途径和分化相关的变化。定量RT-PCR将用于验证差异表达基因。这些数据应该阐明CollVII在老化表皮中的功能。1.Craven NM等。人体皮肤光损伤的临床特征与胶原蛋白VII的减少有关。中国生物医学工程杂志，2003;31(3):344- 344。2.Giangreco等人。人类皮肤老化与干细胞标记物表达减少有关。J投资皮肤，2009年9月24日。按3。陈玉青等。培养的人皮肤成纤维细胞和角质形成细胞表达VII型胶原基因：供体年龄和细胞因子反应的影响。中国生物医学工程学报，2009;32(2):559 - 561。4.Martins等。皮肤鳞状细胞癌中BM型胶原蛋白缺失的侵袭性行为增加。中国生物医学工程学报，2009;22(2):391 - 391。5.Woodley DT等。用局部维甲酸治疗光老化皮肤会增加表皮-真皮锚定原纤维。《美国医学协会杂志》上。1990; 263(22): 3057 - 9。

Type VII collagen (CollVII) is a basement membrane (BM) protein in human epithelia that is the main component of anchoring fibrils (AF) structures involved in epidermal-dermal adherence. In ageing skin, cumulative ultraviolet light exposure results in aberrant differentiation, reduced expression of stem cell markers and damage to the BM(1,2). Keratinocytes and fibroblasts from older individuals are known to secrete less CollVII and secreted CollVII is less responsive to cytokine modulation(3). Our recent work showed that knock-down of CollVII in squamous cell carcinoma cells results in markedly disorganized differentiation of epithelium in 3-D cultures reversible by recombinant CollVII(4). Topical tretinoin (Vitamin A) is known to double human AF density, possibly by inhibiting collagenase expression(5). We will explore the hypothesis that reduction of ColVII during normal human ageing results in aberrant differentiation and cell fate determination and that increasing endogenous vitamin A might reverse this process. Primary keratinocytes will be derived from redundant skin from young (n=5< 30 years) and older individuals (n=5 >65 years), with appropriate ethical approval. CollVII from cells and media will be quantitated by immunoblotting. Organotypic 3-D epidermal cultures (OTC) will be generated on dispase-treated (removes CollVII) de-epidermalised dermis (DED). Immunostaining will be performed for proliferation, interfollicular stem cell, apoptotic and early and late differentiation markers. Targeted siRNA to completely deplete CollVII will be performed in young and old cells to see if older cells are more susceptible to aberrant differentiation. Recombinant CollVII will also be injected into the DED of old cell OTCs to see if this reverses the process. Retinoic acid metabolism binding agents (RAMBAs), such as liarozole and talarozole, block the catabolism of endogenous vitamin A, thus increasing endogenous cellular vitamin A secretion. Young and old primary keratinocytes will be incubated with increasing doses of the RAMBAs to see if this increases CollVII secretion differentially in old versus young keratinocytes. Retinoic acid will be used as an additional control. Similar experiments will be performed with keratinocytes on DED to see if there is increased deposition of CollVII at the basement membrane and altered differentiation as assessed by immunostaining. As epigenetic mechanisms may play a role in retinoid-induced differentiation, triplicate biological replicates of young and old cells will be exposed to talarozole and whole-genome methylation will be assessed using the Illumina Infinium HumanMethylation 27 BeadChip Array. DNA will be bisulphite modified prior to hybridization to the chip. In parallel, whole genome expression arrays will be performed. Data will be analysed using Illumina software and Ingenuity Pathway analyses, screening specifically for changes related to collagens, nuclear hormone receptor pathways and differentiation. Quantitative RT-PCR will be used to verify differentially expressed genes. These data should shed light on the function of CollVII in ageing epidermis. 1.Craven NM et al. Clinical features of photodamaged human skin are associated with a reduction in collagen VII. Br J Dermatol. 1997;137(3):344-50. 2.Giangreco et al. Human Skin Aging Is Associated with Reduced Expression of the Stem Cell Markers. J Invest Dermatol. 2009 Sep 24. in press 3.Chen YQ et al. Type VII collagen gene expression by human skin fibroblasts and keratinocytes in culture: influence of donor age and cytokine responses. J Invest Dermatol. 1994;102(2):205-9. 4.Martins VL et al. Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of BM type VII collagen. J Cell Sci. 2009;122:1788-99. 5.Woodley DT et al. Treatment of photoaged skin with topical tretinoin increases epidermal-dermal anchoring fibrils. JAMA. 1990;263(22):3057-9.