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

III型胶原(CollVII)是人体上皮细胞中的一种基底膜(BM)蛋白，是参与表皮-真皮黏附的锚定纤维(AF)结构的主要成分。在老化的皮肤中，累积的紫外线暴露会导致异常分化，干细胞标记物的表达减少，并对BM(1，2)造成损害。已知来自老年人的角质形成细胞和成纤维细胞分泌较少的CollVII，并且分泌的Collvii对细胞因子调节的反应较差(3)。我们最近的工作表明，在3-D培养中，下调鳞状细胞癌细胞中的Collvii基因会导致明显的上皮细胞无序分化，这是重组Collvii(4)可逆的。外用维甲酸(维生素A)可能通过抑制胶原酶的表达，使人体房颤密度增加一倍。我们将探索一种假设，即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，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从年轻人(n=5&lt；30岁)和老年人(n=5&gt；65岁)多余的皮肤中获得，并获得适当的伦理批准。细胞和培养液中的胶原蛋白VII将通过免疫印迹进行定量。器官型3-D表皮培养(OTC)将在经Disdisase处理(去除Collvii)的去表皮真皮(DED)上产生。将进行增殖、滤泡间干细胞、凋亡以及早期和晚期分化标志物的免疫染色。在年轻和年老的细胞中将进行靶向siRNA以完全耗尽CollVII，以查看年老的细胞是否更容易发生异常分化。重组的CollVII也将被注射到旧细胞OTCs的DED中，看看这是否逆转了这一过程。维甲酸代谢结合剂(RAMBA)可阻断内源性维生素A的分解代谢，从而增加细胞内源性维生素A的分泌。年轻的和老年的原代角质形成细胞将与增加剂量的RAMBA一起孵育，看看这是否会增加老年和年轻角质形成细胞中的Collvii分泌。维甲酸将被用作额外的对照。DED上的角质形成细胞将进行类似的实验，看看基底膜上是否有更多的Collvii沉积，以及免疫染色评估是否有分化改变。由于表观遗传机制可能在维甲酸诱导的分化中发挥作用，年轻和老年细胞的三重生物复制将暴露于他拉唑，全基因组甲基化将使用Illumina Infinium Human甲基化27珠芯片阵列进行评估。DNA将在与芯片杂交之前进行亚硫酸氢盐修饰。同时，将进行全基因组表达阵列。数据将使用Illumina软件和独创性路径分析进行分析，专门筛选与胶原蛋白、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的基因。这些数据应该有助于阐明Collvii在衰老表皮中的功能。1.Craven NM等人。人类皮肤光损伤的临床特征与III型胶原减少有关。1997；137(3)：344-50。2.Giangreco等人。人类皮肤老化与干细胞标记物的表达减少有关。J·维斯特·皮马托。2009年9月24日出版社3.陈永强等人。培养的人皮肤成纤维细胞和角质形成细胞VII型胶原基因的表达：供体年龄和细胞因子反应的影响。J·维斯特·皮马托。1994年；102(2)：205-9。4.Martins VL等人。伴有BM III型胶原缺失的皮肤鳞状细胞癌的侵袭行为增加。J细胞科学。2009年；122：1788-99。5.Woodley DT等人。外用维甲酸治疗光老化皮肤可增加表皮-真皮锚定纤维。贾玛1990年；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.