【作者】 唐桦；

【导师】 谢红付；

【作者基本信息】 中南大学 ， 皮肤病与性病学， 2008， 博士

【摘要】 衰老是指机体器官对环境改变的适应性随年龄增长而逐渐下降,进而易患疾病或引起死亡的一种现象。衰老一方面受机体预定的基因程序调控,另一方面由内外“磨损和消耗”所引起,二者同时表现于细胞和分子水平。皮肤由于位于体表,其衰老主要分为自然衰老和光老化两种形式,其分子机制非常复杂,特别是对皮肤自然衰老机制的研究并不深入。皮肤自然衰老机制代表性的学说有:基因调控学说、自由基学说、代谢失调学说。而遗传基因改变是皮肤内源性衰老的根本。皮肤自然衰老临床表现的一大特点就是皮肤干燥、脱屑、脆性增加,修复功能减退;组织学上则表现为皮肤变薄,表皮更新减慢,角质层屏障功能下降,以及角质层水合能力下降。正常角质层含水量保持在10%-20%之间,使皮肤柔软,不发生干燥、皲裂现象,但老年人皮肤含水量仅是正常的75%,所以表现为皮肤干燥。同时皮肤组织细胞的水分减少,细胞皱缩、老化、使皮肤出现细小皱纹,酶的活性降低。因此,皮肤的衰老与皮肤含水量、角质层的水合能力及屏障功能有关。水通道蛋白(AQP)是上世纪90年代发现的一类能快速转运水的膜整合蛋白。目前发现AQP在哺乳动物中有13个亚型,不同AQP亚型的分布具有组织、细胞、亚细胞特异性。根据这些水通道蛋白转运功能特性的差异,将其分为两个亚家族:水选择通道亚家族(aquaporins),这类通道对水分子的通透性具有高度选择性;另一个亚家族是水-甘油通道(aquaglyceroporins),这类通道除对水分子通透外,还对甘油和尿素等中性小分子也具有通透性。皮肤上主要分布的水通道蛋白是AQP3,它属于水-甘油通道蛋白亚家族。目前发现AQP3参与多种生理、病理过程,它与尿液的浓缩、气道表面的液体平衡、肠道水的吸收均有密切关系。而在皮肤的病理生理过程中,AQP3同样可以转运水、甘油到达表皮,因此对于表皮的屏障作用、水的保存有很大的意义。有研究者发现,哺乳动物皮肤AQP3的缺失可导致皮肤干燥,角质层水合作用下降,皮肤弹性下降,以及皮肤屏障修复延迟,生物合成功能受损,伤口愈合延迟。这些便提示我们AQP3参与皮肤衰老的过程。为了验证AQP3与皮肤衰老的关系,我们首先运用RT-PCR及Western blot研究不同年龄组非曝光部位皮肤组织中AQP3 mRNA、蛋白的表达量。结果显示:1.青年组AQP3 mRNA表达水平为0.654±0.104,中年组AQP3 mRNA表达水平为0.685±0.143,老年组AQP3 mRNA表达水平为0.316±0.109;2.青年组AQP3蛋白表达水平为1.936±0.250,中年组1.813±0.224,老年组1.044±0.271。我们发现老年组皮肤中AQP3 mRNA、蛋白的表达量较青年组和中年组均显著性减少(P值均＜0.01)。第二部分,我们利用消化分离的方法原代培养了正常人非曝光部位皮肤角质形成细胞和成纤维细胞,进行了细胞鉴定后分别检测了两种细胞不同年龄段AQP3的表达,结果显示:1.角质形成细胞中,青年组AQP3 mRNA表达水平为1.240±0.205,中年组AQP3 mRNA表达水平为0.889±0.136,老年组AQP3 mRNA表达水平为0.392±0.083。AQP3mRNA表达随年龄增加而减少。角质形成细胞中AQP3蛋白表达水平为:青年组1.303±0.231,中年组0.980±0.164,老年组0.360±0.142,AQP3蛋白表达亦随年龄增加而减少。2.成纤维细胞中,青年组AQP3mRNA表达水平为0.881±0.118,中年组AQP3 mRNA表达水平为0.757±0.167,老年组AQP3 mRNA表达水平为0.285±0.143。免疫细胞化学示AQP3蛋白表达水平为:青年组:0.378±0.123,中年组:0.355±0.112,老年组:0.171±0.060。WB示成纤维细胞中AQP3蛋白表达水平为:青年组0.792±0.092,中年组0.692±0.155,老年组0.375±0.112。老年组成纤维细胞AQP3 mRNA、蛋白的表达量较青年组和中年组均显著性减少(P值均＜0.01)。3.青年组角质形成细胞AQP3mRNA、蛋白表达明显高于青年组成纤维细胞(P值均＜0.01),中年组角质形成细胞AQP3蛋白表达明显高于中年组成纤维细胞(P＜0.05),老年组角质形成细胞AQP3 mRNA、蛋白表达与老年组成纤维细胞无显著性差异。综上所述:AQP3在皮肤组织及体外原代培养的角质形成细胞、成纤维细胞中的表达与年龄有关。AQP3的表达下降与皮肤自然衰老有关。更多还原

【Abstract】 Aging means that organs’ adaptability decreases accompanied with years of age, and human is liable to diseases or death. Aging is regulated by presumptive genes, as well as exterior and interior’s abrasions and consume, which are both shown in cell and molecular level. Skin is located in body surface, skin aging comprise natural aging and photoaging. Mechanism of natural aging is not clear, the representative theory include gene regulation theory、free radical theory、metabolic disorder theory. Gene regulation is fundamental of skin natural aging.One of feature of the clinical manifestation of skin natural aging is xerosis cutis、desquamating、fragility increasing, and recovery hypofunction; the histological manifestation include skin thinning、epidermal renewal stepping down、stratum corneum’s barrier and hydration function descending. The normal water content of stratum corneum retained between 10%-20%, which make skin soft; but in old man, the water content of skin is 75% compared normal content, which cause xerosis cutis. Meanwhile,water content of skin cells decrease, which cause tiny shrinkage and enzymatic active decrease. Thus, skin aging is concerned with water content、stratum corneum’s barrier and hydration function.AQP is a kind of membrane integral protein which can fast transport water. AQP has 13 hypotype in mammalian, which are distributed in different tissue and cell. According to the different function of AQPs, AQPs are divided into two subfamily: aquaporins and aquaglyceroporins. The former transport only water; aquaglyceroporins transport not only water but also glycerol. AQP3 is a aquaglyceroporin which is located in skin. AQP3 participate in several patho-physiological functions including urine concentration、water balance of air tube surface、intestinal tract’s water absorption. In dermal patho-physiological course, AQP3 can transport water and glycerol to epiderm, so AQP3 is significant to epidermal barrier and water retention. Researchers found AQP3-/- mouse display xerosis cutis、stratum corneum’s barrier function descending、skin elasticity descending、skin barrier repairing delayed、and biosynthesis damage、wound healing delay. All of these hint that AQP3 participate skin aging.In order to identify the relationship between AQP3 and skin aging, we detect the expression of AQP3 mRNA and protein of different ages by RT-PCR and Western blot. The result display: 1 .AQP3 mRNA in teenage group: 0.654±0.104, in middle-age group: 0.685±0.143,in old- age: 0.316±0.109. 2.The expression of AQP3 protein in in teenage group: 1.936 +0.250, in middle-age group: 1.813±0.224,in old- age: 1.044 + 0.271. We found AQP3 mRNA and protein in old-age group significantly lower than the level of middle-age group (P<0.01), and teenage group(P＜0.01)In the second part, normal human epidermal keratinocytes and skin fibroblast of different ages were cultured; the expression of AQP3 was investigated in keratinocytes and fibroblast respectively. The result display: 1 .in keratinocytes, AQP3 mRNA in teenage group: 1.240±0.205, in middle-age group: 0.889±0.136,in old- age: 0.392±0.083. The expression of AQP3 protein in in teenage group: 1.303±0.231, in middle-age group: 0.980±0.164,in old- age: 0.360 + 0.142. AQP3mRNA and protein in old-age group keratinocytes decreased with the increase of age. 2. In fibroblast, AQP3 mRNA in teenage group: 0.881±0.118, in middle-age group: 0.757±0.167,in old- age: 0.285±0.143. The expression of AQP3 protein by WB in teenage group: 0.792±0.092, in middle-age group: 0.692±0.155,in old- age: 0.375 + 0.112. AQP3 mRNA and protein in old-age group fibroblast decreased significantly (P<0.01). 3. AQP3 mRNA and protein in keratinocytes of teenage group is signify-cantly higher than fibroblasts of teenage group(P＜0.01). AQP3 protein in keratinocytes of middle-age group is significantly higher than fibroblasts of middle-age group(P<0.05). AQP3 mRNA and protein of old-age group in keratinocytes and fibroblasts is not significantly different.ALL findings suggest the expression level of AQP3 in the human skin is associated with age, and decreases as the age grow. AQP3 may play a important role in aging of human skin.更多还原