【作者】 宣敏；

【导师】 程飚；

【作者基本信息】 南方医科大学 ， 整形外科学， 2014， 博士

【摘要】 衰老(aging)又称老化,是机体在退化时期功能下降及生理紊乱的综合表现,以多器官系统进行性的功能结构减退为特征。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,二者发生于细胞和分子水平,表现为DNA、蛋白质、脂类物质以及细胞器等的损伤和有害修饰积累。既往认为,机体细胞的发育过程皆由未分化到分化、从分化到衰老、再由衰老至凋亡的过程。伴随自噬(autophagy)概念的出现,这一生命科学领域热门的研究方向让人们开始重新认识衰老及相关疾患机制。细胞的自稳是通过对细胞内成分合成和降解的精确调节而实现。无论细胞处于正常生理还是病理状态下,自噬作为机体一种重要的防御和保护机制,它能够维持细胞的自我稳态或保证细胞的生存。研究发现,细胞自噬在各种真核生物衰老进程中发挥重要的调节机制,但自噬功能随着年龄增长而衰退,这种衰退可能是由于年龄相关的细胞结构改变及功能下降所致。皮肤组织作为生物体有机整体的一部分,其老化也可能与自噬有关。国外学者发现,年长女性的皮肤真皮中成纤维细胞的自噬体量相对年轻女性升高。体外实验表明,自噬降解步骤在年长供体的皮肤成纤维细胞中被抑制,进而导致自噬体的积累,这提示自噬功能在老年皮肤成纤维细胞中的受损,可能导致皮肤的完整性和皮肤强度受影响。细胞应激反应是胞外的条件下的变化或波动的反应造成大分子结构和功能的损坏。不同的应激因子触发不同的细胞反应,即诱导细胞修复机制,诱导一些应激因子短暂适应引起的细胞反应,诱导自噬或触发细胞死亡。创面的修复是一个复杂的过程,主要依靠修复细胞、炎性细胞、胞外基质和生长因子的协同作用来重建受损的软组织。在衰老哺乳动物,自噬系统的功能发生障碍,当遭遇创伤、感染、缺氧等打击后,由于自噬功能改变,可能无法维持细胞稳态,而向着延期愈合的方向发展。目前自噬对衰老皮肤创而愈合产生怎样的影响及机制尚未有相关研究,本项研究有助于了解自噬在老年创面愈合中扮演的角色,真正阐明衰老皮肤愈合的机制。第一部分自噬在不同年龄组大鼠皮肤急性创面愈合过程中的表达研究目的：通过研究不同年龄组大鼠急性创而愈合过程中,自噬相关基因LC3、Beclin-1、 p62的表达变化,观察老年和年轻大鼠正常皮肤中及在创面愈合过程中的自噬活动差异。方法：1.建立SD大鼠急性切创皮肤全层缺损模型,对不同年龄组创面组织进行HE和Masson三色染色,观察创面组织炎症、真皮微血管形成、胶原沉积、附属器发育以及再上皮化情况。2.透射电子显微镜观察不同年龄组大鼠正常皮肤成纤维细胞及角质形成细胞自噬体形态、结构。3.采用Envision免疫组化法和qRT-PCR、 Western blotting法检测LC3、 Beclin-1、p62在不同年龄组创面愈合中各时相点(Od、4d、7d、14d、21d)的表达差异。结果：1.光镜下情况：正常皮肤中,年轻组(3月龄)大鼠表皮较厚,皮下组织与真皮界限不十分清楚。老年组(18月龄)大鼠表皮厚度变薄,表皮与真皮连接变平坦、松弛。真皮中胶原纤维、弹性纤维数量减少,排列不规则,且常断裂成碎片。4d时,年轻组真皮可见毛细血管明显扩张,有大量中性粒细胞、巨噬细胞浸润,已有大量肉芽组织形成；老年组中性粒细胞浸润及真皮毛细血管数量均较年轻组少。7d时,年轻组可见创口已基本为肉芽组织填平,炎性细胞减少；老年组创口尚未被肉芽组织填平,仍有大量炎性细胞浸润。21d时,年轻组创口基本愈合,肉芽组织纤维化,向瘢痕组织转变。Masson三色染色可见胶原纤维数量较老年组增多,排列规则。老年组在21天后才完全或接近愈合,21d时仍可见少量炎性浸润,新生的真皮胶原积累松散,数量较少,排列不规则,多呈碎片弥漫状,规则积累的胶原蛋白较少。3.电镜观察情况：在年轻组和老年组大鼠正常皮肤的角质形成细胞及成纤维细胞中,均可见胞浆内双层膜或多层膜的自噬泡,其内可见细胞器如线粒体、内质网、核糖体残留,老年组自噬泡数量较年轻组多。4.免疫组织化学染色：LC3、 Beclin-1在年轻组和老年组大鼠正常皮肤的表皮及真皮均有表达,在创缘处表皮层、真皮层及毛囊、新生肉芽组织血管内皮细胞、巨噬细胞及成纤维细胞中均可见阳性表达。创伤前老年组两种蛋白在皮肤的表达均多于年轻组,LC3、 Beclin-1蛋白的表达均随时间呈上升趋势。在4d、7d、14d、21d时LC3的表达在年轻组较多,而Beclin-1则是老年组表达较多。5.不同年龄组创面愈合过程中LC3、 Beclin-1、 p62的mRNA表达情况：(1)不同年龄组大鼠LC3皮肤创面的mRNA表达变化量的比较在各时相点,两组LC3的表达增加均有显著性差异(P<0.05),均为4d<14d<7d<21d;在14d,年轻鼠组LC3的表达增加显著高于老年鼠组(P<0.05)。(2)不同年龄组大鼠皮肤创面Beclin-1的mRNA表达变化量的比较在各时相点,两组Beclin-1的表达增加有显著性差异(P<0.05),其中4d<14d<7d<21d;在各时相点,年轻鼠组中Beclin-1的表达增加均显著低于老年鼠组(P<0.05)。(3)不同年龄组大鼠皮肤创面的p62mRNA表达变化量的比较在各时相点,两组p62的表达减少有显著性差异(P<0.05),均为4d>7d>14d>21d;在4d、7d,年轻鼠组和老年鼠组的表达减少无显著性差异(P>0.05)；在14d、21d,年轻鼠组p62表达减少显著低于老年鼠组(P<0.05)。6. Western blotting检测不同年龄组创面愈合过程中LC3、Beclin-1、p62的蛋白表达情况：两组在创伤4d、7d、14d、21d时,LC3-Ⅱ Beclin-1蛋白表达均是逐渐升高的,而p62蛋白表达是逐渐下降的。LC3-Ⅱ Beclin-1p62的蛋白水平表达与基因水平表达相一致。结论：1.在正常衰老皮肤中自噬相关基因的表达高于正常年轻组皮肤,提示自噬与皮肤衰老密切相关,可能与衰老皮肤细胞的自噬降解能力下降,造成自噬体的蓄积,进而加快了皮肤老化进程有关。2.在皮肤创面愈合过程中,年轻及衰老皮肤细胞自噬相关蛋白的表达均有变化,提示自噬参与了皮肤创面愈合。年轻皮肤能够维持细胞的稳态,将自噬活动控制在正常范围内,从而促进创伤修复。而衰老皮肤由于本身自噬系统调控功能发生障碍,导致出现与自身不相适应的过度的自噬发生,从而造成细胞不可逆的损伤而延缓了创面愈合。第二部分富血小板血浆(PRP)对老年大鼠皮肤急性创面愈合过程中细胞自噬的影响目的：制作大鼠背部急性全层皮肤缺损模型,观察富血小板血浆(PRP)对创面愈合的疗效,并检测创面愈合过程中LC3、Beclin-1、 p62和CD31的表达情况,探讨PRP对自噬的影响,及自噬和创面愈合的相互关系。方法：1.建立SD大鼠急性切创皮肤全层缺损模型,分为生理盐水组(对照组)、富血小板血浆组(PRP组)、少血小板血浆组(PPP组),取第Od、4d、7d、14d皮肤创面标本,进行HE和Masson三色染色,观察创面愈合情况。2.透射电镜观察三组大鼠在各时相点的皮肤细胞自噬体形态、结构。3.比较三组创面愈合率。4.采用Envision免疫组化法、qRT-PCR、 Western-blotting法检测LC3、Beclin-1、 p62、 CD31在不同年龄组创面愈合中各时相点(4d、7d、14d、21d)的表达差异,在mRNA水平上分析各个基因之间的相关性。结果：1.光镜观察三组在各时相点的创面愈合情况(1)胶原纤维情况：在4d时,三组创面中心的成纤维细胞数量均增加,14d开始减少。7d时,增生的肉芽组织中有少量胶原纤维。14d时,PRP组不仅胶原纤维数量较多,而且胶原纤维的排列比较整齐,而在对照组中,上皮连接仍留有间隙,胶原纤维不成熟,数量较少,排列不规则。PPP组的胶原纤维生成情况介于两组之间。(2)新生血管及附属器情况：创伤4d三组创面组织中均可见少量血管内皮细胞及成纤维细胞。血管内皮细胞可见少量CD31表达,而对照组几乎未观察到血管内皮细胞及成纤维细胞。7d时,PRP组肉芽组织增长优于其他两组,三组的血管内皮细胞、成纤维细胞及新生毛细血管数量均增多,CD31表达量增多。在14d,PRP组出现新生毛细血管,管腔增大,可见血管平滑肌细胞,并初步形成毛细血管网。21d时PRP组毛细血管较多,毛细血管网结构正常,腺体增多。PPP组毛细血管及腺体数量相对PRP组较少,对照组则在三组中最少。2.电镜观察情况：在创伤4d,成纤维细胞和角质形成细胞内的自噬泡数量均增加,PRP组的自噬体数量增加相对于PPP组和对照组较少。3.三组创面愈合率的比较：PRP组愈合时间最短,再上皮化速度最快,优于另外两组,有显著性差异(P<0.05)。4.免疫组织化学染色：LC3、 Beclin-1蛋白在创面的表达特点同前,两个蛋白在PRP组的表达弱于其他两组。5.三组创面愈合过程中LC3、 Beclin-1、 p62的mRNA表达情况：LC3在各组内的各时相点表达量均有显著性差异(P<0.05),在14d、21d时三组之间有显著性差异(P<0.05)。 Beclin-1的表达量在7d、14d、21d时各组间均有显著差异(P＜0.05), PRP组表达量在这三个时相点均低于其他两组。p62的表达在三组均呈下降趋势,PRP在4d、7d时的表达量均小于正常对照组。相关性分析显示,在4d和7d时,CD31与Beclin-1呈负相关,与p62呈正相关。6. Western blotting检测三组创面愈合过程中LC3、 Beclin-1、p62的蛋白表达情况：在三组不同处理组中,LC3-Ⅱ、 Beclin-1、 CD31蛋白表达均随时间逐渐升高,p62蛋白表达随着时间变化逐渐下降。LC3-Ⅱ、Beclin-1、CD31、p62的蛋白水平表达与基因水平表达一致。结论：1.PRP在老年皮肤创面愈合中可能通过对细胞自噬的影响,参与促进创面愈合的作用。2.在衰老皮肤创面愈合过程中,自噬相关蛋白Beclin-1、 p62与CD31相关,提示自噬参与了创面愈合中新生血管、再上皮化等不同阶段。更多还原

【Abstract】 Senescence, also known as aging, is performance of during the body function decline and the degradation of overall with physiological disorders, characterized by the functional structure dysfunction of progressive multi-organ system. Expressed as DNA, proteins, lipids and the like modified cell damage and harmful accumulation, the body’s aging process predetermined by gene regulation, on the other hand, it is caused by internal and external "wear and tear", which both occurred in the cellular and molecular level. It is common that the development of the body rests undifferentiated cells to differentiate, from differentiation to aging, and then by the process of aging to apoptosis. But with emergence of the concept about "autophagy", people began to re-understanding of the aging process and its mechanisms in this popular field of life sciences research.Cell homeostasis is achieved by precisely regulating the synthesis and degradation of cellular components. There are two degradation pathways of intracellular substances, one is the ubiquitin-proteasome system (UPS), specifically responsible for the degradation of the majority of cellular proteins, mainly short-lived protein, which is a highly efficient protein degradation pathway. Another one is autophagy, the major protein degradation and long life organelles. Autophagy is important for the degradation of eukaryotic cells, also known as type Ⅱ cell death and it is a process of cell autophagy-related gene in the regulation of the use of lysosomal degradation of damaged cells and macromolecules. There are three main types of autophagy in cell, namely giant autophagy (Macroautophagy), micro-autophagy (Microautophagy) and chaperone-mediated autophagy. Autophagy is the main way of the the cycle of giant cytoplasmic constituents, commonly referred to as autophagy. The common process of the autophagy is that protein degradation are all achieved within lysosomes. It uses its lysosomal degradation damaged cells and macromolecules to achieve the cell management and quality control of proteins and organelles. Cells "junk", including damage or aging organelles, long-lived proteins, errors or misfolded protein synthesis, etc., must be promptly removed by autophagy.Whether the cells are in a normal physiological or pathological status,autophagy is always an important body defense and protection mechanisms to maintain cellular homeostasis or self-assurance cell survival. The study found that autophagy aging process in a variety of eukaryotic organisms plays an important regulatory mechanism. Autophagy function will decline with aging,which may be due to the changes in cell structure and the age-related decrease of function. The study said that autophagy plays a key role in the aging process in organisms, and as part of an organic whole organism, aging autophagy of skin tissue may also be relevant with it. Foreign scholars found that older women’s skin own a higher amount of fibroblasts than young women during the dermis autophagy.The results showed that the degradation process autophagy was inhibited with older donor skin fibroblasts, leading to the accumulation of autophagic, which indicated that autophagy fibroblasts damaged skin in the elderly, leading to skin integrity and strength of the skin damage.The cell stress in vitro is a change or fluctuation of extracellular conditions, which causes the structure and function of macromolecules damage. Different stress factors trigger different cellular responses that called induce cell repair mechanism, which induce transient adaptation of a number of stress factors to cause the cell response, induce autophagy or trigger cell death. Irreparable damage or exposure to prolonged stress may lead to aging. In aging mammals, since the function of autophagy system disorders, autophagy as a holding cell homeostasis auto repair process that increase with age becomes less effective. The aging body suffers from trauma, infection and hypoxia blow may be cause the decline of autophagy that may not maintain cell homeostasis, and then toward worsening or delayed healing.Wound repair is a complex process, rely mainly on repair cells, inflammatory cells, growth factors and extracellular matrix synergy damage to soft tissue reconstruction. Aged-skin wound is the difficult healing, which the pathogenesis is quite complex and subject to multiple factors. The aging of cells and the whole body system, local recurrent ischemia-reperfusion injury, bacterial colonization may lead to delayed healing of wounds. As already mentioned,aged skin autophagic activity decreased with aging, and activity increased autophagy has anti-aging effects, but autophagy senile skin wound healing and what impact mechanism is not related to research, this study helps to understand the impact and mechanisms of autophagy plays a role in wound healing of the elderly, which can at the same time to promote wound healing by regulating autophagy.part1the expression of autophagy in different age groups of rats with acute skin wound healing processObjective To study the expression of autophagy-related genes LC3、 Beclin-1、 p62in cutaneous wound healing between the different age groups rats.Methods1. A full-thickness excisional lesion were performed on the rats back. We observed and compared wound tissue inflammatory conditions, dermal remodeling,collagen accumulation condition and adnexal development status by HE and masson’s stained methods.2. Observe the ultrastructure of autophagy in the skin of the old and young rats with electron microscopy.3. We established new monoclonal antibodies against the LC3、 Beclin-1、 p62used them for the specific western blot and envision immunohistochemistry detection at different time points (0,4,7,14,21d). Another, we used qRT-PCR detection the gene expression of LC3,Beclin-1,p62at different time points (0,4,7,14,21d).Results1. HE staining showed that the microstructures of skin were differente between the young and the old rats. The young rats normal epidermis and dermis fibrous connective tissue were more maturational. The subcutaneous tissue of the young rats were thicker, however, the boundaries were not very clear. The older rats normal epidermis were thinner, the connection of epidermis and dermis flattened, relaxed. The number of elastic fibers and collagen fibers were reducing, irregular and breaked into pieces. The small blood vessels were significant vasodilation, a large number of neutrophils, macrophages infiltration on4th day after wounding. There was a large number of granulation tissue formation on4th day after wounding in the younger group, in addition there were large number of fibroblasts. Granulation tissue had been substantially filled on7th day after wounding, fewer inflammatory cells. The wounds were completely healed on21th day after wounding on young rats. The wound were not completely healed on21th day after wounding on old rats.2. Masson trichrome staining showed:In the old rats21th days after injury, still shows a small amount of inflammatory infiltration, newborn dermal collagen accumulation loose, fewer, irregular, mostly filled with debris like, less collagen accumulation rule. All the wounds were healed in the young group21th day after injury, the number of collagen fibers increased and arranged regularly.3. Observed with electron microscopy, there were autophagy in keratinocytes and fibroblasts. The autophagy had bilayer or multilayer film. And there were residual organelle such as mitochondria, endoplasmic reticulum, ribosomes residue. There were more autophagy in older group.4. Immunohistochemistry:LC3,Beclin-1in the epidermis and dermis were expressed in normal skin group of young and old rats. The edge of the wound, the epidermis, dermis and hair follicles showed positive staining. In the newborn granulation tissue vascular endothelial cells, macrophages and fibroblasts are visible expression. Before wounding, two protein expression in the skin were more in the older group.LC3,Beclin-1protein expression were upward trend over time after wounding. LC3expression were more in the younger group, and Beclin-1expression is higher in older group on4,7,14,21th day afer wounding.5. qRT-PCR(1) LC3mRNA expressed in cutaneous wound healing between the different age groups rats.LC3mRNA expression were significant differences (P<0.05) on7d,14d,21d after wounding,4d<14d<7d<21d.The expression of LC3was significantly higher in the young rats (P<0.05).(2) Beclin-1mRNA expressed in cutaneous wound healing between the different age groups rats.Beclin-1mRNA expression were significant differences (P<0.05) on4d,7d,14d,21d after wounding,4d<14d<7d<21d. The expression of LC3was significantly less in the young rats.(3) p62mRNA expressed in cutaneous wound healing between the different age groups rats.p62mRNA expression were significant differences (P<0.05) on4d,7d, 14d,21d after wounding,4d>7d>14d>21d.On4d,7d, the expression of p62had no significant difference, the expression of p62was significantly less in the young rats.6. Western blottingLC3-II, Beclin-1protein expression was gradually increased4d,7d,14d,21d after wounding in the two groups, and the p62protein expression is gradually declining, that consistented with mRNA qRT-PCR results of the three genesConclusions1. Autophagic degradation decreased, resulting in the accumulation of autophagy and the destruction of the integrity and strength of the skin.2. In cutaneous wound healing, autophagy ability was enhanced. The skin of young rats can maintain steady self, and control autophagy in the normal range, thereby promoting wound healing. Regulatory function was disorder, resulting in the excessive autophagy itself occur, resulting in irreversible damage and delay the healing process in old rats.Part2Platelet-rich plasma (PRP) effect on the cell autophagy in the skin wound healing in aged rats with acute.Objective:platelet rich plasma (PRP) is extracted by centrifugation of blood out from the autologous platelet concentrate, after activated can form a gelatinous substance (platelet gel, PG). Due to the rich variety of growth factors, active peptide and other substances, mainly PDGF, TGF-β,VEGF, EGF, IGF.these cytokines can reduce wound inflammation and promote vascularization to accelerate healing, but also to promote the proliferation of epidermal and dermal cells to restore normal dermal matrix remodeling skin structure. Establish the rat model of acute back full-thickness skin defect to observe the efficacy of PRP to promote wound healing, and the detection process of autophagy genes and CD31expression, and investigate the effect of PRP on autophagy and also study the relationship between autophagy and wound healing.Method:1. Establish the SD rat model of acute back full-thickness skin defect. To use saline (control group), platelet-rich plasma group (PRP group), less PRP group (PPP group) to treated wounds respectively. And then take the wound skin specimens at0.4,7,14and21th day, wound tissue after section for HE and Masson staining was observed.2. Using transmission electron microscope to observe the skin autophagy morphology and structure in three groups at different times.3. Comparison the rate of wound healing of three groups.4.Using Envision immunohistochemical method, qRT-PCR and western-blotting assay the expression of LC3, Beclin-1, p62, CD31at different times in different age groups.Results1. The number of fibroblasts were increased at4d and then started to decrease at14d after wounding. There were a few collagen fibers in granulation tissue at7d. Epithelialization and collagen synthesis improved, the number of collagen fibers increased and arranged regularly in PRP group on14d. In control group, epithelial connection was still a gap, collagen fibers had not be mature, irregular arrangement. The number and state of collagen fibers of PPP group fall in between the other two groups.2. There were a small amount of vascular endothelial cells and fibroblasts in granulation tissue on4d. In control group, There were few vascular endothelial cells and fibroblasts. On7d, granulation tissue developed best in PRP group. Vascular endothelial cells, capillaries and fibroblasts increased. The expression of CD31increased. On14d,PRP group had new capillaries, lumen increased, showed that the initial formation of vascular smooth muscle.On21d, there were most capillaries and sebaceous glands,capillary network architecture was normal in PRP group. There were the lowest capillaries in control group.3. The number of autophagy in keratinocytes and fibroblasts increased on4d. The autophagy had bilayer or multilayer film. And there were residual organelle such as mitochondria, endoplasmic reticulum, ribosomes residue. There were fewer autophagy in PRP group.4. The wound healing rate of PRP group was significantly higher than the other groups(P＜0.05) on4d,7d,14d,21d.5. LC3mRNA expression were significant differences on14d,21d after wounding (P<0.05). On14d, the expression of LC3was highest. Beclin-1mRNA expression were significant differences (P<0.05) on4d,7d,14d,21d after wounding. The expression of Beclin-1was significantly less in the PRP group. The expression of p62was significantly less in the PRP group. Correlation analysis of data showed that the expression CD31of were correlated with p62and Beclin-1, on the4d and7d,negatively correlated with Beclin-1, was positively correlated with p62.6. LC3Ⅱ, Beclin-1and CD31protein expression was gradually increased after wounding, and the p62protein expression is gradually declining in the three groups. It was consistent with mRNA qRT-PCR results of the three genes.Conclusions1. PRP inhibited autophagy activity in wound healing in old rats and improve wound healing.2. Expression CD31of were correlated with autophagy related gene p62and Beclin-1,suggested that autophagy participated in the angiogenesis during wound healing.更多还原