【作者】 程琳；

【导师】 周黎明；

【作者基本信息】 四川大学 ， 药学， 2022， 硕士

【摘要】 研究背景:21世纪是抗衰老的世纪,紫外线是导致衰老的重要原因之一。但是随着环境污染的加剧,臭氧层遭到破坏,导致到达地球表面的紫外线增多。人体皮肤是人体最大的器官,也是人体的主要保护屏障。它长期暴露在阳光下,但是阳光中的紫外线辐射会对皮肤造成伤害。其中中波紫外线(Ultraviolet B,UVB)(280-320nm)能穿过表皮到达真皮上层,从而导致暴露于UVB辐射下的皮肤出现DNA损伤,同时因活性氧(Reactive Oxygen Species,ROS)的增加和内源性抗氧化剂的减少而出现严重的氧化应激,进一步加重DNA的损伤。UVB引起的DNA损伤和氧化应激会引起皮肤细胞的一系列病理性改变,如晒伤,红斑,水肿等。这类副效应会导致皮肤光老化,是皮肤癌发生发展和免疫系统功能下降的关键风险因素。再加上人类对皮肤美容的关注度日益增加,如何修复紫外线损伤的皮肤是人类皮肤健康关注的焦点。干细胞拥有自我更新及分化成不同细胞和组织的能力,是一种有活力的细胞。同时,干细胞分泌的多种物质在细胞通讯中发挥重要的自分泌/旁分泌作用。间充质干细胞(Mesenchymal Stem Cells,MSCs)是一种重要的成体干细胞。MSCs细胞广泛应用于肌肉和骨衰退性疾病、肝病、脊髓和脑神经损伤、老年痴呆、免疫性疾病、心脑血管疾病等。人脐带来源的间充质干细胞(Human Umbilical Cord MSCs,HUCMSCs)与其他来源的MSCs细胞相比,具有非侵入性分离方法、无伦理问题、较低的免疫原性、更高的增殖能力和更稳定的倍增时间等优势,从而具有良好的应用前景。有文献报道,HUCMSCs细胞来源的细胞外囊泡(Extracellular Vesicles,EVs)减少紫外线辐射诱导的ROS产生,减轻氧化应激和DNA损伤,减少衰老和凋亡细胞,上调I型胶原蛋白的表达和促进细胞增殖。HUCMSCs细胞的条件培养基能减少人类患者的皱纹,增加小鼠皮肤中的超氧化物歧化酶和谷胱甘肽过氧化物酶活性,下调丙二醛的表达。尽管人们已经认识到HUCMSCs细胞具有抗光损伤作用,但是从细胞上清液中分离EVs的方法复杂,并且时间和材料成本高。据此,我们希望通过透析袋浓缩HUCMSCs细胞上清液(Concentrated HUCMSCs Supernatant,CHS)的方法,因其方法制备工艺绿色简单高效、普适性好。综上所述,HUCMSCs细胞在抗光损伤方面前景无限,而且来源于脐带的间充质干细胞,是安全的,再加上CHS的制备方法高效简单。基于以上分析,本课题将通过体内外实验探讨CHS对UVB损伤是否具有修复作用。研究方法:1.通过组织块贴壁法分离HUCMSCs细胞。通过流式细胞术检测HUCMSCs细胞表面标记物。通过成骨、成脂和成软骨实验检测HUCMSCs细胞分化能力。通过透析袋浓缩HUCMSCs细胞的培养上清液。2.建立UVB损伤人永生化角质形成细胞(The immortalized human keratinocyte cell line,HaCaT)模型,通过CCK8检测细胞活力,通过细胞衰老相关β-半乳糖苷酶(SA-β-Gal)染色鉴定衰老细胞。3.研究CHS对UVB损伤HaCaT细胞的修复作用。通过CCK8检测细胞活力,划痕实验检测细胞迁移能力,PI单染流式细胞术检测细胞周期,SA-β-Gal染色鉴定衰老细胞,AO/EB荧光染色观察细胞存活情况,免疫印迹检测Beclin1、LC3、PI3K、p-PI3K、AKT、p-AKT、m TOR、p-m TOR蛋白的表达,细胞自噬染色法(MDC法)检测细胞自噬囊泡。4.研究CHS对UVB损伤的小鼠皮肤的修复作用。通过观察皮肤状态检测CHS的外观效果,通过血象和脏器系数检测CHS的安全性,通过HE染色观察皮肤的结构,通过Masson染色检测胶原蛋白的表达,SA-β-Gal染色鉴定衰老细胞。研究结果:1.HUCMSCs细胞形状呈梭形。P2代HUCMSCs细胞高表达标记物CD73、CD90和CD105均高于99.94%,低表达标记物CD11b、CD19和HLA-DR低于0.77%。HUCMSCs细胞具有分化为骨、脂肪和软骨细胞的能力。HUCMSCs细胞染色体核型正常。CHS总蛋白浓度为183.18 mg/m L。2.100 m J/cm~2的UVB剂量辐射HaCaT细胞,对细胞活力有一定损伤作用,且衰老细胞明显增加。因此,选择100 m J/cm~2作为UVB损伤细胞模型的剂量。3.CHS促进UVB损伤的HaCaT细胞活力,增加UVB损伤后细胞迁移力,改善UVB辐射造成的G1期阻滞,减少UVB损伤后衰老和死亡细胞的数量。4.CHS上调HaCaT细胞Beclin 1和LC3-II蛋白的表达水平。CHS上调UVB损伤HaCaT细胞Beclin 1和LC3-II蛋白的表达水平。CHS上调UVB损伤HaCaT细胞中p-AKT和p-m TOR蛋白的表达水平。CHS增加UVB损伤HaCaT细胞自噬水平。5.将CHS涂敷于被UVB破坏的小鼠皮肤后,皮肤弹性及皮肤光泽度有所改善;真皮层胶原蛋白表达增加;皮肤SA-β-Gal表达下调。研究结论:1.CHS对UVB损伤的HaCaT细胞有修复作用:促进细胞活力,促进细胞迁移力,减少衰老和死亡的细胞,促进自噬,激活PI3K/AKT/m TOR信号通路。2.CHS对UVB损伤的小鼠皮肤有修复作用:皮肤变亮,胶原蛋白表达增加,衰老细胞减少。更多还原

【Abstract】 Research Background:The 21st century is the century of anti-aging,and ultraviolet is one of the major causes of aging.However,with the increase in environmental pollution,the ozone layer has been destroyed,resulting in an increase in ultraviolet reaching the earth’s surface.Human skin is the largest organ of the body and the main protective barrier of the body.It is exposed to sunlight for long periods of time,but ultraviolet radiation from sunlight can cause damage to the skin.Among them,medium-wave ultraviolet B(UVB)(280-320 nm)penetrates the epidermis to reach the upper dermis,resulting in DNA damage to the skin exposed to UVB radiation and severe oxidative stress due to increased reactive oxygen species(ROS)and reduced endogenous antioxidants,further aggravating DNA damage.UVB-induced DNA damage and oxidative stress can cause a series of pathological changes in skin cells,such as sunburn,erythema,and edema.Such side effects can lead to photoaging of the skin,which is a key risk factor for skin cancer development and decreased immune system function.Coupled with the increasing concern for skin beauty,the repair of ultraviolet-damaged skin is a major concern for human skin health.Stem cells possess the ability to self-renew and differentiate into different cells and tissues.At the same time,stem cells secrete a variety of substances that play an important autocrine or paracrine role in cellular communication.Mesenchymal stem cells(MSCs)are an important type of adult stem cells and MSCs are widely used in muscle and bone decline diseases,liver diseases,spinal cord and brain nerve injuries,Alzheimer’s disease,immune diseases,cardiovascular diseases,etc.Human umbilical cord MSCs(HUCMSCs)have advantages over other MSCs,such as non-invasive isolation method,no ethical issues,lower immunogenicity,higher proliferation capacity and more stable ploidy time,thus have good application prospects.It has been reported that Extracellular Vesicles(EVs)derived from HUCMSCs reduce ultraviolet radiation-induced ROS production,attenuate oxidative stress and DNA damage,reduce senescent and apoptotic cells,upregulate type I collagen expression and promote cell proliferation.Conditioned medium of HUCMSCs reduces wrinkles in human patients,increases superoxide dismutase and glutathione peroxidase activities and downregulate malondialdehyde expression in mouse skin.Although it has been recognized that HUCMSCs have anti-photodamaging effects,the isolation of EVs from cell supernatants is complex and costly in terms of time and materials.Accordingly,we hope to use the method of concentrated HUCMSCs supernatant(CHS)by dialysis bag because of its green,simple,efficient and universal preparation process.In summary,HUCMSCs are promising in anti-photodamage,and are derived from umbilical cord MSCs,which are safe,plus the CHS preparation method is efficient and simple.Based on the above analysis,this project will explore whether CHS has a repairing effect on UVB damage through in vivo and vitro experiments.Research methods:1.HUCMSCs were isolated by tissue block apposition method.Surface markers of HUCMSCs were detected by flow cytometry.The differentiation ability of HUCMSCs was detected by osteogenic,lipogenic and chondrogenic assays.The supernatant of HUCMSCs was concentrated by dialysis bag.2.A UVB-injured immortalized human keratinocyte cell line(HaCaT)model was established,cell viability was detected by CCK8,and senescent cells were identified by cellular senescence-associatedβ-galactosidase(SA-β-Gal)staining.3.To study the repair effect of CHS on UVB-damaged HaCaT cells.Cell viability was detected by CCK8,cell migration ability by scratch assay,cell cycle by PI single-stain flow cytometry,identification of senescent cells by SA-β-Gal staining,cell survival by AO/EB fluorescence staining,immunoblotting for Beclin 1,LC3,PI3K,p-PI3K,AKT,p-AKT,m TOR,p-m TOR protein.The expression of autophagic vesicles was detected by cellular autophagy staining(MDC method).4.To study the repair effect of CHS on the skin of UVB-injured mice.The cosmetic effect of CHS was detected by observing the skin condition.The safety of CHS was detected by blood and organ coefficients.The structure of skin was observed by HE staining.The expression of collagen was detected by Masson staining.The senescent cells were identified by SA-β-Gal staining.Research result:1.The shape of HUCMSCs cells was spindle-shaped.The high expression markers CD73,CD90 and CD105 of P2 generation HUCMSCs were all higher than 99.94%,and the low expression markers CD11b,CD19 and HLA-DR were lower than 0.77%.HUCMSCs had the ability to differentiate into bone,adipose and chondrocytes.The karyotype of HUCMSCs was normal.The total protein concentration of CHS was183.18 mg/m L.2.The UVB dose of 100 m J/cm~2 irradiated HaCaT cells,which had a certain damage effect on cell viability,and the senescent cells were significantly increased.Therefore,100 m J/cm~2 was chosen as the dose for the UVB damage cell model.3.CHS promoted UVB-injured HaCaT cell viability,increased cell migration ability after UVB injury,improved G1 phase arrest caused by UVB radiation,and reduced the number of senescent and dead cells after UVB injury.4.CHS upregulated the expression levels of Beclin 1 and LC3-II proteins in HaCaT cells.CHS upregulated the expression levels of Beclin 1 and LC3-II proteins in UVB-injured HaCaT cells.p-AKT and p-m TOR proteins in UVB-injured HaCaT cells were upregulated by CHS.CHS increased the level of autophagy in UVB-damaged HaCaT cells.5.After applying CHS to the skin of mice damaged by UVB,the skin elasticity and skin gloss were improved.CHS increased the expression of collagen in the dermis.CHS down-regulated the expression of SA-β-Gal in the skin.Research conclusion:1.CHS has repair effects on UVB-damaged HaCaT cells:promotes cell viability,promotes cell migration,reduces senescent and dying cells,promotes autophagy,and activates PI3K/AKT/m TOR signaling pathway.2.CHS has a repairing effect on UVB-damaged mouse skin:skin brightens,collagen expression increases and senescent cells decrease.更多还原