【作者】 孙青；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2016， 博士

【摘要】 皮肤指身体表面包在肌肉外面的组织,是人体最大的器官,主要承担着保护身体、排汗、感觉冷热和压力等功能。皮肤覆盖全身,它使体内各种组织和器官免受物理性、机械性、化学性和病原微生物性的侵袭。当由于外界损伤或疾病等因素造成皮肤缺损时,其危害可以是致命的。皮肤组织工程是利用生物学和工程学原理,构建出用于修复、维持和改善损伤组织功能的替代物,形成人工再生的皮肤等同物,移植于需要修复、重建的皮肤病损处。随着干细胞和组织工程研究不断深入,干细胞作为种子细胞在组织工程中的价值得到逐步体现。寻找新型种子细胞在国内外都是皮肤损伤修复研究领域的核心和热点。羊水细胞由于其所处的特殊的解剖位置,长期以来受到了人们的重视。对于羊水细胞的研究最早可追溯至20世纪初,随着羊膜穿刺技术和细胞培养条件的日趋成熟,羊水细胞的培养成功率也得到了提高,羊水细胞的研究也得到了迅速的发展。羊水细胞在基因突变导致的染色体异常、遗传病和先天性代谢异常等疾病的产前诊断中发挥着重要作用。近年来研究发现羊水细胞中含有多种干细胞的标记物,且在体外通过特殊的诱导微环境能够分化成多种不同类型的细胞,这使羊水来源干细胞成为干细胞研究的一个新的热点。因此羊水作为干细胞的一个新的来源,将为干细胞的研究揭开一个新的研究领域,为组织工程提供新的种子细胞来源。本研究皆在建立人羊水干细胞体外分离和培养方法,并分析其生物学特性,利用羊水干细胞的高增殖和多向分化潜能,开展羊水干细胞体外向表皮角质细胞的分化研究,并在基因、蛋白、超微结构等方面验证;在此基础上进一步利用人羊水干细胞的低免疫原性的特征,构建小鼠皮肤损伤模型,展开羊水干细胞对皮肤损伤修复的研究,观察植入细胞的存活、迁移、分化及小鼠创面的修复,探讨其加快小鼠创面修复的关键分子机制。同时,初步探讨羊水干细胞在真皮层汗腺损伤修复中的作用,为皮肤组织工程提供理想的种子细胞,也为临床治疗大面积烧伤病人提出新方案,具有研究的创新性和潜在的应用价值。第一部分人羊水干细胞的体外培养方法和生物学特征鉴定及其向表皮角质细胞分化的研究目的:建立体外分离、提取、培养和纯化人羊水干细胞的方法,并对其生物学特性进行鉴定,在此基础上,定向诱导羊水干细胞分化为表皮角质细胞,并对其生物学特性及功能进行鉴定。方法:选取孕19-22周健康妇女羊膜穿刺之羊水组织,在无菌条件下用cd117磁珠分选,分选出阳性细胞,1200rpm离心后加入羊水干细胞培养基,置于37℃、5%co2培养箱内培养,每天全量换液。倒置显微镜观察体外培养的羊水干细胞的形态,增殖情况。rt-pcr检测不同传代次数羊水干细胞、胚胎干细胞、成体干细胞标志基因的表达;流式细胞术检测其表面标志物;细胞连续培养计数分析羊水干细胞的传代增殖能力。待细胞生长至汇合率为70%时,进行传代培养。取第三代的羊水干细胞,以2×104个/皿的密度接种于6cm细胞培养皿中,收集培养过人角质细胞的kgm2培养基,用0.22μm的滤器过滤,收集到的培养基为条件培养基cm(conditionedmedium)及添加诱导因子的角质化细胞培养的基础培养基kbm2为诱导培养基,每天更换诱导培养基;待诱导后的细胞生长至汇合率为70%-80%时,对细胞消化传代,在倒置显微镜下观察诱导分化细胞的形态变化,待细胞出现上皮样克隆时,在诱导培养基中额外添加bmp4和维生素c,待诱导分化30天后得到羊水干细胞来源的的角质细胞。rt-pcr检测、免疫荧光染色分别在基因、蛋白水平检测诱导后细胞的生物学特征;流式细胞术检测羊水干细胞的分化率;透射电镜技术观察细胞内部超微结构;三维气液组织培养方法及h&e染色检测分化后的羊水干细胞形成复层上皮的能力,免疫组织化学分析诱导分化的羊水干细胞形成复层上皮中各个不同层次的标志蛋白的表达。结果:(1)羊水干细胞表达表皮干细胞标志(k19和β1-integrin)、胚胎干细胞的标志(oct4、sox-2、c-myc、klf4、rex-1和nanog)和上皮细胞的标志(k8和k18)及b7家族负性分子b7h1、b7h2、b7h3和b7h4及btla,但并不表达角质细胞的标志(k14)和正性共刺激分子cd40、cd80和cd86;t淋巴细胞体外增殖实验显示,与羊水干细胞混合培养的t淋巴细胞增殖较前明显减缓,而加入b7h1和b7h4阻断性抗体后,t淋巴细胞数量明显增加,且阻断b7h4后的作用更为显著;(2)羊水干细胞诱导分化获得的角质细胞呈典型的上皮样形态,排列规则且紧密,边界清晰,类似铺路石状,在基因和蛋白水平均表达正常角质化细胞的标志k5和k14,且分化率约为35%;(3)羊水干细胞分化而来的角质细胞保留了羊水干细胞的高核质比,且具有羊水干细胞不具有的特殊细胞器:张力原纤维;(4)三维气液培养及h&e染色结果显示羊水干细胞来源的角质细胞能够进一步分化成熟,形成复层上皮,表达角质细胞成熟的标志k10,involucrin。结论:从羊膜穿刺健康样本,经过cd117磁珠分选后,成功获得羊水干细胞,其干性介于胚胎干细胞与成体干细胞之间,具有向表皮角质细胞的分化潜能,表达b7家族负性分子,具有免疫调节作用及高增殖能力。体外诱导羊水干细胞获得的角质细胞在基因和蛋白水平表达角质化细胞标志k5和k14,并具有角质细胞典型的细胞器:张力原纤维,并且能进一步分化成熟,形成复层上皮,表达角质细胞成熟的标志。因此,羊水干细胞可以做为皮肤损伤修复的有价值的种子细胞。第二部分人羊水干细胞参与小鼠皮肤损伤修复的研究及初步机制探讨目的:建立小鼠背部皮肤损伤模型,利用羊水干细胞进行移植修复,观察移植后实验动物皮肤损伤修复情况,羊水干细胞在小鼠体内的存活,细胞的增殖及分化并分析其可能的修复机制,为进一步深入研究羊水干细胞在皮肤损伤修复中的作用奠定基础,也为临床治疗提出新方案。方法:利用稳定传代的羊水干细胞,用慢病毒转染了gfp报告基因,病毒滴度为6.5×106iu/ml,且流式检测其转化效率,确保后期实验的准确性。取鼠龄为4-6周的雄性balb/c小鼠,将其背部的毛发用电动理发器剔除后在构建直径为1cm的圆形皮肤缺损创面,在创面边缘注射入转染gfp的羊水干细胞,羊水干细胞的数量为5x106,将转染gfp的相同数量的成纤维细胞及假手术组作为对照,并将手术后的小鼠单独饲养,观察至小鼠完全苏醒并能自由活动,每隔一天观察,并未出现异常及明显的免疫排斥反应。用uthscsaimagetool软件计算小鼠创面大小,并作图分析;分别取小鼠创面4天的肉芽组织及14天的修复组织进行h&e染色,观察炎症细胞、血管的新生及创面的修复情况;流式细胞术及免疫荧光染色技术检测植入小鼠创面细胞的存活情况及分化情况;real-timepcr分别检测不同天数小鼠创面皮肤修复因子及炎症相关因子的表达。利用小分子rna干扰技术下调b7h4分子在羊水干细胞中的表达;制造皮肤损伤模型,在创面边缘注射入下调b7h4的羊水干细胞,其数量为5x106,将转染gfp的成纤维细胞及正常羊水干细胞组作为对照,分别取不同组别的小鼠创面进行免疫化学、流式细胞术检测浸润的t淋巴细胞数量;real-timepcr分别检测不同天数不同组别小鼠创面皮肤修复因子及炎症相关因子的表达,采用spss10.0统计学软件进行统计学分析。计量资料以均数±标准差(x±s)表示,组间两两对比进行t检验,以p<0.05具有统计学意义。结果:(1)羊水干细胞组在移植细胞7天后,小鼠背部创面面积明显小于成纤维细胞组和假手术组,术后14天,羊水干细胞组小鼠背部创面修复明显加快,对照组创面愈合也较为明显;术后21天,羊水干细胞组小鼠背部创面完全愈合,对照组创面较前缩小,但背部仍显明显创面;(2)羊水干细胞移植组新生表皮较成纤维细胞组和假手术组更厚,并且有比较多的新生血管和成纤维细胞,未见浸润的炎症细胞,而成纤维细胞移植组和假手术组新生表皮较薄,有较多的炎症细胞;术后14天,羊水干细胞移植组出现明显的真皮乳头结构和毛囊等皮肤附属器;(3)随着修复的进行,羊水干细胞在体内的存在逐渐较少。荧光检测结果显示,羊水干细胞组和成纤维细胞组在术后7天仍有部分的gfp阳性细胞存在;术后21天,羊水干细胞组仅有个别gfp阳性细胞存在;(4)在修复早期,修复相关因子(bfgf,vegf,cxcl12,tgf-β1,kgf)在羊水干细胞中的表达明显高于成纤维细胞和假手术组,随着修复的进行,它们的表达逐渐下降,21天时,表达量与正常小鼠皮肤的表达接近,而成纤维细胞组和假手术组的修复因子在7天时表达明显低于羊水干细胞组,21天才到达最高;而早期的炎症相关因子(il-1β,il-6,tnf-α,cox2及mac3)在成纤维细胞组和假手术组的表达明显高于羊水干细胞组,随着修复的进行,各组的表达都逐渐下降,到达21天,羊水干细胞中的表达接近于正常小鼠皮肤的表达,而成纤维细胞组和假手术组在21天时仍然高于羊水干细胞组;(5)经小分子rna干扰后,羊水干细胞b7h4的表达明显下降,与下调b7h4的羊水干细胞共培养的t淋巴细胞增殖数量明显增加,由此显示羊水干细胞表达的共刺激分子b7h4是下调t细胞体内外增殖的关键因子;(6)而免疫组化实验结果也显示,下调b7h4的羊水干细胞组有较多的浸润t淋巴细胞,流式细胞仪检测了浸润t淋巴细胞的比例发现,成纤维细胞移植组、下调b7h4羊水干细胞移植组浸润的t淋巴细胞数量明显高于羊水干细胞组;(7)相应生物因子检测结果表明,在修复早期,修复相关因子在羊水干细胞中的表达明显高于成纤维细胞和下调b7h4羊水干细胞移植组,随着修复的进行,它们的表达逐渐下降,而成纤维细胞组和下调b7h4羊水干细胞移植组因子的表达在21天时才到达最高;同时还发现,在早期,炎症相关因子在成纤维细胞组和下调b7h4羊水干细胞组的表达明显高于羊水干细胞组,随着修复的进行,各组的表达都逐渐下降,而成纤维细胞组和下调b7h4羊水干细胞组的表达在21天时仍然高于羊水干细胞组。结论:羊水干细胞能够明显加快小鼠创面的愈合,促进血管形成,调节局部炎症反应,提前形成皮肤附属器;在修复早期,羊水干细胞能够在体内直接诱导分化成为表皮角质细胞,表达角质细胞标志;随着修复的完成,羊水干细胞在小鼠体内逐渐消失。修复后期,羊水干细胞可通过微环境,募集小鼠自身的干细胞参与修复。其通过微环境加快小鼠背部创面的愈合可能是通过b7h4的介导作用。下调b7h4在羊水干细胞的表达,可介导创面修复减缓,损伤微环境中t淋巴细胞浸润数量增加以及炎症反应加剧。我们的实验发现负性b7家族分子-b7h4是介导羊水干细胞免疫调节的关键分子。羊水干细胞通过表达负性分子b7h4,建立有利于创面修复的炎症反应微环境,从而加快创面的愈合。第三部分人羊水干细胞向真皮汗腺细胞分化的研究目的:定向诱导羊水干细胞分化为真皮汗腺样细胞,并对其生物学特性及功能进行鉴定,在此基础上,进一步探讨羊水干细胞来源汗腺细胞对小鼠汗腺损伤修复。方法:用传统方法将羊水干细胞培养后,real-timepcr检测不同株羊水干细胞在汗腺相关基因的表达,分析其向汗腺细胞分化的潜能。取第三代的羊水干细胞,以2×104个/皿的密度接种于6cm细胞培养皿中,收集培养过人汗腺细胞的条件培养基,用0.22μm的滤器过滤,收集到的培养基为cm及添加shh的汗腺细胞培养基sgm为诱导培养基,两者比例为1:1,每天更换诱导培养基;待诱导后的细胞生长至汇合率为70%-80%时,对细胞消化传代,在倒置显微镜下观察诱导分化细胞的形态变化,分别在7天,14天,21天,28天时对诱导的羊水干细胞进行检测。real-timepcr、免疫荧光染色、分别在基因、蛋白水平检测诱导后细胞的生物学特征;流式细胞术检测羊水干细胞的分化率;透射电镜技术观察细胞内部超微结构;三维气液组织培养方法及h&e染色检测分化后的羊水干细胞形成汗腺样结构的能力,免疫荧光染色分析诱导分化的羊水干细胞形成的汗腺样结构在汗腺相关标志的表达。在裸鼠肩部构建汗腺损伤模型,将经过生物学功能鉴定的羊水干细胞来源的汗腺细胞注射入损伤部位,注射入的细胞量为2x105。将pbs注射组,同体对侧不做任何处理设为空白对照组。结果:(1)羊水干细胞在汗腺相关基因k18,cd24及k19有比较高的表达,弱表达eda,edar,k8,但不表达cea;(2)羊水干细胞诱导分化获得的汗腺细胞形成不同直径的紧密排列的多层的扁平细胞,呈“鹅卵石”样,在基因和蛋白水平均表达cea,且eda,edar及k8的表达也有显著提高,流式检测其分化率约为30%以上;(3)透射电镜结果显示,羊水干细胞分化而来的汗腺细胞具有羊水干细胞不具有的特殊细胞器:微绒毛,乙酰胆碱实验证明该汗腺的分泌功能;(4)三维气液培养及h&e染色结果显示羊水干细胞来源的汗腺细胞能够进一步分化成熟,形成汗腺样结构,表达汗腺腺体的标志:eda,edar,cea,k8,k18,na-k-atp及sma;(5)汗腺肩部损伤部位能够形成汗腺腺体样结构,表达k8,cea等标志。结论:羊水干细胞具有向汗腺细胞的分化潜能。体外诱导羊水干细胞获得的汗腺细胞在基因和蛋白水平表达汗腺细胞标志eda,edar,k8,cea,其分化率约为30%,分化获得的汗腺细胞具有汗腺的典型特征:微绒毛,并且能进一步分化形成汗腺样结构,表达汗腺腺体的标志,能够在体外修复损伤汗腺,形成汗腺腺体结构。综上所述,本研究建立的羊水干细胞体外培养和扩增体系,并检测了其生物学特征,在此基础上,体外分化形成表皮角质细胞不仅在细胞水平,更在分子、超微结构上证实了分化而来的角质细胞不仅具有正常角质细胞的形态,而且能进一步成熟形成复层上皮,同时也通过体内实验证实羊水干细胞表达的负性B7H4分子介导其免疫调节作用,使小鼠皮肤创面修复效果显著。并在此基础上,初步研究了羊水干细胞向真皮层汗腺细胞的分化,证明了羊水干细胞不仅能在体外诱导分化形成汗腺细胞,并且能在体内形成汗腺样结构,这为进一步探讨羊水干细胞移植治疗大面积深度烧伤及其他免疫系统疾病的临床运用提供了新的理论基础,具有研究的创新性及良好的应用价值。更多还原

【Abstract】 Skin is the body’s largest organ, is mainly responsible for the protection of the body, sweating, feeling hot and cold and pressure function. Skin covering the whole body, it makes the body of various tissues and organs from physical, mechanical, chemical and pathogenic microbial invasion. When the skin defect caused by external injury or disease, the harm can be fatal. Skin tissue repair and functional reconstruction are the core and focus of bio-medical research both at home and abroad. Amniotic fluid cells due to their special anatomical location, received the people’s attention for a long time. The study of amniotic fluid cells can be traced back to the early 20 th century. With the growing maturity of amniocentesis and cell culture conditions, the success rate for culture of amniotic fluid cells has also been improved, amniotic fluid cell research has also been a rapid development. Amniotic fluid cell plays an important role in chromosomal abnormalities, genetic disease and congenital metabolic disorders, and other diseases in the prenatal diagnosis. Recent studies have found that amniotic cells contain a variety of stem cell markers, h AFSCs can be induced to a variety of different types of cells by special induction micro-environment, and Amniotic fluid-derived stem cells become a new hot spot for stem cell research. So as a new source of stem cells, h AFSCs will open a new field of stem cell study and provide a new source of seed cells for tissue engineering.Our study is aim to establish a method to isolate and culture human amniotic fluid stem in vitro and to analyze its biological characteristics. According to the high proliferation and differentiation potential of amniotic fluid stem cells, a study on amniotic fluid stem cell differentiation was carried out. We will verify the h AFSCs derived kerationcytes in m RNA, protein and ultra-structural level. Based on above results, combined with the low immunogenicity of h AFSCs, we will study on the repair of skin damage by amniotic fluid stem cells. We will also use a mouse excisional wound model to observe the survival, migration and differentiation of the implanted cells and to explore the key molecular mechanism of wound healing in mice. The research will provide ideal seed cells for skin tissue engineering, and will put forward new protocol for the clinical treatment of large area burn patients, which will have innovative and potential application value. Part I The cultivation, identification and differentiation of human amniotic fluid stem cells into keratinocytes in vitroObjective: To establish a method to isolate, extract and purify of human amniotic fluid stem cells and to identify its biological characteristics. On the basis of this, to differentiate of amniotic fluid stem cells into keratinocytes, and to identify its biological characteristics.Methods: Samples of amniotic fluid were obtained following routine amniocentesis carried out on pregnant women after 19-22 weeks of gestation. After amniocentesis, immunoselection with an antibody specific for human c-Kit(CD117) was used to isolate h AFSCs. Cultures of h AFSCs were maintained in a humidified incubator under 5% CO2 at 37°C. RT-PCR detected the expression of embryonic stem cells and adult stem cell marker of different passages of amniotic fluid stem cells. Third-passage h AFSCs were seeded in 6-well plates at a density of 2 ×104 cells per well. In the experimental group, inducing medium keratinocyte basal medium-2 supplemented with factors and keratinocyte-conditioned medium at a ratio of 1:1 was used. BMP4 and vitamin C were added additionally when the epithelial clone formed. RT-PCR and immunofluorescence staining were used to detect the gene and protein level of AFS-K respectively. Flow cytometry were to detect the differentiation rate of h AFSCs. The transmission electron microscope was used to observe the ultrastructure of cells. H&E staining analyze the ability to form a completely pluristratified epithelium in 3D air-liquid tissue cultures. Immunohistochemical analysis the markers of different layers.Results:(1) h AFSCs express K19 and β1-integrin(markers of epithelial stem cells) as well as K8 and K18(markers of epithelium). In addition, h AFSCs expressed marker genes for embryonic stem cells, including Oct4, Sox2, C-Myc, Klf4, Rex-1 and Nanog. After 10 passages, subcultures of h AFSCs maintained a gene expression pattern similar to that of early-passage cells. h AFSCs were negative for the positive co-stimulatory molecules CD40, CD80 and CD86 but showed strong expression of the negative co-stimulatory molecules B7H1, B7H2, B7H3, B7H4 and BTLA. h AFSCs can inhibit the activity of human and mouse T lymphocytes. The h AFSCs B7H4-blocking antibody can significantly inhibit the suppression of T lymphocytes by h AFSCs.(2) Keratinocytes derived from h AFSCs(AFS-K) formed spontaneously with typical pavementous epithelial morphology. RT-PCR results identified h AFS-K as K5 and K14 positive. Flow cytometry analysis of K5- and K14-positive h AFS-K cells showed the differentiation rate to 35%, and immunostaining confirmed that the cells express the K5 and K14 proteins.(3) h AFS-K cells display features of keratinocyte precursor cells, with tonofibrils distributed in the cytoplasm, but these cells also have a prominent nucleolus and a high nucleus-to-cytoplasm ratio.(4) H&E staining of organotypic cultures of h AFS-K showed a pluristratified epithelium. Immunohistochemistry showed that K14 and K5 were present in the basal compartment. A reciprocal pattern was found for K10, for involucrin, and for markers of keratinocytes in suprabasal layers, which was present only in layers overlying the basal layer.Conclusions: h AFSCs have the potential to differentiate into epithelium, showed strong expression of the negative co-stimulatory molecules suggests that they may be involved in inhibiting lymphocyte activation and suppressing inflammatory responses. h AFS-K cells express the K5 and K14 proteins and have similar biological characteristics to keratinocytes. h AFS-K cells can form a completely pluristratified epithelium in 3D air-liquid tissue cultures. Taken together, the present study identifies h AFSCs may be a novel source for seed cells for epidermal regeneration. Part II The effects of h AFSCs transplantation in reparation of skin damage and its preliminary mechanismObjective: An injury model was generated and the h AFSCs were transplanted to the edge of the wound area. We aim to observe the recovery of the mice and the proliferation, differentiation of the h AFSCs and analyze the possible mechanism of the reparation of wound.Methods: A lentivirus containing the GFP gene was obtained at a titre of 6.5 ×106 IU/ml. An injury model was generated in BALB/c male mice. A full-thickness skin wound(1 cm diameter) was made on the back of the mouse. The wound received a total of 5×106 GFP-positive h AFSCs or fibroblasts, and then housed the mice individually. The wounds were measured using the UTHSCSA Image Tool. 4 days of granulation tissue and 14 days of repair tissue were harvested and then stained with H&E. We observed the inflammatory cells and neonatal vascular. Flow cytometry and immunofluorescence staining were to detect the survival and differentiation of the cells. Real-Time PCR analyzed the expression of repair-related and inflammatory factors in different days. On such a basis, the interfering RNA for B7H4 down-regulation was constructed. The same injury model was generated in BALB/c male mice. The wound received a total of 5×106GFP-positive B7H4 down-regulated h AFSCs, normal h AFSCs or fibroblasts. Immunohistochemistry and flow cytometry were to detect the amounts of infiltration of T lymphocytes. Real-Time PCR analyzed the expression of repair-related and inflammatory factors in different days. The data, which are presented as the mean± SD, were analyzed using the t-test and considered statistically significant at P < 0.05.Results:(1) Wounds treated with h AFSCs exhibited accelerated wound closure when compared to fibroblast-treated wounds or sham groups at day 7. At day 21, the wounds in h AFSCs-treated mice achieved almost complete wound closure, whereas no completely closed wounds were observed in the fibroblast-treated or sham group.(2) Histological analysis of wounds revealed enhanced re-epithelialization in h AFSCs-treated wounds when compared with fibroblast-treated wounds or the sham group. Analysis of wounds at day 4 indicated that granulation tissue in h AFSCs-treated wounds contained more fibroblasts and capillary-like structures but fewer infiltrated inflammatory cells. According to the analysis of wounds at day 14, h AFSCs-treated wounds appeared to enhance the formation of skin appendage-like structures.(3) A large number of GFP-positive cells were present in h AFSCs-treated wounds at day 7, with this proportion declining at day 14 and day 21. Immunofluorescence analysis revealed that the number of cells(including h AFSCs and fibroblasts) in the wound declined over time.(4) The m RNA levels of repair-related factors were highest in the h AFSCs group at day 7 and much higher than in the fibroblast and sham group but then reduced gradually through days 14 and 21. In contrast, the expression of factors in the fibroblast and sham groups reached a maximum at day 21 before the repair was complete, while the expression of inflammatory cytokines in the fibroblast and sham groups was much higher than in the h AFSCs-treated group on the first day. By the end of wound healing, the expression of factors in h AFSCs groups was the same as its expression in normal mouse skin.(5) B7H4 expression was downregulated in h AFSCs by using a lentiviral vector that carries an interfering RNA for B7H4. B7H4 downregulation in h AFSCs relieved the suppression of T-cell activation. The h AFSCs group repaired the skin damage faster than the B7H4-downregulated and fibroblast groups.(6) The fibroblast and B7H4-downregulated groups had more visible infiltrated CD3+ T cells than the h AFSCs group.(7) The expression of repair-related factors in h AFSCs was higher than in the B7H4-downregulated h AFSCs and fibroblasts by day 7, whereas the B7H4-downregulated h AFSCs and fibroblast groups reached the highest expression of these factors at days 14 or 21. However, the expression of inflammatory factors in h AFSCs was slightly higher than in B7H4-downregulated h AFSCs at day 7. In later stages, the expression of inflammatory factors in the h AFSCs group was more moderate than in the other two groups.Conclusion: h AFSCs exhibited accelerated wound closure, provide a moderate inflammation reaction micro-environment and enhance the formation of skin appendage-like structures. B7H4-downregulated h AFSCs relief of accelerated repair and had more visible infiltrated T cells. B7H4 can regulate low immunogenicity and lead h AFSCs to create a mild repair micro-environment to effectively promote wound repair.Part III The differentiation of human amniotic fluid stem cells into sweat gland cellsObjective: To differentiate of amniotic fluid stem cells into sweat gland cells, and to identify its biological characteristics. On the basis of it, we further explored the sweat gland injury repairation in vivo.Methods: The amniotic fluid stem cells were cultured as previously described. Real-time PCR detected several different lines of amniotic fluid stem cells in sweat gland related genes, and analyze its differentiative potential into sweat gland cells. Third-passage h AFSCs were seeded in 6-well plates at a density of 2 ×104 cells per well. When the sweat gland cells reached confluence, the conditioned medium(CM) was collected every day. The collected CM was filtered through a 0.22μm filter before use. The sweat gland cells differentiation medium consists of a 1:1 ratio of SGM and CM supplemented with 50 ng/ml of EGF and 20ng/ml of Shh. The morphologic changes of differentiated cells were observed under inverted microscope. The differentiated amniotic fluid stem cells were analyzed at day7, 14, 21 and 28 respectively. Real-time PCR and immunofluorescence staining were used to detect the gene and protein level of AFS-SG respectively. Flow cytometry were to detect the differentiation rate of h AFSCs. The transmission electron microscope was used to observe the ultrastructure of cells. H&E staining analyze the ability to form a sweat gland-like structure in 3D tissue cultures. Immunofluorescence staining analysis sweat gland related markers. Sweat gland damage model is constructed on the shoulder part of the nude mice. 2X105 AFS-SG cells were injected. PBS injection group and consubstantial to side without any treatment as blank control groupResults:(1) h AFSCs express K18, CD24 and K19 highly and express EDA, EDAR and K8 weakly, while negative for CEA.(2) During the differentiation process, the h AFSCs exhibited changes in their morphology from mesenchymal like cells to epithelioid cells. The observed h AFS-SG cells were larger in size than the cells before culture in this medium, and observation shows obvious cell-cell contacts, similar to SG cells. The expression of the EDA, EDAR, K8 and CEA m RNAs was significantly increased. Flow cytometry was used to quantify the differentiation efficiency of the h AFS-SG cells which to be 30%.(3)Transmission electron microscopy confirmed that microvilli could be observed on the membrane of the h AFS-SG cells. These results suggest that the AFS-SG cells have the secretory function.(4) h AFS-SG cells formed sweat gland-like structures which expressed the sweat gland secretory markers EDA, EDAR, CEA, K8, K18, Na-K-ATPase and SMA.(5) The injury area can form the sweat gland like structure, expression of K14, K8, and CEA.Conclusions: h AFSCs have the potential to differentiate into sweat gland cells. AFS-SG cells express the EDA, EDAR, K8 and CEA proteins and have similar biological characteristics to sweat gland cells. AFS-SG cells had a typical cellular structure for sweat gland cells: microvilli. The AFS-SG cells have the ability to form the entire tubular structure of a sweat gland in vitro and in vivo.更多还原