【作者】 孙晓燕；

【导师】 谢臻蔚；

【作者基本信息】 浙江大学 ， 临床医学（专业学位）， 2019， 硕士

【摘要】 研究背景压力性尿失禁(Stress Urinary Incontinence,SUI)是好发于孕产妇及中、老年妇女的慢性病,严重影响患者生活质量,被称为“社交癌”。近年来,随着二胎政策开放和人口老龄化,SUI患者数剧增,引起医学界重视;但该病发病机制欠清,缺乏理想的临床防治措施。现已公认,SUI主要由尿道支撑组织薄弱所致;进一步研究认为,成纤维细胞主导的尿道支撑组织微环境细胞外基质(Extra-cellular Matrix,ECM)重构障碍是该病发病的关键,但其具体调控机理尚未明确。外泌体是近年来备受关注的胞间通讯方式,参与多种组织局灶ECM微环境结构及功能调控;通过探讨外泌体在尿道支撑组织中的表达及其在SUI发病中的作用,有望为该病发病机制研究提供可行方向;然而,国内外文献罕见相关报道。目的本研究旨在明确尿道支撑组织局灶微环境中是否存在外泌体调控方式;阐明SUI患者是否存在尿道支撑组织外泌体异常表达;并在体外细胞水平,探讨该外泌体对尿道支撑组织成纤维细胞功能的影响。研究方法1.收集女性尿道支撑组织,分为SUI组和非SUI组,每组各8例;匹配两组间年龄、绝经时间、孕产次、分娩方式及体重指数(Body Mass Index,BMI)。2.采用沉淀法提取尿道支撑组织来源外泌体,联合电镜、纳米颗粒示踪分析技术及蛋白免疫印迹法,鉴定其形态特征、粒径分布范围及外泌体标志蛋白CD63的表达。3.采用纳米颗粒示踪分析技术及BCA蛋白定量法,检测SUI组和非SUI组尿道支撑组织来源外泌体含量,比较组间差异。4.获得女性尿道支撑组织原代成纤维细胞,共培养细胞与尿道支撑组织来源外泌体,分为F+Sexo组(成纤维细胞与SUI组尿道支撑组织来源外泌体共培养)、F+Nexo组(成纤维细胞与非SUI组尿道支撑组织来源外泌体共培养)和F组(即对照组,成纤维细胞与培养基共培养),采用CCK-8增殖实验、划痕实验、蛋白免疫印迹法及反转录酶-聚合酶链锁反应法,观察各组成纤维细胞增殖、迁移和胶原合成功能,比较组间差异。研究结果1.女性尿道支撑组织中提取到沉淀物质,透射电镜显示其形态为类圆形杯口状,蛋白免疫印迹法显示其外泌体特异性标志蛋白CD63表达呈现阳性结果,纳米颗粒示踪分析技术显示其直径主要分布于50-200nm区间,鉴定为外泌体。2.SUI组和非SUI组单位质量(g)尿道支撑组织来源外泌体蛋白含量分别为1606.02 ug 和 1216.75 ug,外泌体数量分别为 2219x10^8 个和 982 x10^8 个,SUI组外泌体蛋白含量和数量均显著高于非SUI组(p均<0.05)。3.F+Sexo组成纤维细胞在24h、48h及72h时间点细胞的增殖率分别为30%、52%及 214%,F+Nexo 组分别为 16%、32%及 105%;F+Sexo 组和 F+Nexo 组各时段细胞OD值均较对照组显著增加(p均<0.05),F+Sexo组各时段细胞OD值显著高于 F+Nexo 组(p均<0.05)。4.24h、48h及72h的细胞划痕愈合率F+Sexo组分别为19%、26%及27%,F+Nexo组分别为38%、61%及77%,对照组分别为59%、100%及100%;经统计学分析,F+Sexo组和F+Nexo组各时段细胞划痕愈合率均显著低于对照组(p均<0.05),F+Sexo组各时段细胞划痕愈合率均显著低于F+Nexo组(p均<0.05)。5.F+Sexo组细胞Ⅰ型和Ⅲ型前胶原mRNA及蛋白表达水平显著高于对照组(p均<0.05);F+Nexo组细胞Ⅰ型和Ⅲ型前胶原的mRNA和蛋白水平与对照组相比无统计学差异。结论1.尿道支撑组织局灶微环境存在外泌体调控方式。2.SUI组尿道支撑组织外泌体的表达量显著高于非SUI组。3.尿道支撑组织来源外泌体可能通过影响成纤维细胞增殖、迁移及胶原合成能力参与SUI发病。更多还原

【Abstract】 BackgroundAs a common chronic disease,Stress Urinary Incontinence(SUI)usually occurs in maternal,middle-aged and elderly women,which severely plagued the patients.In recent years,with the opening of the second child policy and the aging of the population,the number of SUI patients has increased dramatically,which has attracted the attention of the medical profession.However,the pathogenesis of the disease is not clear,and there is no ideal clinical prevention and treatment measures.The SUI is mainly caused by the weakness of urethral supporting tissue have been recognized,and the recent studies have shown that extra-cellular matrix(ECM)remodeling disorder of urethral supporting tissue micro-environment dominated by the fibroblast is the key to the pathogenesis of SUI,but the detailed mechanism is still in the exploratory stage.Exosome is a highly concerned inter-cellular communication,which involved in the formation and functional regulation of ECM micro-environment in various tissues.It is expected to provide a feasible direction for the study of the pathogenesis of SUI by investigating the expression of exosomes in urethral supporting tissues and their role in the pathogenesis of SUI.However,there are few related reports at home and abroad.ObjectiveThe purpose of this study was to clarify whether there is the regulation of exosome in the local micro-environment of urethral supporting tissue,to clarify whether there is abnormal expression of exosome in urethral supporting tissue in SUI patients,and to explore the effect of exosome on the function of fibroblasts in urethral supporting tissue at the cell level in vitro.Method1.Female urethral supporting tissues(about 2 cm from the urethral orifice)were collected and divided into SUI group and non-SUI group with 8 cases in each group.Age,menopausal time,times of pregnancy and delivery,mode of delivery and body mass index(BMI)were matched between the two groups.2.The exosomes from human urethral supporting tissues were extracted by the method of precipitation.The morphological characteristics,the distribution of the particle size and expression of CD63(exosomes’ marker protein)were identified by electron microscopy,nanoparticle tracking analysis and Western blot.3.The quantity of exosomes extracted from unit mass of urethral supporting tissues in SUI and non-SUI groups were compared by nanoparticle tracking analysis and BCA protein quantification.4.Fibroblasts were co-cultured with urethral supporting tissues-derived exosomes,dividing into F+Sexo group(co-culture of fibroblasts and urethral supporting tissue-derived exosomes from SUI group),F+Nexo group(co-culture of fibroblasts and urethral supporting tissue-derived exosomes from non-SUI group)and F group(equates control group,co-culture of fibroblasts and culture medium).The effects of exosomes on the proliferation,migration and collagen synthesis of fibroblasts were analyzed by CCK-8 increment test,scratch test,protein immunoblotting and reverse transcriptase-polymerase chain reaction.Results1.Precipitation was successfully extracted from primary urethral supporting tissues.The transmission electron microscopy showed that the morphology of the sediment was round cup-shaped.Western-blot showed that the expression of CD63,a specific marker protein of the exosome,was positive.Nanosight analysis showed that the diameter of the sediment was mainly distributed between 50 and 200 nm.2.The protein content of primary urethral supporting tissues-derived exosomes in SUI group and non-SUI group was 1606.02ug/g and 1216.75 ug/g respectively,and the number of exosomes was 2219 x 10 ^ 8 particles/g and 982 x 10 ^ 8 particles/g,respectively.There were significant differences between the two groups.The protein content and the number of exosomes in SUI group were significantly higher than those in non-SUI group(all P<0.05).3.The increase rate of OD value of cells in the group of F+Sexo was 30%in 24 hours,52%in 48 hours and 214%in 72 hours;in the group of F+Nexo was 16%in 24 hours,32%in 48 hours and 105%in 72 hours.Statistical analysis showed that the OD values of cells at 24,48 and 72 hours increased in both F+Sexo and F+Nexo groups(all P<0.05).The OD values of cells at 24,48 and 72 hours in F+Sexo group were significantly higher than those in F+Nexo group(all P<0.05).4.Scratch healing rate of cells at 24h,48h and 72h were 59%,100%and 100%in control group,respectively;were 19%,26%and 27%in F+Sexo group and were 38%,61%and 77%in F+Nexo group.Statistical analysis showed that the scratch healing rates of F+Sexo group and F+Nexo group at 24h,48h and 72h were significantly lower than those of control group(all P<0.05).Scratch healing rates of F+Sexo group at 24h,48h and 72h were significantly lower than that in F+Nexo group(all P<0.05)5.The expression of collagen I and collagen III of cells in F+Sexo group was significantly higher than that in control group both in m RNA and protein levels(all P<0.05).While there weren’t significant difference between F+Nexo group and control group of the expression of collagen I and collagen III of cells both in m RNA and protein levelsConclusion1.Exosomes were existed in the micro-environment of urethral supporting tissues.2.Urethral supporting tissue-derived exosomes are correlated with SUI.3.The exosomes from urethral supporting tissues may participate in the pathogenesis of SUI by regulating the proliferation,migration and collagen synthesis ability of fibroblasts.更多还原