毛囊保存技术在自体毛发移植中的作用机制及疗效的影响研究

目的研究不同毛囊保存技术对离体毛囊活性的影响,并对自体毛发移植中的毛囊活性进行评估和比较。方法将毛囊放置于0℃~4℃生理盐水(A组)、0℃~4℃醋酸平衡盐(B组)、0℃~4℃DMEM培养基(C组)、胰岛素加氢化可的松加DMEM培养基(D组)、D组加10%自体血清(E组)、E组加表皮生长因子(F组)各缓冲液6 h后,在培养箱中培养6 d。测量毛囊生长长度(hair shaft elongation,HSE),HE染色观察毛囊生长状态及自体移植后20周时行梳发实验测定疗效。结果 HSE结果表明,A、B、C组之间无显著差异,D、E、F组HSE明显高于A、B、C组;F组明显优于D、E组,差异具有统计学意义(P0.05)。A、B、C组毛囊的毛母质、外毛根鞘中凋亡细胞明显增多,F组中仅可见散在凋亡细胞,毛囊的生长状态最好。A+B+C组和D+E+F组的有效率分别为60.0%和86.67%,差异有统计学意义(P0.05)。结论 Williams E液作为毛囊存放的缓冲液,有利于保持毛囊的活性。     

成人睾丸支持细胞分离方法的建立及在胰岛移植中的应用

目的:建立成人睾丸支持细胞简便高效分离方法,并应用于胰岛移植。方法:A组采用胰蛋白酶、DNA酶、胶原酶、透明质酸酶一步消化法;B组采用胰蛋白酶和DNA酶第一步消化,胶原酶和透明质酸酶第二步消化;C组采用胰蛋白酶和DNA酶第一步消化,透明质酸酶第二步消化,胶原酶第三步消化。采用形态学观察和免疫组化鉴定支持细胞;MTT法和流式细胞术(FCM)测定3组支持细胞的活性和纯度;Western blot检测3组支持细胞Fas-L的表达;比较3组支持细胞与胰岛共移植至糖尿病鼠的效果。结果:经鉴定,分离的细胞具有支持细胞的特征;方法A和B分离的支持细胞数量较多,方法B和C分离的支持细胞杂质较少;MTT法检测结果显示,B组支持细胞活性显著高于A组和C组(P0.05);Western blot结果显示,B组支持细胞Fas-L的表达水平显著高于A组和C组(P0.05);FCM检测3种方法分离的S支持细胞纯度分别为(85.2±1.8)%、(92.3±2.5)%、(93.1±2.6)%,B组和C组的支持细胞纯度显著高于A组(P0.05);将3组支持细胞与胰岛共移植至糖尿病鼠模型肾包膜下,B组胰岛移植物存活时间显著高于单独胰岛移植组和A、C两组(P0.05)。结论:采用两步消化法能够获得纯度和活性较高的支持细胞,其与胰岛共移植能够显著延长移植物存活。     

免疫抑制剂在非清髓性造血干细胞移植预处理中的应用

非清髓性造血干细胞移植(non-myeloablative stem celltransplantation,NST)已经广泛应用于血液系统恶性疾病、某些实体器官肿瘤以及非恶性疾病的治疗,在某些疾病方面有取代标准移植的趋势.NST的预处理方案中应采用较强的免疫抑制剂,以促进供者细胞的植入,通过移植物抗肿瘤效应(GVT)来清除受者造血细胞及肿瘤细胞[1].随着对NST研究的深入,越来越多的免疫抑制剂被用于NST预处理方案中,现就NST预处理中常用的免疫抑制剂的作用机制、研究进展及临床应用、不良反应作一综述.     

富血小板血浆联合微粒皮移植在高原地区老年慢性小创面中的临床研究

目的探究在高原地区老年慢性小创面治疗中使用富血小板血浆(PRP)联合微粒皮移植的治疗效果。 方法选取2018年2月至2022年2月青海省人民医院烧伤整形科收治的老年慢性小创面患者60例。根据患者入院时间不同将患者分为观察组和对照组,观察组采用PRP联合微粒皮移植,对照组采用PRP联合刃厚邮状皮移植。采集患者的外周静脉血,采用二次离心法制备PRP;2组患者均于局部浸润麻醉下应用电动取皮机自大腿前内侧根据创面需要取合适大小刃厚皮片,皮片厚度约0.2 mm,观察组供受区比例为1∶4~1∶8,将所取皮片剪碎后切割成直径<1 mm²的微粒皮备用;对照组供受区比例为1∶1~1∶2,将所取皮片制备为大小约1 cm×1 cm的刃厚邮状皮备用。创面清创、止血后,观察组将制备好的微粒皮均匀涂抹在创面后以PRP填充创面,用聚氨酯泡沫敷料或凡士林纱布覆盖创面避免PRP流失及失活,清洁敷料覆盖并固定;对照组将制备好的PRP填充于创面,再将制备好的刃厚邮状皮片移植于PRP之上,用凡士林或聚氨酯泡沫敷料覆盖清洁敷料加压包扎。2组术后均常规行抗感染等治疗,术后第6、11、16、21、26天创面换药,直至创面完全愈合。术后第21天,统计2组患者的治疗有效率;统计2组患者的创面愈合时间;创面愈合后6个月,以温哥华瘢痕评估量表(VSS)对2组患者的瘢痕情况进行评估。数据比较采用Mann-Whitney U检验和χ²检验。 结果术后第21天,观察组治疗有效27例,无效3例,治疗有效率为90.00%;对照组治疗有效20例,无效10例,治疗有效率为66.67%,2组比较差异有统计学意义(χ²＝6.278,P＝0.043)。观察组创面愈合时间为为26.0(24.0, 27.0) d,短于对照组[43.0(39.8, 47.3) d],2组比较差异有统计学意义(Z＝－6.531,P<0.05)。创面愈合后6个月,观察组VSS评分中色素沉着、瘢痕厚度、血管分布和柔韧性评分分别为0(0, 0)、0(0, 0)、0(0, 0)、1(0, 1)分,均分别低于对照组[2.0(1.0, 2.3)、1.5(0.8, 2.0)、1(1, 2)、2(1, 3)分],2组比较差异均有统计学意义(Z＝－6.310、－4.838、－5.624、－4.431,P<0.05)。 结论在高原地区,对于老年慢性小创面的治疗,PRP联合微粒皮移植的创新技术具有提高治疗有效率、缩短创面愈合时间的疗效,为患者提供更多治愈的选择性。     

医用胶原蛋白海绵及负压封闭引流联合自体刃厚皮复合移植在足部毁损伤难愈性创面中的应用

目的观察医用胶原蛋白海绵及负压封闭引流(VSD)联合自体刃厚皮复合移植修复足部毁损伤难愈性创面的效果。方法选取2017年1月至2019年10月海南省人民医院烧伤与皮肤修复外科收治的足部毁损伤难愈性创面患者18例,其中合并足背肌腱外露12例,跖骨骨外露6例。创面清创后,予医用胶原蛋白海绵覆盖肌腱外露、骨外露创面,并安装VSD装置,待创面覆盖肉芽组织后,再给予自体刃厚皮移植修复创面。记录治疗时间、外露肌腱或骨质坏死率、皮片成活率,术后随访6个月,以Maryland足部功能评分标准评估外观及功能。结果18例患者足部均得以保存,外露的肌腱和骨质均保持活性,皮片成活率85%~95%,平均(90±5)%。残余创面给予间断换药治疗后全部愈合,治疗时长14.0~45.0 d,平均(29.5±15.5)d。出院后6个月复诊以Maryland足部功能评分标准评估足部功能,优3例,良4例,中6例,差5例。结论医用胶原蛋白海绵及VSD在促进肌腱、骨质外露创面肉芽组织生长的同时可保留外露的肌腱及骨组织活性,联合自体刃厚皮移植,能较好地修复足部毁损伤难愈性创面。     

毛囊干细胞的分离及培养方法研究

目的建立一种分离效率高、创伤小的毛囊干细胞分离方法，筛选毛囊干细胞体外培养的最优条件，考察毛囊干细胞的体外增殖活性。方法获取兔胡须部皮肤全层，比较2种不同分离方法获得细胞的贴壁、增殖及克隆形成情况。通过免疫组化的方法检测毛囊干细胞β1整合素、CK19表达情况。选用MTT法和乳酸脱氢酶来间接反映毛囊干细胞的增殖能力。分析细胞周期考察细胞增殖过程中各期细胞的分布。观察血清浓度梯度条件培养液对毛囊干细胞增殖的影响。结果“两步酶”法的分离能获得大量细胞且细胞迅速贴壁，克隆迅速形成。获取的毛囊干细胞胞浆中有β1整合素、CK19的阳性表达。毛囊干细胞生长曲线及细胞周期都表明毛囊干细胞有高增殖能力。毛囊干细胞增殖表现出血清浓度的依赖。结论“两步酶”法是一种分离效率高、创伤小的方法，能够获取大量高表达β1整合素、CK19的毛囊干细胞。毛囊干细胞在适当条件下表现为高增殖能力，其生长有一定的血清依赖性。     

Lectin histochemistry of glycoconjugates in the feline hair follicle and hair

The distribution of glycoconjugate in the feline hair follicle and hair was studied by light and electron microscopic histochemical methods. The hair apparatus was found to contain considerable amounts of complex carbohydrates with different saccharide residues (alpha-D-mannose, beta-D-glucose, alpha-L-fucose, beta-N-acetyl-D-glucosamine). Variations of those were detected in the plasma membrane of the hair follicle cells during the course of their differentiation and keratinization, namely, alph-D-glucose, alpha-L-fucose and beta-N-acetyl-D-glucosamine in the suprabulbar and bulbar regions. The reaction level of sialic acid residues in the plasma membrane decreased in some cell layers during the course of differentiation. The results obtained from the present study indicated that interaction between saccharide residues of neutral carbohydrates and sialyl groups during the anagen phase might contribute to cell keratinization in hair follicles and hairs. It is discussed whether the existence of glycogen in outer root sheath cells might enable these cells to provide other hair apparatus cells with energy when necessary. Moreover, it became obvious from variations in sialyl residue distribution that cell differentiation processes terminate first of all in Huxley's and Henle's layers within the suprabulbar region of the hair follicle, as followed by the hair cortex.     

血管内皮生长因子在毛囊生物学中的研究现状

血管内皮生长因子(vascular endothelial growth factor,VEGF)又名血管通透性因子(vascular permeability factor,VPF),是重要的血管生成正性调节因子。作为毛乳头细胞的一种自分泌生长因子,其对毛囊的生长亦有重要作用。血管内皮生长因子不仅能促进毛囊的生长,还参与毛囊生长周期的调控。在内皮细胞中,血管内皮生长因子发挥作用主要是通过与其受体的结合,诱导受体二聚体化和自身磷酸化,从而激活胞内信号转导通路,但在毛囊细胞中是否如此,仍需进一步研究。     

Immunohistochemical study of hair follicle stem cells in regenerated hair follicles induced by Wnt10b

The regulation of the periodic regeneration of hair follicles is complicated. Although Wnt10b has been reported to induce hair follicle regeneration, the characteristics of induced hair follicles, especially the target cells of Wnt10b, have not yet been clearly elucidated. Thus, we systematically evaluated the expression and proliferation patterns of Wnt10b-induced hair follicles. We found that Wnt10b promoted the proliferation of hair follicle stem cells from 24 hours after AdWnt10b injection. Seventy-two hours after AdWnt10b injection, cells outside of bulge area began to proliferate. When the induced hair follicle entered full anagen, although the hair follicle stem cells were normal, canonical Wnt signaling was maintained in the hair precortex cells. Our results reveal that the target cells that overexpressed Wnt10b included hair follicle stem cells, hair precortex cells, and matrix cells.     

毛囊细胞--一种新的皮肤组织工程种子细胞

毛囊的上皮细胞和真皮细胞与皮肤的表皮角朊细胞和真皮成纤维细胞具有很大的相似性,但其具有更强的增殖分化能力和更多的生物学特性,并且毛囊真皮细胞具有干细胞的一些特性,作为皮肤组织工程的种子细胞具有更独特的优势,在构建带有皮肤附属器的组织工程皮肤上有潜在的前景.     

Histopathologic diagnosis of alopecia: clues and pitfalls in the follicular microcosmos

In recent years the increased number of scalp biopsies in alopecia has afforded the dermatopathologist improved confidence in distinguishing the histopathologic characteristics of ‘classic’ scarring and non-scarring alopecias, as well as the spectrum of their presentation. Occasionally, however, histopathologic ‘dogmas’ have been disproven. The loss of sebaceous glands considered to be a clue to scarring alopecia or the peribulbar lymphoid cell infiltrate diagnostic of alopecia areata may present in unconventional scenarios, and thus, represent pitfalls. Clues to diagnose histopathologically ‘silent’ (invisible) alopecias, the identification of multiple etiologies, and awareness of the ‘time-factor’ are herein discussed.     

毛囊真皮鞘细胞中过表达Noggin对皮肤及毛囊生长的作用

背景:Noggin蛋白是一个抑制BMP信号通路的重要分子,可以与BMP2/4结合形成复合物,从而阻断BMP信号,影响生物有机体的正常发育过程。研究认为真皮鞘细胞是一类能够长期自我更新的真皮干细胞,参与毛囊再生过程中真皮毛乳头和真皮鞘的形成。在毛囊发育过程中,真皮毛乳头中Noggin参与毛囊的起始,Noggin的缺失会导致毛囊数量的减少和毛囊生长缓慢,但人们对于Noggin蛋白在毛囊真皮鞘中的作用所知甚少。目的:研究Noggin蛋白在毛囊真皮鞘中的生物学功能。方法:利用真皮鞘特异的αSMA-Cre ERT2工具鼠在真皮鞘中特异过表达Noggin蛋白,分别在出生后8,9 d对实验组αSMA-CreER;pMES-Noggin小鼠和对照组αSMA-CreER小鼠注射4-羟基他莫昔芬,在出生后21,23,28 d获取皮肤组织,苏木精-伊红染色观察毛囊生长情况和皮下脂肪层厚度,免疫组化染色分析表型。结果与结论:通过苏木精-伊红染色结果发现毛囊生长并没有受到影响,但毛囊皮下脂肪组织生长发育受到严重影响,小鼠皮下脂肪层变薄。免疫组化结果说明BMP信号降低可能是导致毛囊脂肪层变薄的原因。这一发现拓展了人们对于真皮鞘细胞和Noggin蛋白的认识,也为人们更加准确理解毛囊再生和组织生长机制提供了重要依据。     

Effect of preantral follicle isolation technique on in-vitro follicular growth,oocyte maturation and embryo development in mice

BACKGROUND: The use of mechanical and enzymatic techniques to isolate preantral follicles before in-vitro culture has been previously described. The aim of this study was to assess the effect of the isolation procedure of mouse preantral follicles on their subsequent development in vitro. METHODS: Follicles were isolated either mechanically or enzymatically and cultured using an individual non-spherical culture system. Follicular development and steroidogenesis, oocyte in-vitro maturation and embryo development were assessed for both groups. RESULTS: After 12 days of culture, follicles isolated mechanically had a higher survival rate but a lower antral-like cavity formation rate than follicles isolated enzymatically. Enzymatic follicle isolation was associated with a higher production of testosterone and estradiol compared with mechanical isolation. A stronger phosphatase alkaline reaction was observed after enzymatic isolation, suggesting that follicles isolated enzymatically had more theca cells than those isolated mechanically. However, both isolation techniques resulted in similar oocyte maturation and embryo development rates. CONCLUSIONS: Enzymatic follicular isolation did not affect theca cell development. Follicular steroidogenesis was enhanced after enzymatic isolation but the developmental capacity of oocytes was comparable to that obtained after mechanical isolation.