Screening and analysis of differentially expressed genes of dermal papillae cells with aggregative behavior

Objective: To screen and clone differentially expressed genes of dermal papillae cells (DPC) with aggregative behavior, and to explore the molecular mechanism of their aggregation. Methods: Total RNAs were extracted from DPC with and without aggregative behavior and double strand cDNAs were synthesized by using SMART cDNA synthesis, respectively. The cDNA fragments of differentially expressed genes in DPCs with aggregative behavior were isolated by suppression subtractive hybridization. Positive clones were screened by PCR method and verified by cDNA dot blot, Northern blot and then analyzed through homologous retrieving. Results: A subtractive cDNA library of DPC with aggregative behavior has been successfully constructed. The result of screening and cloning of the library showed that, DPC with aggregative behavior could expresse genes related to homologous aggregation, proliferation and cycle control, including known genes (capping protein, paladin, vascular endothelial growth factor), hematopoietic stem/progenitor cells (HSPC) related clone (HSPC011 and HSPC016) and a new gene. Conclusion: The construction of subtracted library of DPC lays solid foundation for screening and cloning new and specific genes related to aggregative behavior of DPC. Several genes might be cooperatively involved in the homologous aggregation, proliferation and cycle control of DPC. Among these genes, capping protein and palladin might be closely related to the aggregative behavior of dermal papilla cells, and VEGF and HSPC related clone would be responsible for the status of higher proliferation of dermal papilla cells.

Flavonoids in safflower extract reduce cisplatin-induced damage to human follicle dermal papilla cells by inhibiting DNA damage and Rad17/Chk1/Cdc25C signaling

Background:Cisplatin is a chemotherapeutic agent commonly used clinically for the treatment of various human cancers.Patients often reduce the use of cisplatin due to its side effects,which in turn affects its treatment.This study explored the mechanism of action of safflower extract as an adjuvant traditional Chinese medicine for chemotherapy.Methods:Primary human follicle dermal papilla cells(HFDPCs)were used as target cells for cisplatininduced damage to hair cells.Western blotting was used to investigate the molecular targets of cisplatin and safflower extract in causing HFDPCs damage.Cell survival and cell cycle were analyzed by mitochondrial staining reagent WST-1 and propidium iodide.Results:Cisplatin could reduce the viability of HFDPCs without causing cell death.Cisplatin increased the level of phospho-Rad17 in HFDPCs and activated the Chk1/Cdc25C signaling to reduce the expression of Cdc2 protein,thereby arresting the cells in the G2/M phase.The combination of safflower extract and the flavonoids could effectively inhibit the signal transduction of Rad17/Chk1/Cdc25 in cisplatin-treated cells and reduce the cell population in the G2/M phase.Finally,we also confirmed that safflower extract could effectively inhibit the damage to HFDPCs caused by cisplatin,mainly at the level of reducing the DNA damage caused by cisplatin.Conclusions:Safflower extract can be used as an adjuvant Chinese medicine for chemotherapy to reduce the damage caused by chemotherapy to normal hair follicle cells.

Cloning and bioinformatic analysis of HSPC016 gene in dermal papilla cells

Objective： To clone the full-length cDNA sequence of HSPC016 gene, an aggregative growth related gene in dermal papilla cells （DPC）, and analyze its characteristics and predict its biological function. Methods： Rapid amplification of cDNA ends （RACE） technology was entailed to amplify the 5＇ and 3＇ sequences of HSPC016. The amplified fragments were TA-cloned, sequenced and spliced together to obtain the full-length cDNA. Its chromosome localization, domain and possible function were analyzed by bioinformatic methods. Results.. Two isoforms, 400 bp and 493 bp, were obtained. The gene was mapped on chromosome 3q21. 31, and was conservative on evolution. HSPC016, a 64aa protein, belongs to PD053992 protein family and its functional domain was homologous to T2FA gene. Conclusion： PISPC016 may be related to transcriptional regulation and its protein product may act as a subunit of a transcriptional complex and play a role on DPC growth and differentiation through facilitating or suppressing other genes＇ transcription within the nucleus.

Epidermal stem cells and skin tissue engineering in hair follicle regeneration

The reconstitution of a fully organized and functional hair follicle from dissociated cells propagated under defined tissue culture conditions is a challenge stillpending in tissue engineering. The loss of hair follicles caused by injuries or pathologies such as alopecia not only affects the patients' psychological well-being, but also endangers certain inherent functions of the skin. It is then of great interest to find different strategies aiming to regenerate or neogenerate the hair follicle under conditions proper of an adult individual. Based upon current knowledge on the epithelial and dermal cells and their interactions during the embryonic hair generation and adult hair cycling, many researchers have tried to obtain mature hair follicles using different strategies and approaches depending on the causes of hair loss. This review summarizes current advances in the different experimental strategies to regenerate or neogenerate hair follicles, with emphasis on those involving neogenesis of hair follicles in adult individuals using isolated cells and tissue engineering. Most of these experiments were performed using rodent cells, particularly from embryonic or newborn origin. However, no successful strategy to generate human hair follicles from adult cells has yet been reported. This review identifies several issues that should be considered to achieve this objective. Perhaps the most important challenge is to provide threedimensional culture conditions mimicking the structure of living tissue. Improving culture conditions that allow the expansion of specific cells while protecting their inductive properties, as well as methods for selecting populations of epithelial stem cells, should give us the necessary tools to overcome the difficulties that constrain human hair follicle neogenesis. An analysis of patent trends shows that the number of patent applications aimed at hair follicle regeneration and neogenesis has been increasing during the last decade. This field is attractive not only to academic researchers but also to the companies that own almost half of the patents in this field.

Application of extracellular vesicles from mesenchymal stem cells promotes hair growth by regulating human dermal cells and follicles

BACKGROUND Dermal papillae(DP)and outer root sheath(ORS)cells play important roles in hair growth and regeneration by regulating the activity of hair follicle(HF)cells.AIM To investigate the effects of human mesenchymal stem cell-derived extracellular vesicles(hMSC-EVs)on DP and ORS cells as well as HFs.EVs are known to regulate various cellular functions.However,the effects of hMSC-EVs on hair growth,particularly on human-derived HF cells(DP and ORS cells),and the possible mechanisms underlying these effects are unknown.METHODS hMSC-EVs were isolated and characterized using transmission electron microscopy,nanoparticle tracking analysis,western blotting,and flow cytometry.The activation of DP and ORS cells was analyzed using cellular proliferation,migration,western blotting,and real-time polymerase chain reaction.HF growth was evaluated ex vivo using human HFs.RESULTS Wnt3a is present in a class of hMSC-EVs and associated with the EV membrane.hMSC-EVs promote the proliferation of DP and ORS cells.Moreover,they translocateβ-catenin into the nucleus of DP cells by increasing the expression ofβ-catenin target transcription factors(Axin2,EP2 and LEF1)in DP cells.Treatment with hMSC-EVs also promoted the migration of ORS cells and enhanced the expression of keratin(K)differentiation markers(K6,K16,K17,and K75)in ORS cells.Furthermore,treatment with hMSC-EVs increases hair shaft elongation in cultured human HFs.CONCLUSION These findings suggest that hMSC-EVs are potential candidates for further preclinical and clinical studies on hair loss treatment.

JAM-A facilitates hair follicle regeneration in alopecia areata through functioning as ceRNA to protect VCAN expression in dermal papilla cells

The dermal papilla cells in hair follicles function as critical regulators of hair growth.In particular,alopecia areata(AA)is closely related to the malfunctioning of the human dermal papilla cells(hDPCs).Thus,identifying the regulatory mechanism of hDPCs is important in inducing hair follicle(HF)regeneration in AA patients.Recently,growing evidence has indicated that 3 untranslated regions(3 UTR)of key genes may participate in the regulatory circuitry underlying cell differentiation and diseases through a socalled competing endogenous mechanism,but none have been reported in HF regeneration.Here,we demonstrate that the 3 UTR of junctional adhesion molecule A(JAM-A)could act as an essential competing endogenous RNA to maintain hDPCs function and promote HF regeneration in AA.We showed that the 3 UTR of JAM-A shares many microRNA(miRNA)response elements,especially miR-221–3p,with versican(VCAN)mRNA,and JAM-A 3 UTR could directly modulate the miRNA-mediated suppression of VCAN in self-renewing hDPCs.Furthermore,upregulated VCAN can in turn promote the expression level of JAM-A.Overall,we propose that JAM-A 3 UTR forms a feedback loop with VCAN and miR-221–3p to regulate hDPC maintenance,proliferation,and differentiation,which may lead to developing new therapies for hair loss.

Extracellular vesicles from 3D cultured dermal papilla cells improve wound healing via Krüppel-like factor 4/vascular endothelial growth factor A-driven angiogenesis

Background:Non-healing wounds are an intractable problem of major clinical relevance.Evidence has shown that dermal papilla cells(DPCs)may regulate the wound-healing process by secreting extracellular vesicles(EVs).However,low isolation efficiency and restricted cell viability hinder the applications of DPC-EVs in wound healing.In this study,we aimed to develop novel 3D-DPC spheroids(tdDPCs)based on self-feeder 3D culture and to evaluate the roles of tdDPC-EVs in stimulating angiogenesis and skin wound healing.Methods:To address the current limitations of DPC-EVs,we previously developed a self-feeder 3D culture method to construct tdDPCs.DPCs and tdDPCs were identified using immunofluorescence staining and flow cytometry.Subsequently,we extracted EVs from the cells and compared the effects of DPC-EVs and tdDPC-EVs on human umbilical vein endothelial cells(HUVECs)in vitro using immunofluorescence staining,a scratch-wound assay and a Transwell assay.We simultaneously established a murine model of full-thickness skin injury and evaluated the effects of DPC-EVs and tdDPC-EVs on wound-healing efficiency in vivo using laser Doppler,as well as hematoxylin and eosin,Masson,CD31 andα-SMA staining.To elucidate the underlying mechanism,we conducted RNA sequencing(RNA-seq)of tdDPC-EV-and phosphate-buffered saline-treated HUVECs.To validate the RNA-seq data,we constructed knockdown and overexpression vectors of Krüppel-like factor 4(KLF4).Western blotting,a scratch-wound assay,a Transwell assay and a tubule-formation test were performed to detect the protein expression,cell migration and lumen-formation ability of KLF4 and vascular endothelial growth factor A(VEGFA)in HUVECs incubated with tdDPC-EVs after KLF4 knockdown or overexpression.Dual-luciferase reporter gene assays were conducted to verify the activation effect of KLF4 on VEGFA.Results:We successfully cultured tdDPCs and extracted EVs from DPCs and tdDPCs.The tdDPC-EVs significantly promoted the proliferation,lumen formation and migration of HUVECs.Unlike DPC-EVs,tdDPC-EVs exhibited significant advantages in terms of promoting angiogenesis,accelerating wound healing and enhancing wound-healing efficiency both in vitro and in vivo.Bioinformatics analysis and further functional experiments verified that the tdDPC-EV-regulated KLF4/VEGFA axis is pivotal in accelerating wound healing.Conclusions:3D cultivation can be utilized as an innovative optimization strategy to effectively develop DPC-derived EVs for the treatment of skin wounds.tdDPC-EVs significantly enhance wound healing via KLF4/VEGFA-driven angiogenesis.

半胱氨酸、蛋氨酸对体外培养绒山羊次级毛囊生长及毛乳头细胞增殖的影响

旨在探讨半胱氨酸(Cys)、蛋氨酸(Met)对体外培养燕山绒山羊次级毛囊(SF)生长及毛乳头细胞(DPCs)增殖凋亡的影响。本研究选取1只1周岁左右健康雄性燕山绒山羊皮肤若干,完整分离的次级毛囊随机分成Cys试验组和Met试验组,每组各浓度3个重复,每个重复8根毛囊,Cys试验组添加浓度分别为0、40、80、120、160和200μg·mL^(-1);Met试验组添加浓度分别为0、10、15、20、25和30μg·mL^(-1)。毛囊培养7 d,每隔24 h观察毛囊生长形态并测量毛囊生长长度。从燕山绒山羊次级毛囊中提取、纯化DPCs,细胞计数并绘制DPCs生长曲线,将DPCs随机分为Cys试验组和Met试验组,每组各浓度6个重复,Cys试验组添加浓度分别为0、20、40、60、80、100μg·mL^(-1);Met试验组添加浓度分别为0、2、4、6、8、10μg·mL^(-1)。培养2 h后用CCK-8试剂盒进行细胞增殖活力测定,确定燕山绒山羊DPCs中Cys和Met的最适培养浓度,利用qRT-PCR技术检测不同浓度Cys和Met的DPCs增殖相关基因(PCNA、CCND1、CDC42、CDK 4)、凋亡相关基因(P21、P53、Bax、Caspase-3、Bcl-2)、皮肤细胞分化相关基因(IVL)及角蛋白相关基因(K10、K 14)mRNA表达水平。结果表明,与对照组相比,添加80和120μg·mL^(-1) Cys与10、15、20和25μg·mL^(-1) Met均可显著影响次级毛囊生长速率和累计生长长度(P<0.01),且120μg·mL^(-1) Cys和20μg·mL^(-1) Met促生长效果最好;添加20、40和60μg·mL^(-1) Cys可以显著影响DPCs增殖(P<0.05),添加40μg·mL^(-1) Cys显著上调PCNA、CCND1、CDK4、Bcl-2、K10、K 14和IVL基因mRNA表达量(P<0.05);添加6μg·mL^(-1) Met可以显著影响DPCs增殖(P<0.05),并显著上调PCNA、CCND1、CDK4、P21、Bcl-2、K 10和K 14基因mRNA表达量(P<0.05)。综上,添加Cys和Met可以显著促进长绒期燕山绒山羊次级毛囊生长,最适添加量分别为120μg·mL^(-1)和20μg·mL^(-1);添加Cys和Met可通过上调细胞增殖、分化、角蛋白等基因mRNA、下调细胞凋亡基因mRNA表达,促进乳头细胞增殖、抑制细胞凋亡进而促进燕山绒山羊次级毛囊生长。

基于转录组测序分析SOX18在湖羊毛囊毛乳头细胞中的功能

旨在初步探究SOX18基因在湖羊毛囊毛乳头细胞中的功能。本研究首先采用免疫荧光染色试验对从湖羊毛囊中分离的细胞进行鉴定,然后构建SOX18基因过表达载体并通过qRT-PCR和Western blot检测其过表达效率。将经过鉴定且生长状态良好的毛乳头细胞分为两组,分别转染SOX18过表达载体和空载质粒,每组3个重复。采用Trizol法提取6个样本的总RNA并构建cDNA文库,利用Illumina Novaseq6000平台进行测序,然后对样本相关性和差异表达基因进行分析,并对差异表达基因进行GO功能分类和KEGG通路注释,寻找SOX18基因调控的相关候选基因和通路。为确认转录组数据的准确性,随机选取9个差异表达基因进行qRT-PCR验证。免疫荧光结果显示,毛乳头相关基因在所分离的细胞中高表达,表明所分离的毛乳头细胞纯度高。qRT-PCR和Western blot检测发现,SOX18基因过表达能够增加毛乳头细胞中SOX18的mRNA和蛋白水平,表明构建的SOX18基因过表达载体可用于后续试验。转录组分析结果表明样本间相关性好,SOX18过表达组和对照组相比共发现157个基因差异表达,其中103个基因在SOX18过表达后上调,54个基因下调,qRT-PCR分析表明转录组数据准确性高。GO功能分析显示,一些差异基因富集于细胞进展和毛囊发育过程。KEGG富集分析表明,一些差异表达基因富集于细胞增殖和毛囊发育相关信号通路。GO功能分析和KEGG富集分析表明SOX18在毛乳头细胞的增殖和毛囊发育过程中起作用。本研究通过转录组测序分析发现SOX18可能通过影响细胞增殖和毛囊发育相关的基因和信号通路来影响毛乳头细胞增殖和毛囊生长发育,研究结果为解析SOX18基因在湖羊毛乳头细胞和毛囊中的分子调控机制提供了理论基础。

ATG14调控家兔毛囊毛乳头细胞自噬进程的功能探究

旨在探究自噬相关蛋白14(autophagy-related protein 14,ATG14)调控家兔毛囊毛乳头细胞(dermal papilla cells, DPCs)自噬进程对毛囊发育生长的影响。本试验选取健康6月龄长毛兔,采集背部皮肤分离培养DPCs,通过克隆ATG14基因的编码序列(coding sequence, CDS),利用生物信息学对ATG14的生物学特性进行初步分析,在家兔DPCs中过表达或敲减ATG14对自噬相关蛋白和毛囊生长发育相关基因的表达及DPCs增殖水平的影响进行探究。结果表明,ATG14基因CDS长度为1 479 bp,共编码492个氨基酸,不存在潜在信号肽及跨膜区,属于定位于细胞核的不稳定蛋白,在不同哺乳动物中存在同源性。家兔DPCs中过表达或敲减ATG14后,WB结果显示ATG14能够上调自噬标志蛋白LC3和Beclin1的蛋白表达,抑制自噬抑制蛋白P62表达水平。ATG14能够增加细胞中pEGFP-LC3B荧光表达,表明ATG14能够激活细胞中LC3B的表达。同时,在DPCs中过表达ATG14能够显著上调CCND1、FGF2、LEF1、BCL2和WNT2的mRNA表达水平,显著下调SFRP2和TGFβ-1的基因表达水平(P<0.05),敲减ATG14能够显著下调CCND1、FGF2、LEF1、BCL2和WNT2的基因表达水平,显著上调SFRP2和TGFβ-1的mRNA水平(P<0.05)。ATG14能够上调LEF1和CCND1的蛋白表达水平。此外,DPC中过表达ATG14能够显著促进DPCs细胞增殖(P<0.01)。本研究通过对家兔ATG14基因调控DPCs自噬进程的功能进行分析,为阐明家兔毛囊生长发育的调控机制提供理论依据。

人脐带干细胞外泌体对人毛乳头细胞增殖的影响

目的探究人脐带间充质干细胞外泌体(hUCMSC-Exos)对人毛乳头细胞(HDPCs)增殖的影响,并对hUCMSC-Exos促毛发生长作用的机制进行初步探索。方法二步酶法分离HDPCs并进行体外培养,培养人脐带间充质干细胞(hUC-MSCs),收集细胞培养上清,通过超高速离心法提取外泌体,并对其进行电镜、粒径及表面标记物鉴定。双氢睾酮(dihydrotestosterone,DHT)诱导HDPCs建立雄激素性脱发细胞模型。将hUCMSC-Exos与HDPCs共培养,采用细胞增殖试验(EdU)检测诱导的HDPCs的相对活力。Real-time qPCR检测碱性磷酸酶(ALP)表达水平、Western blot检测β-catenin、Wnt10b、GSK-3β在蛋白水平的表达。结果所获取的HDPCs、h UC-MSCs和hUCMSC-Exos均符合毛乳头细胞、间充质干细胞及外泌体特征。EdU阳性细胞数显著增加,外泌体可有效促进HDPCs增殖(P<0.05),增强HDPCs活力,减轻DHT造成的损伤(P<0.05)。Real-time qPCR显示外泌体可增强ALP基因表达水平(P<0.05),增强毛囊诱导能力;Western blot证实β-catenin、Wnt10b、GSK-3β在蛋白水平的表达均有差异(P<0.05)。结论hUCMSCExos可促进DHT诱导的HDPCs增殖,增强其毛囊再生修复能力,其机制可能与激活Wnt/β-catenin信号通路有关。

雪松醇抑制二氢睾酮诱导胎鼠真皮成纤维细胞雄激素受体活化

目的探究雪松醇对体外培养胎鼠真皮成纤维细胞二氢睾酮(DHT)诱导雄激素受体(AR)活化的抑制作用。方法分离第18.5天(E18.5)C57BL/6J胎鼠真皮,建立原代胎鼠真皮成纤维细胞培养;用倒置显微镜观察胎鼠成纤维细胞凝集生长;用碱性磷酸酶(ALP)试剂盒和荧光染色技术检测胎鼠成纤维细胞的ALP活性和CD133表达。将指数生长期胎鼠真皮成纤维细胞分为4组,即空白对照组、DHT组、雪松醇组和雪松醇+DHT联合处理组。不同浓度(0~300mol/L)雪松醇和DHT处理细胞48h后,用细胞计算试剂盒(CCK8)测定细胞存活率;用免疫荧光法、蛋白质印迹法(Westernblotting)和实时荧光定量聚合酶链反应(qRT-PCR)法检测各组AR及其共激活因子四个半LIM结构域2(FHL2)的表达水平和AR核定位;用ALP染色法检测各组细胞ALP活性变化。结果30μmol/L DHT处理胎鼠真皮成纤维细胞,48 h后观察到AR主要分布在细胞核,胞核/胞质荧光强度比值为(3.39±0.04)/高倍镜视野(HPF)。然而,雪松醇+DHT联合处理组胎鼠真皮成纤维细胞AR着色分布在胞浆和胞核,胞核/胞浆荧光强度比值为(1.24±0.02)/HPF,二者比较差异有统计学意义(P<0.05),提示雪松醇可抑制DHT诱导的AR核转位;此外,笔者还观察到,DHT可以下调ALP表达并上调AR共激活因子FHL2的表达,而雪松醇可以逆转DHT对成纤维细胞的作用。结论雪松醇除阻止DHT诱导AR核转位以外,还能抑制共激活因子FHL2的表达,双靶点下调DHT-AR信号活性。外用雪松醇治疗雄激素源性脱发(AGA)值得进一步研究。

DDIT4介导的自噬在毛乳头细胞氧化应激损伤中的保护作用

目的探索DNA损伤诱导转录因子4(DNA damage induced transcription factor 4,DDIT4)在毛乳头细胞氧化应激损伤中的保护作用及其机制。方法使用长波紫外线(ultraviolet radiation A,UVA)和过氧化氢(H 2 O 2)建立毛乳头细胞氧化应激模型;CCK-8检测不同处理条件下的细胞活力,利用2′,7′-二氯荧光素二乙酸酯(2′,7′-dichlorofluorescein diacetate,DCFH-DA)试剂检测细胞内活性氧(ROS)的变化;电镜观察自噬小体;Western blot观察DDIT4及自噬相关指标微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、泛素结合蛋白(ubiquitin-binding protein P62,P62)、雷帕霉素哺乳动物靶蛋白(mammalian target of rapamycin,mTOR)、p-mTOR的表达情况。结果UVA和H 2 O 2导致毛乳头细胞内ROS升高,活力下降(P<0.05);氧化应激环境下毛乳头细胞内的DDIT4表达增加(P<0.05),而抗氧化剂N-乙酰基-L-半胱氨酸(N-acetyl-L-cysteine,NAC)可有效抑制此效应(P<0.05);UVA或H 2 O 2处理后毛乳头细胞内自噬水平升高,表现为自噬体数量增多,LC3Ⅱ/Ⅰ比值增高(P<0.05),P62表达下降(P<0.05),3-甲基腺嘌呤(3-methyladenine,3-MA)阻断自噬可观察到细胞活力进一步降低(P<0.05),细胞内ROS增多(P<0.05),反之,雷帕霉素(rapamycin,RAPA)可增加自噬水平提升氧化条件下毛乳头细胞活力,减少细胞内ROS生成(P<0.05);下调DDIT4表达可使氧化应激环境下毛乳头细胞自噬减弱,LC3Ⅱ/Ⅰ减少(P<0.05),p-mTOR/mTOR、P62增加(P<0.05),细胞活力下降(P<0.05),细胞内ROS增多(P<0.05)。结论DDIT4可能通过自噬缓解毛乳头细胞氧化应激损伤。

防脱育发草本精粹对毛发生长及其抗氧化、抗炎的性质研究

目的 探究防脱育发草本精粹防脱育发能力。方法 通过细胞计数试剂盒8测定防脱育发草本精粹对真皮乳头细胞(DPC)增殖与抗氧化能力作用,利用实时荧光定量PCR检测防脱育发草本精粹对DPC抗氧化、抗炎症关键基因的表达影响,通过动物实验验证其对于毛发生长的作用。结果 防脱育发草本精粹(250μg/ml)能显著促进DPC增殖与抗氧化能力,显著抑制白细胞介素-6(IL-6)和促进抗氧化相关基因:谷氨酸-半胱氨酸连接酶修饰剂亚基基因(GCLM)、血红素加氧酶1基因(HMOX1)、铁蛋白重链1基因(FTH1)、铁蛋白轻链基因(FTL)、NAD(P)H:醌氧化还原酶1(NQO1)的表达,差异有统计学意义(P <0.05)。动物实验验证显示,防脱育发草本精粹缩短了小鼠毛囊的休止期。结论 该防脱育发草本精粹显著促进DPC的增殖、抗氧化及抗炎症能力,缩短毛囊的休止期和促进毛发生长。

毛乳头细胞凝集性生长与细胞外基质分泌关系的组织化学研究

目的 研究毛乳头细胞的凝集性生长方式与其产生的细胞外基质之间的关系。方法 用人头皮分离的低传代毛乳头细胞与毛发上皮细胞共同培养形成的毛乳头细胞凝集性生长区及非凝集生长区的毛乳头细胞进行阿新蓝、过碘酸雪夫氏染色 ,观察染色反应的强弱。结果 新分离的毛乳头 ,阿新蓝和过碘酸雪夫氏染色均呈强阳性 ,培养的毛乳头细胞凝集性生长区阿新蓝—过碘酸雪夫氏染色亦显示强阳性 ,而非凝集区的毛乳头细胞显色呈弱阳性。结论 培养的毛乳头细胞凝集性生长后 。

毛乳头细胞诱导毛囊形成的研究

目的 探讨培养的毛乳头细胞在体内外条件下诱导毛囊形成的可能性。方法 采用酶消化法获得毛乳头细胞、真皮鞘细胞、毛囊上、下段及球部细胞 ,进行毛囊组织工程重建 ,或用游离细胞混合移植于裸鼠 ,组织学观察毛囊形成情况。 结果 毛囊间表皮细胞、毛囊上段上皮细胞、下段上皮细胞和球部细胞在间质细胞凝胶上均可形成双层结构的组织工程皮肤 ,在真皮鞘细胞胶原凝胶上毛囊的上、下段上皮细胞形成了毛囊结构 ,移植于裸鼠后 8周毛乳头细胞胶原凝胶诱导毛囊上、下段细胞形成了毛囊。低代毛乳头细胞与毛囊上皮细胞混合移植形成了数量较多、结构典型的毛囊 ,并有肉眼可见的毛发纤维产生。结论 毛囊的真皮成分细胞即毛乳头细胞、真皮鞘细胞在体内、外均具有诱导毛囊形成的能力 ,通过与毛囊上皮细胞之间的相互作用 ,可诱导毛囊形成。

毛乳头细胞凝集性生长对诱导毛囊样结构形成能力的影响

目的 以细胞外基质成分纤维连接蛋白促进培养的人头皮毛乳头细胞凝集性生长行为 ,观察毛乳头细胞的凝集性生长对诱导毛囊形成能力的影响。方法 传代培养的人头皮毛乳头细胞经细胞外基质纤维连接蛋白预处理后 ,促进形成凝集性生长团 ,再与毛囊上皮细胞共同移植于裸鼠皮下 ,分别于 8、12周进行组织学检查 ,观察毛囊形成及分化程度。结果 8周后纤维连接蛋白预处理的毛乳头细胞组可见有毛囊样结构形成 ,未处理组仅见松散细胞团 ;12周后两组均可见有毛囊样结构形成 ,但纤维连接蛋白预处理组较未处理组诱导形成毛囊样结构分化更成熟 ;真皮成纤维细胞组未见有毛囊样结构形成。结论 毛乳头细胞的凝集性生长特性对其诱导毛囊形成的能力有密切的关系 ,细胞呈凝集性生长后 ,其生物学功能增强。

人毛乳头细胞在不同传代时期某些细胞因子表达的变化

目的观察毛乳头细胞在体外培养条件下的生长特性,为毛囊重建提供参考资料。方法采用二步酶消化法分离正常人头皮毛囊的毛乳头细胞,在体外进行传代培养,用免疫组化方法观察不同传代毛乳头细胞生长因子表达的差异。结果高代培养毛乳头细胞逐渐丧失其凝集性生长的特性,生长因子的表达逐渐消失。结论毛乳头细胞的生长及其特性的保持受许多因素的影响,体外培养的毛乳头细胞与体内有差异。低代毛乳头条件培养基可恢复毛乳头细胞部分生长特性。

毛囊真皮细胞的培养和组织化学研究

目的为了提高毛乳头细胞培养的成功率和工作效率，并观察体外培养条件下毛乳头细胞的组织化学性质。材料与方法采用0．2％胶原酶D直接消化人产砂皮毛囊下部真皮鞘组织，分离出来的毛乳头予以悬浮培养，真皮鞘细胞和毛乳头细胞分别用DMEM培养基进行传代培训和多种组织化学染色。结果消化法显著降低了劳动强度，提高了培养成功率，加快了毛乳头细胞的生长，组织化学染色表明，毛乳头细胞阿新兰、甲苯胺O和PAS染色阳性，有异染性，龙是细胞凝集区和多层化细胞团中呈强阳性，而真皮勒细胞对阿新兰和甲苯胶蓝O染色呈阳性，无异染性，PAS阴性。结论消化法是一种简捷快速的高效培养毛乳头细胞的新方法，而毛乳头细胞是一类高度特殊化了的成纤维细胞，真皮鞘细胞与之关系紧密。

人毛囊细胞来源双层皮肤替代物的构建和组织学特征

目的探索人毛囊细胞构建的双层皮肤替代物的组织学特征。方法用毛乳头细胞、真皮鞘细胞分别与外根鞘细胞构建复合壳多糖双层皮肤替代物,分析其体外和移植至大白鼠后的组织学特征。结果毛囊来源细胞构建的皮肤替代物中表皮细胞排列更紧密、角化突出,真皮内可见与表皮相连的上皮细胞柱及球形膨大细胞团形成。结论毛囊细胞是构建皮肤替代物的良好细胞来源。