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.

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.

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.

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.

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。

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。

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。

Hair growth-promotion effects and antioxidant activity of the banana flower extract HappyAngel^(■):double-blind, placebo-controlled trial

Banana flowers contain various bioactive components, including several antioxidants with anti-inflammatory effects. However, it is unclear whether they can reduce and prevent hair loss. This study examines the effect of banana flower extracts on preventing hair loss and strengthening hair roots. The banana flower extract(HappyAngel^(■))was used to treat human hair follicle dermal papilla cells(HFDPCs)and the expression of reactive oxygen species(ROS), dihydrotestosterone(DHT), and hair-related genes(SRD5A1, SRD5A2, AR, and KROX20)were monitored. Fifty subjects were divided into a placebo group and a banana flower group. The experimental group consumed banana flower extract daily for twelve weeks and then underwent hair testing, hair-related genes analysis, collection of hair loss, and questionnaires. The results showed that the banana flower extract significantly increased hair cell growth and decreased the expression of ROS, DHT, and hair follicle growth inhibition-related SRD5A1, SRD5A2, and AR genes, and significantly increased the expression of hair growth-related KROX20 gene in HFDPCs. Consuming banana flower extract for twelve weeks increased the hair root diameter and reduced hair loss and scalp redness compared to the placebo group. Thus, banana flower extract(HappyAngel^(■))can stimulate hair growth and inhibit the activation of hair loss genes.

KRT16在长毛兔毛囊发育过程中的表达规律及功能探究

旨在探究KRT16在长毛兔毛囊发育过程中的表达规律及功能。本试验选取12只健康的6月龄皖系长毛兔,于剪毛后在生长期、退行期和休止期选取背部皮肤采样。通过克隆得到兔KRT16基因的编码序列,利用生物信息学软件对KRT16编码序列(CDS)的生物学特性进行初步分析。实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)分析KRT16在毛囊不同时期表达量。在毛囊毛乳头细胞(dermal papilla cell, DPC)中过表达和敲低KRT16,探究KRT16对毛囊生长发育相关基因的调控作用,以及对DPC增殖的影响。结果显示,KRT16基因的编码序列全长1 431 bp,可编码476个氨基酸,在不同哺乳动物中表现出高度同源性。实时荧光定量PCR结果显示,在毛囊发育周期中,KRT16在毛囊发育周期呈现出不同的表达水平,在生长期高表达。通过在兔毛乳头细胞中过表达与敲减KRT16,检测毛囊发育相关基因的表达水平变化,过表达KRT16后,SFRP2和TGFβ1基因的mRNA表达量极显著下降(P<0.01),BCL2、CCND1、EGF、LEF1和CTNNB1基因的mRNA表达量极显著提高(P<0.01);而敲降KRT16后,SFRP2和TGFβ1基因的表达量极显著提高(P<0.01),BCL2、CCND1、EGF、LEF1和CTNNB1基因的表达量极显著下降(P<0.01)。同时,KRT16能够显著促进毛乳头细胞增殖。综上表明,KRT16参与毛囊周期性发育过程,并能够调控毛囊生长发育相关基因,促进毛乳头细胞增殖。该研究通过对KRT16基因功能的初步研究,为长毛兔毛囊发育的基础研究提供参考,同时为毛囊发育相关基因的功能研究提供依据。

小鼠毛乳头细胞体外分离培养方法的优化和评价

目的 :探索小鼠毛乳头细胞(DPCs)体外分离培养的改良新方法。方法 :选用雌性C57BL/6小鼠,以精细显微解剖获取毛乳头部位,联合胶原酶消化,并添加10 ng/mL碱性成纤维细胞生长因子(bFGF)以优化培养体系,建立体外分离培养小鼠DPCs的改良新方法;以光镜下观察、细胞计数、碱性磷酸酶(ALP)特异性染色及荧光定量PCR鉴定DPCs的ALP mRNA表达水平,与传统方法比较进行相关评价。结果 :与传统方法比较,改良方法的毛乳头基本全部贴壁,细胞可更快迁出及生长融合,所获得的细胞数量是传统方法的5.85倍;经传代后传统方法的低代次DPCs有聚集性生长现象,随代次增加,细胞形态逐渐变大衰老,增殖明显迟缓,聚集性生长愈发不明显,至第5代聚集性现象消失。而改良方法的DPCs经传代后,第1、2和5代形态仍呈长梭形及多角形的特点,至第5代仍保有聚集性生长的特点;且具有更强的ALP活性,尤其是高代次(第5代)细胞更为明显,而传统方法的高代次细胞则几乎无ALP活性;改良方法的ALP mRNA表达水平与传统方法相比也明显上调,尤其是第5代细胞更加明显。结论 :改良方法可获取更多数量、并具有较好功能的小鼠DPCs,为开展毛囊相关研究提供可靠的细胞来源。

毛囊微型化发生的机制与最新研究进展

雄激素性脱发(androgenetic alopecia,AGA)又称为脂溢性脱发,是临床上最常见的脱发类型,毛囊微型化是该病发生发展过程中的重要特征。目前研究已发现有很多原因可诱导毛囊微型化的发生,一方面通过促使患者头皮毛囊真皮乳头细胞(dermal papilla cell,DPC)分泌毛囊抑制因子、DPC发生凋亡和自噬障碍,另一方面通过毛周微循环形成障碍、毛囊干细胞的过度耗竭及真皮鞘细胞的丢失与纤维化进一步加剧了AGA的发生发展,最终导致患者的毛发由最初的终毛逐渐转换为细小的毳毛。本综述从不同角度探讨毛囊微型化的病因与机制,为未来对AGA的有效治疗提供帮助。

头发与头皮护理的科学基础(Ⅷ)——防脱生发体外评价方法以及植物防脱原料研究进展

随着科技发展和人们生活水平的改善,大众的审美亦逐渐提高,人们对于外貌尤其是头发方面愈加关注,防脱、生发也成为了大家关注的焦点。脱发,是指头发脱落的一种生理现象,分为生理性脱发和病理性脱发。毛囊(HF)在毛发形态的形成和生长中起着重要作用,不同的细胞信号通路和生长因子参与调控毛囊生长周期。本文基于人真皮乳头细胞(HDPCs)模型,综述了几种体外应激诱导模型,通过模型中标记物的变化,以此来考察和筛选防脱原料的功效。此外,还列举了几种常见的植物防脱原料,并概括其在毛囊中的作用机理,希望为今后防脱生发理论的研究和植物防脱原料开发提供参考。

马尾松松针提取物对人毛乳头细胞的保护作用及机制研究

研究了马尾松松针提取物对人毛乳头细胞(Human dermal papilla cells,HDP)的保护作用及机制。通过制备并分析不同溶剂松针提取物中主要活性成分含量;建立二氢睾酮(Dihydrotestosterone,DHT)诱导HDP细胞损伤模型,采用CCK8法检测细胞相对活力,qPCR检测细胞中血管内皮生长因子(Vascular endothelial growth factor,VEGF)、转化生长因子-β2(Transforming growth factor-β2,TGF-β2)、β-连环蛋白(β-Catenin)和蓬乱蛋白1(Dishevelled 1,DVL1)基因相对表达量,探究松针提取物对Wnt/β-Catenin信号通路的影响;最后采用UPLC-QTOF/MS分析鉴定提取物成分。结果表明,松针50%醇提物可显著提高HDP细胞相对活力,最高达88.3%(P<0.05);显著提高VEGF mRNA表达量(P<0.05),降低TGF-β2 mRNA相对表达量(P<0.01);并显著促进Wnt/β-Catenin信号中DVL1 mRNA和β-Catenin mRNA表达(P<0.05,P<0.01)。从松针50%醇提物中共鉴定出36种成分,包括黄酮类、木脂素类、生物碱和萜类等化合物。马尾松松针提取物对人毛乳头细胞具有保护作用,其机制可能与Wnt/β-Catenin信号传导途径有关。

毛囊再生修复机制及其研究进展

本文对毛囊再生机制及毛囊再生的研究进展进行综述。毛囊是皮肤的基本结构之一,是人体皮肤中生长毛发的重要组织。毛囊的损失或损伤可以导致头发稀疏、白发、脱发等问题,严重影响生活质量。因此,毛囊再生的研究受到了广泛关注。本文详细介绍了毛囊的结构和功能,重点阐述毛囊再生的机制和过程,总结了已有的关于毛囊再生的研究成果。毛囊再生技术具有重要的临床应用价值,如治疗脱发、实现个性化美容等。本文强调毛囊再生研究的重要性,并展望未来的研究方向和应用前景。

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。

硫酸软骨素、硫酸肝素对毛乳头细胞粘附及生长的影响

目的 探讨细胞外基质在毛囊生长发育中的作用 ,寻找毛乳头细胞 (DPC)生长的调节因素。方法 用两步酶消化法分离培养DPC ,并通过免疫组化α 平滑肌肌动蛋白 (Actin)染色证实。测定硫酸软骨素A ,硫酸软骨素C及硫酸肝素对DPC粘附及生长的影响。结果 两步酶消化法分离培养DPC效率高 ,纯度高 ,方法简单易行。硫酸软骨素A和硫酸肝素明显促进DPC粘附及生长 ,而硫酸软骨素C作用不明显。结论 某些细胞外基质可调节DPC的生长 ,从而影响毛囊的生长发育。

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。