中波紫外线诱导小鼠皮肤光损伤中miRNA141表达的研究

【摘要】 目的 探讨小室移植法重建小鼠毛囊,观察细胞成分对毛囊再生的影响。 方法 取0 ～ 2 d的C57BL/6乳鼠背部皮肤,胰酶消化后分离表真皮,再分离出毛囊上皮细胞。实验分表皮细胞混合毛囊胚芽组、真皮细胞组、表皮细胞混合毛囊胚芽 + 真皮细胞组、毛囊上皮细胞 + 真皮细胞组。用小室移植法接种于裸鼠背部,并于移植后1、2、4、8周观察变化,HE染色观察毛囊组织学形态。 结果 小鼠毛囊细胞移植1周时,背部小室开始脱落,伤口结痂;2周时除表皮细胞组外,另3组均长出了短小的毛发,镜下可见毛囊样结构;4周及8周时除表皮细胞组外,均长出了正常的毛发,表皮细胞与真皮细胞混合组及毛囊上皮细胞与真皮细胞混合组毛发生长情况良好,优于单独的真皮细胞组。 结论 毛囊细胞小室移植后可形成新的毛囊,上皮细胞及真皮细胞在毛囊重建中具有重要的作用。 【关键词】 毛囊; 毛囊重建; 鼠科     

毛囊混合细胞植入胶原/壳聚糖多孔支架诱导裸鼠毛发生长及支架内形成血管样结构

目的 探讨利用毛囊混合细胞植入胶原,壳聚糖多孔支架内重建毛囊的可行性与支架内血管形成状态.方法 用注射法将体外培养的C57BL/6J近交系乳鼠背部皮肤毛囊混合细胞接种至胶原,壳聚糖多孔支架,培养2周后,将含毛囊混合细胞胶原/壳聚糖多孔支架植入裸鼠皮下,肉眼观察裸鼠背部毛发形成情况.6周后取移植区皮肤组织经10%甲醛固定后行组织学观察(HE染色).结果 移植至裸鼠皮下5周后,裸鼠背部在含毛囊混合细胞的胶原,壳聚糖多孔支架移植区皮肤出现毛发,丰长,6周后取皮肤组织作HE染色发现有分化成熟的毛囊形成,且支架内有血管样结构形成.而空白胶原/壳聚糖多孔支架移植区皮肤未发现毛生长,组织检杳也未发现有毛囊形成,且只有在支架表浅部位有少黑血管样结构形成.结论 毛囊混合细胞植入胶原/壳聚糖多孔支架内可诱导裸鼠毛发的形成,促进支架血管样结构的形成.     

毛囊混合细胞在胶原/壳聚糖多孔支架上重建毛囊样结构

目的探讨利用毛囊混合细胞植入胶原／壳聚糖多孔支架内体外重建毛囊的可行性。方法用滴加法或注射法将体外培养的C57BL／6J近交系乳鼠背部皮肤毛囊混合细胞以不同传代数、不同细胞密度接种至胶原／壳聚糖多孔支架，倒置显微镜下观察支架表面或浅层的细胞生长或毛囊形成情况。将支架经10％甲醛固定后行组织学观察（H-E染色）。另用共聚焦激光扫描显微镜观察支架内活细胞的生长以及毛囊样结构的形成。结果在一定传代次数和细胞密度下，在支架内可形成具有毛干的毛囊样结构。激光共聚焦扫描显微镜发现团块内细胞排列呈同心圆状，整个三维结构似一长颈花瓶，且该结构仅见于注射法接种细胞的支架内。结论毛囊混合细胞植入胶原／壳聚糖多孔支架内体外可形成具有毛干的毛囊样结构。     

小鼠皮肤移植细胞有效重建毛发

目的研究将新鲜分离的新生小鼠皮肤细胞移植到免疫缺陷鼠上有效重建毛囊的新技术。方法新生C57小鼠取其皮肤,将表皮和真皮细胞分别分离并混合,做成高浓度的细胞悬液,移植到免疫缺陷鼠NU/NU,CB-17 SCID,NOD SCID上,观察皮肤再生情况。结果细胞移植2周之后,伤口处肉眼可见黑色皮肤形成,3周后长出大量毛发,8周后观察重建的毛发维持良好状态。没有移植细胞的对照观察到伤口愈合但没有毛发形成。组织学分析显示再生的皮肤含有成熟的毛囊和毛干并连有皮脂腺及真皮毛乳头,并且新形成的皮肤不仅有表皮层和真皮层,还有皮下脂肪层,表明移植的细胞可重建完整的皮肤。细胞移植到三种不同的免疫小鼠NU/NU,CB-17 SCID和NOD SCID上,毛囊重建的结果相似。结论创建了一套分离和移植皮肤细胞的技术,可在免疫缺陷鼠上高效地重建毛发。该技术可以用于毛囊再生的研究,并具有潜在的临床应用价值。     

不同剂量刺蒺藜对小黑鼠毛囊黑皮素1受体表达的影响

目的观察不同剂量的刺蒺藜对C57BL/6 J小鼠毛囊黑皮素1受体(MC-1R)表达的影响,探讨不同剂量刺蒺藜在毛囊黑素细胞活化过程中的作用。方法选择SPF级C57BL/6 J小黑鼠为实验动物,采用免疫组化技术观察用不同剂量中药刺蒺藜的水提物灌服后小鼠皮肤中MC-1R水平的变化,以正常小黑鼠皮肤为对照。结果MC-1R的表达定位于表皮基底部黑素细胞、角质形成细胞胞浆;在各组皮片中均呈阳性表达,刺蒺藜用量越大表达越明显;MC-1R在各组皮片中的表达与正常对照组皮肤比较差异均有显著性(P<0.05)。结论不同剂量刺蒺藜都能够有效上调MC-1R的表达,MC-1R在刺蒺藜作用下的表达可能是通过扩增生物学效应的方式使黑素合成增强,从而起到调节黑素代谢的作用。     

真皮干细胞在毛囊周期中的作用

毛囊由表皮(上皮)及真皮(间充质)组成,它们之间的相互作用在毛囊的形态发生及生长中发挥重要作用,二者之间相互作用是毛囊成功重建的关键因素.在毛发形成过程中,真皮细胞是诱导者,上皮细胞是应答者.真皮鞘和毛乳头内存在毛囊真皮干细胞,属于成体干细胞,具有慢周期、未分化、自我更新和体外增殖能力强的特点.真皮鞘中的真皮干细胞较长寿,可以历经几个毛囊周期,重建真皮鞘.在毛囊周期的生长期,真皮鞘中的干细胞产生新细胞提供给毛乳头;在退行期,真皮干细胞子代移出毛乳头或死亡.毛囊真皮细胞对于损伤和疾病之后的毛囊重建及修复具有重要意义.     

毛囊毛发的病理生理学

<正>0001小鼠皮肤移植细胞有效重建毛发郝泽芳(济南市妇幼保健院妇产科),潘婧,吴训伟//中国皮肤性病学杂志.-2015,29(11).-1130~1133,1144新生C57小鼠取其皮肤,将表皮和真皮细胞分别分离并混合,做成高浓度的细胞悬液,移植到免疫缺陷鼠NU/NU,CB-17SCID,NODSCID上,观察皮肤再生情况。结果:细胞移植2周之后,伤口     

毛囊干细胞与组织工程研究进展

毛囊干细胞研究取得突破性进展的标志是由孙同天、Lavker和Cotsarelis等提出了隆突激活假说,明确了其定位的部位,是少数几种成体干细胞首先被明确定位的干细胞之一,并逐步得到了验证。毛囊干细胞不仅可以分化为毛囊上皮细胞,而且可以分化为皮脂腺细胞、表皮角质形成细胞,其分化过程是干细胞分化为短暂倍增细胞,最后分化为终末分化细胞。近年来进一步证明了毛囊隆突部也是多种干细胞的居住地,包括黑素干细胞,和毛囊干细胞存在同步化激活的机制。Wnt/β-catenin是毛囊发育、再生循环的基本信号通路,使毛囊在再生过程出现着色一致的毛发。诱导和重建毛囊形成是组织工程皮肤最主要的目标之一,毛乳头细胞的凝集性生长特性是诱导毛囊形成的条件,因此,维持毛乳头细胞凝集性生长特性主要有细胞团块法、低黏附性培养板(或高分子材料膜)法、悬滴培养法和胶原凝胶成球法,以实现体外重建毛乳头的目的。毛囊组织工程在体外还不能真正诱导形成,现有的毛囊再生或新生的成功方法还是须移植到动物体内,才能形成完整毛囊并产生毛发。主要方法有移植小室法、混合游离细胞注射法和皮瓣法。诱导毛囊形成比较成功的细胞是啮齿类动物来源的胚胎或新生鼠毛囊细胞和人胚胎毛囊细胞,成年人头皮毛囊来源细胞目前仍未能诱导出毛囊新生或再生。     

甲状旁腺激素对小鼠毛发周期和毛发生长的影响

以C57BL6小鼠作为动物模型,观察甲状旁腺激素(PTH)的多肽片段PTH(7～34)和PTH(1～34)对毛发周期和毛发生长的影响。用松香/蜡混合物拔毛法诱导C57BL6小鼠毛发由休止期进入生长期,拔毛同时腹腔注射PTH(7～34),持续19天,观察毛发周期的变化。结果,PTH(7～34)组小鼠在拔毛后第5天毛发进入生长期,比对照组提前24h左右;至第19天,对照组小鼠多数毛发进入退行期或休止期,而PTH(7～34)组仍有大部分毛发维持在生长期。C57BL6乳鼠触须毛囊体外培养结果,加PTH(7～34)10-6mol/L浓度组和10-7mol/L浓度组毛干生长速度显著比对照组加快(P<0.01);而PTH(1～34)10-9mol/L浓度组毛干生长明显慢于对照组(P<0.01)。提示PTH的不同片段对毛发生长有不同影响,PTH(7～34)可延长毛发生长期,促进小鼠毛发生长,而PTH(1～34)则对毛发生长有抑制作用。     

UVA诱导抗Ro/SSA血清注射多巴色素异构酶基因敲除小鼠表皮细胞凋亡

目的 探讨人抗Ro/SSA血清腹腔注射多巴色素异构酶基因敲除小鼠(Dct~(-/-))与野生型C57BL/6J小鼠对慢性UVA照射诱导表皮细胞凋亡的易感性.方法 将高滴度人抗Ro/SSA血清间断腹腔注射于Dct~(-/-)、鼠与野生型C57小鼠,随后给予小鼠慢性UVA照射.每次照射剂量为10 J/cm~2,共6次,累计剂量为60 J/cm~2.取尾部照射区域皮肤,行石蜡包埋组织切片,经HE染色和TUNEL标记在光镜下分别计数每个视野100个表皮细胞中日晒伤细胞(SBC)数和凋亡细胞数.结果 经慢性UVA照射后,抗Ro/SSA血清注射Dct~(-/-)-小鼠SBC数为(14±1.0)/100个表皮细胞,凋亡细胞数为(62 ±2.7)/100个表皮细胞.血清注射C57野生型小鼠SBC数为(7±0.6)/100个表皮细胞,凋亡细胞数为(30±1.6)/100个表皮细胞,两者比较差异均有统计学意义(P<0.05).而未注射血清的Dct~(-/-)小鼠SBC数为(6±0.9)/100个表皮细胞,凋亡细胞数为(42±2.5)/100个表皮细胞,与注射血清的Dct~(-/-)小鼠相比差异有统计学意义(P<0.05).结论 当一定浓度的抗Ro/SSA抗体存在时,表皮黑素细胞Dot基因缺陷能增强慢性UVA诱导的表皮细胞凋亡,可能关系到红斑狼疮患者抗Ro/SSA抗体介导皮肤光敏感性发生.     

Kit is expressed by epithelial cells in vivo

In mammalian skin, stem cell factor (SCF) regulates the proliferation and maturation of mast cells and melanocytes, which are thought to be the only cutaneous cells that express the Kit-tyrosine kinase receptor (Kit) and respond to epithelial and mesenchymal-derived SCF. We previously had noted, however, the presence of Kit+ cells in murine hair follicles, in an introepithelial tissue compartment devoid of melanocytes and mast cells. Here we have identified the nature of this Kit+ population of cells in hair follicles of C57BL/6 mice. Anagen hair follicles showed strong Kit immunoreactivity not only in the pigmentary unit above the follicular dermal papilla but also in a much more proximally located, homogenous group of nondendritic, nonmelanized cells. By immunohistochemistry (desmoplakin+/Trp-1-) and electron microscopy (presence of tonofilaments, desmosomes, lack of melanosomes), these Kit+ cells were shown to be hair matrix keratinocytes and were also found in melanocyte-deficient hair follicles (Kit(Sl)/Kit(Sl-d) mice, Kit-neutralizing antibody-treated C57BL/6 mice). Expression of Kit and SCF was strongly hair-cycle-dependent, suggesting a functional role of epithelial Kit expression in hair growth control. This was supported by the observation that mice unable to respond to SCF stimulation (Kit(W)/Kit(W-v)) showed a significant retardation of anagen development compared to their wild-type littermates. The expression of Kit in the most rapidly proliferating compartment of the hair follicle epithelium suggests intriguing, as yet unexplored new functions of Kit signaling in epithelial cell biology.     

转录辅激活因子Mediator 1调控小鼠皮肤毛发再生的作用机制研究

目的:探讨转录辅激活因子Mediator 1（Med1）对小鼠皮肤毛发再生的影响及可能机制。方法:将C57BL/6J品系来源的Med1 flox/flox小鼠与K14-Cre小鼠交配,通过Cre-Loxp系统获得Med1表皮特异性敲除小鼠,即表达K14-Cre的Med1 flox/flox小鼠（...     

Determinants of the direction of hair follicle growth after reconstruction with dissociated cells

Background/objectives: Several hair follicle reconstruction models based on the implantation of dissociated cells have been established. However, little has been said about the direction of growth, which is a very important issue for clinical applications. The aim of this study was to reveal the factors that determine the direction of hair follicle growth after reconstruction with dissociated cells. Methods: We designed an enclosed chamber assay in which a chamber was buried under nude mouse skin that was not exposed to the air, as in a traditional chamber assay. Inductive dermal cells and epithelial cells dissociated from neonatal C57 BL/6 mice were injected into the chamber and dermis for patch assays. The direction of newly formed hair follicles was analysed. Results: Newly formed hair follicles in the enclosed chamber were arranged randomly parallel to the skin on the surface of the panniculus carnosus. Conclusions: These results indicate that developmental space provides the possibility for growth of reconstructed hair follicles and that the air–liquid interface determines the final direction of growth.     

Dissociated skin cells regenerate hair follicles in a microwound, “The Punch Assay”

The purpose of this study was to develop a new, easily executed hair follicle regeneration system and assay, which could be further developed for clinical or cosmetic applications. Dissociated epidermal and dermal progenitor cells, isolated either from neonatal C57BL/6 mice or human foetal scalp tissues, were suspended in (10 μL) F12 medium and pipetted into a 1 or 2 mm-diameter punch biopsy wounds on the back skin of immunodeficient mice. At 3 weeks after transplantation, although pigmented mouse hairs could efficiently form at the injection sites with delivery of mouse cells, none hair formed on the host mouse skin at 3 months after delivery of human cells. Under the same conditions, human follicles could be regenerated when the human skin cells were delivered onto a 2 mm size punch created on a reconstituted human skin (hRSK), which previously generated on the back of an immunodeficient mouse, but the efficiency of hair formation was low. We demonstrated that both mouse and human regenerated follicles showed normal histology and differentiation markers; moreover, the cell chasing experiment confirmed that the regenerated hair follicles were formed from transplanted cells. Compared to other current hair reconstituted assays, the Punch Assay is relatively simple and generates normal hair follicles within a smaller wound. We suggest that the punch assay is a better in vivo assay of cell trichogenicity.