毛囊细胞移植诱导裸鼠毛囊样结构形成的研究

目的:观察毛囊细胞移植诱导裸鼠毛发再生和毛囊重建情况.方法:采用细胞培养技术和裸鼠移植技术,将培养的毛囊毛乳头细胞、真皮鞘细胞和头皮真皮成纤维细胞按比例与毛囊上皮细胞混合,移植到裸鼠皮下,观察毛囊形成情况.结果:在毛囊毛乳头细胞与毛囊上皮细胞混合后移植到裸鼠皮下后可见毛囊样结构形成,而毛囊真皮鞘细胞和头皮成纤维细胞与毛囊上皮细胞混合则不能诱导裸鼠毛囊样结构形成.结论:低传代培养的毛乳头细胞与毛囊上皮细胞混合后在体内可诱导毛囊样结构形成.     

在双层皮肤替代物中诱导毛囊形成的初步研究

目的探索人毛乳头细胞和外根鞘细胞在双层皮肤替代物中诱导毛囊形成的可能性。方法用毛乳头细胞和与外根鞘细胞构建复方壳多糖双层皮肤替代物,观察其体外和大白鼠移植后的组织学特征。结果体外毛乳头细胞和外根鞘细胞构建的皮肤替代物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。但移植后未见肯定的毛囊样结构形成。结论目前的细胞培养技术尚不能在双层皮肤替代物中诱导毛囊形成。     

外源性结缔组织生长因子在人毛囊混合细胞重建组织工程毛囊中的作用

目的 探讨外源性结缔组织生长因子(CTGF)在人毛囊混合细胞重建组织工程毛囊中的作用.方法 采用细胞培养技术体外培养人毛囊外根鞘细胞及人毛乳头细胞,人毛囊外根鞘细胞与人毛乳头细胞以5∶1的比例制备细胞混悬液.流式细胞学检测人毛囊外根鞘细胞中CD200阳性细胞含量.取8只裸鼠用随机数字表法均分为A、B组,制作裸鼠背部创面.A组移植添加20μg/LCTGF的细胞混悬液,B组移植单纯的细胞混悬液.于8周后进行组织学检查,观察毛囊形成情况.采用普通PCR检测移植物组织中人特异性DNA及鼠DNA表达情况.结果 流式细胞学检测结果表明培养的人毛囊外根鞘细胞中CD200阳性细胞占细胞总量19.65％.组织学检查显示A组毛囊样结构形成数量(268±96)显著多于B组(62±20),差异有统计学意义(P＜0.05).PCR结果表明移植物中含有人组织成分.结论 CTGF可促进人毛囊外根鞘细胞与人毛乳头细胞相互作用,诱导毛囊样结构形成.     

人头皮毛乳头细胞体内诱导毛囊形成的实验研究

目的 探讨人头皮毛乳头细胞和毛囊上皮细胞在体内的相互作用，了解毛乳头细胞在体内诱导毛囊形成和调控毛囊生长发育的能力，为毛囊细胞移植奠定实验基础。方法 人头皮毛乳头细胞和毛囊上皮细胞共同移植到无胸腺裸鼠体内，在不同时相取材进行组织切片。H—E、角蛋白免疫组织化学染色。结果 毛乳头细胞和毛囊上皮细胞在体内相互作用，首先形成不规则的混合细胞团、逐渐发展到排列规则、最终形成完整的毛囊结构，并且在移植腔上方的裸鼠皮肤内形成了毛囊结构。这些结构表达毛囊特有的角蛋白。结论 培养的毛乳头细胞与毛囊上皮细胞在裸鼠体内能形成完整的毛囊结构，同时也能诱导裸鼠角质形成细胞、形成毛囊结构。     

毛囊细胞移植法诱导毛囊的初步研究

目的 构建一个可靠有效的移植毛囊细胞诱导毛发发育的模型,以治疗脱发.方法 取自愿捐献的成人头皮标本,联用显微分离与免疫磁珠法获得人毛囊干细胞;消化法获得毛乳头细胞.培养后混合植入裸鼠皮下,观察毛囊形成情况.结果 在裸鼠的皮肤切片中可以看到较为完整的毛囊结构形成.结论 毛囊细胞移植法可以在体内诱导出毛囊样结构,为将来治疗脱发奠定了基础.

Abstract：

Objective To establish a convenient and reliable method for inducing hair regeneration by follicular cell implantation for the treatment of alopecia. Methods The human hair follicle stem cells were separated and purified by micromanipulation and magnetic cell sorting, and human scalp dermal papilla cells were isolated by enzyme digestion. The two cells were mixed and implanted subcutaneously in nude mice to observe the regeneration of the hair follicles. Results Formation of intact hair follicle-like structures was observed in the skin sections of the recipient nude mice. Conclusion Follicular cell implantation can induce hair follicle-like structures in nude mice, which provides a means for efficient hair regeneration for treatment of hair loss.     

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.     

重建毛囊的组织学研究

目的：观察毛囊真皮鞘成分和上皮成分细胞间的相互作用和相互影响。方法：采用细胞三维培养技术将分段毛囊上皮细胞分别与毛乳头细胞、真皮鞘细胞及真皮成纤维细胞进行器官型培养来重建表皮和毛囊。结果：在真皮成纤维细胞凝胶上培养的毛囊间表皮细胞、上段、下段和球部细胞重建出了表皮，而上段细胞形成的表皮最厚，球部细胞形成的表皮最薄；在毛囊真皮鞘细胞胶原凝胶上时上段和下段细胞重建出了毛囊结构。结论：首次成功地证明毛囊     

不同性别间的毛囊诱导作用

成人毛发的生长和毛囊的循环再生受到位于毛囊基底部的真皮与表皮组织之间相互作用的精确调控。在啮齿类动物,将分离的毛囊皮肤细胞重新导入体内可诱导生成新的毛囊,但在人类还未获成功。尽管人类的组织器官移植已取得很大进展,但排斥反应严格地限制了在免疫不相容性个体间的细胞移植,即使毛囊被认为是一组具有“免疫特权”的器官也不例外。本文报道了在人类不同性别的组织不相容个体之间移植毛囊真皮鞘组织可以诱导新毛囊形成的研究结果。     

小鼠触须毛囊外根鞘细胞的体外培养及uPA和uPAR表达研究

目的建立小鼠毛囊外根鞘细胞的体外培养方法及研究尿激酶型纤溶酶原激活剂及其受体(uPA/uPAR)的表达.方法建立以毛囊真皮鞘细胞为滋养层,培养外根鞘细胞并对uPA/uPAR的表达进行免疫细胞化学鉴定.结果外根鞘细胞在此滋养层上生长较好,并有uPA/uPAR的表达.结论毛囊真皮鞘细胞是外根鞘细胞较好的滋养层,毛囊外根鞘细胞具有迁移增殖能力.     

Biological characterization of cultured dermal papilla cells and hair follicle regeneration in vitro and in vivo

Background Dermal papilla cells (DPC) are a group of mesenchyme-derived cells at the base of the hair follicle, where they regulate and control hair follicle growth through the expression and secretion of cytokines. Nevertheless, the role of DPC derived chemokines and other cytokines in the hair follicle biology remain speculative. In this study, we investigated the expression of basic fibroblast growth factor (bFGF), endothelin-1 (ET-1) and stem cell factor (SCF) in different passages of cultured DPC and their effects on the biological behaviour of DPC.Methods The expression of bFGF, ET-1 and SCF in different passages of cultured DPC and their possible effects on the biological behavior of DPC are investigated using in situ hybridization and immunochemistry. In addition, we performed transplantation of hair follicle cells into nude mice. The cultured DPC, dermal sheath cells and fibroblast of human scalp, respectively, were mixed with cells of the hair follicle epithelium in different ratios, and then were cultured in hair follicle organotypic cultures or implanted into the subcutis of nude mice.Results The expression of ET-1 and SCF in early passages of cultured DPC became stronger, but turned weaker and even negative in late passages (>6 passages). Hair follicle-like structures were formed after DPC combined with the cells of hair follicle epithelium cells in hair follicle organotypic cultures. When hair follicle organotypic cultures were implanted into the subcutis of nude mice, the relative intact hair follicles were formed. After the transplantation of hair follicle cells into the nude mice, the hair follicle-like structure was formed in the group that contained DPC mixed with hair follicle epithelium cells. However, no hair follicles were formed in the other two groups. It was found that the higher the expression of ET-1 and SCF in DPC, the stronger the ability of DPC to induce hair follicle regeneration.Conclusions The cultured DPC can induce hair follicle regeneration and sustain hair growth in vivo and in vitro. Moreover, the expression of ET-1 and SCF is correlated with the ability of DPC inducing hair follicle regeneration.     

Biological characterization of cultured dermal papilla cells and hair follicle regeneration in vitro and in vivo

Background Dermal papilla cells （DPC） are a group of mesenchyme-derived cells at the base of the hair follicle, where they regulate and control hair follicle growth through the expression and secretion of cytokines. Nevertheless, the role of DPC derived chemokines and other cytokines in the hair follicle biology remain speculative. In this study, we investigated the expression of basic fibroblast growth factor （bFGF）, endothelin-1 （ET-1） and stem cell factor （SCF） in different passages of cultured DPC and their effects on the biological behaviour of DPC. Methods The expression of bFGF, ET-1 and SCF in different passages of cultured DPC and their possible effects on the biological behavior of DPC are investigated using in sire hybridization and immunochemistry. In addition, we performed transplantation of hair follicle cells into nude mice. The cultured DPC, dermal sheath cells and fibroblast of human scalp, respectively, were mixed with cells of the hair follicle epithelium in different ratios, and then were cultured in hair follicle organotypic cultures or implanted into the subcutis of nude mice. Results The expression of ET-1 and SCF in early passages of cultured DPC became stronger, but turned weaker and even negative in late passages （〉6 passages）. Hair follicle-like structures were formed after DPC combined with the cells of hair follicle epithelium cells in hair follicle organotypic cultures. When hair follicle organotypic cultures were implanted into the subcutis of nude mice, the relative intact hair follicles were formed. After the transplantation of hair follicle cells into the nude mice, the hair follicle-like structure was formed in the group that contained DPC mixed with hair follicle epithelium cells. However, no hair follicles were formed in the other two groups. It was found that the higher the expression of ET-1 and SCF in DPC, the stronger the ability of DPC to induce hair follicle regeneration. Conclusions The cultured DPC can induce hair follicle regeneration and sustain hair growth in vivo and in vitro. Moreover, the expression of ET-1 and SCF is correlated with the ability of DPC inducing hair follicle regeneration.     

GasderminA3基因突变对小鼠毛囊β-catenin表达的影响

目的探讨GasderminA3(GSDMa3)基因突变前后β-catenin在毛囊中表达的变化。方法采用免疫组化S-P法、RT-PCR、Western blot等方法,检测出生后11d(毛囊生长期)、16d(毛囊退化早期)、18d(毛囊退化晚期)、24d(毛囊静止期)等各个时相点的GSDMa3基因突变鼠和C57BL/6(B6)鼠背皮毛囊β-catenin的表达表化。结果GSDMa3基因突变鼠和B6鼠β-catenin的表达均从11～24d逐渐减弱,但GSDMa3基因突变鼠β-catenin的表达在各个时期均强于B6鼠。在生长期,β-catenin表达于内外根鞘和毛母质细胞中,在GSDMa3基因突变鼠的表达强于B6鼠;在退化期,β-catenin主要表达于外根鞘和毛母质细胞中,在突变鼠的表达更强于B6鼠;在静止期,β-catenin主要表达在突变鼠的外根鞘细胞中,而在B6鼠的毛囊中表达很少。结论GSDMa3基因可能通过调节β-catenin的表达,影响毛囊的生长和发育。     

TPA促进小鼠毛囊中TCF3的表达

目的 检测12-O-十四烷酰佛波醋酸酯-13 (TPA)处理C57 BL/6( B6)小鼠背部皮肤后TCF3表达的变化情况.方法 采用免疫荧光染色、RT-PCR和Western blot等技术,检测TPA和丙酮分别处理7周大小的B6鼠背部皮肤1、2和4周后TCF3的表达情况.结果 与丙酮处理组相比,TPA处理组中B6鼠...     

胚胎皮肤细胞裸鼠皮下移植构建毛发发育模型

目的 观察胚胎皮肤细胞裸鼠皮下移植后的毛发发育情况,构建毛发发育模型.方法 从C57BL/6鼠胚胎的皮肤中分离出真皮和表皮细胞,将它们按照一定比例重新混合后采用注射法移植到裸鼠皮下,显微镜下观察毛发形成情况.结果 镜下观察到裸鼠皮下有毛囊及毛发纤维形成并有毛干长出.结论 胚胎皮肤细胞体内移植可以构建毛发发育的完整模型.

Abstract：

Objective To observe the hair development after subcutaneous implantation of embryonic skin cells in nude mice, and construct a model of hair development. Methods Dermal and epidermal cells isolated from embryonic mouse skin were mixed at a given ratio and injected subcutaneously in nude mice, and hair formation after the implantation was observed under a microscope. Results Formation of the hair follicles and fibers under the skin of the recipient nude mice and emergence of the hair shaft were observed microscopically. Conclusion Embryonic skin cells be used to construct a complete model of hair development after implantation in vivo.     

3-甲基腺嘌呤通过抑制自噬促进小鼠毛囊再生的作用研究

为研究自噬在毛囊周期循环和休止期脱发中的作用,以及是否可通过调节毛囊上皮细胞的自噬促进休止期毛囊的再生,采用拔毛法诱导3~4周龄C57BL/6小鼠的毛囊生长期,给予腹腔注射自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)处理,并以空白和自噬诱导剂雷帕霉素(rapamycin,RAPA)为对照,结果发现,经3-MA处理后的毛囊再生明显加快,且新生毛发的数量与质量均明显优于Vehicle组和RAPA组。取处理后的小鼠皮肤组织经HE染色、Western blot、免疫组化和免疫荧光检测,发现经3-MA处理后,毛囊上皮细胞中的Beclin1表达、LC3BⅡ/Ⅰ比值均显著降低,P62和Ki67表达增加,同时以CD34、CD49f为双标记的毛囊干细胞的数目明显增多;但经RAPA处理后的毛囊中Beclin1表达、LC3BⅡ/Ⅰ比值明显增加,但P62和Ki67表达下降,且毛囊干细胞的数目减少。实验结果表明,通过3-MA抑制毛囊上皮细胞的自噬可以促进由拔毛法诱导的C57BL/6小鼠的毛发再生。3-MA或其他相关的自噬抑制剂可能在未来的人类休止期脱发的治疗中发挥重要作用。     

去细胞羊膜负载毛囊干细胞修复裸鼠全层皮肤缺损

目的利用去细胞羊膜负载毛囊干细胞修复裸鼠全层皮肤缺损。方法体外分离和培养人毛囊干细胞,取经慢病毒介导的绿色荧光蛋白（pGC FU-GFP-Lentiviru）标记后第4代毛囊干细胞接种于去细胞羊膜上;负载后第7天,HE染色光学显微镜观察细胞黏附生长情况。于18只C57BL/6裸鼠背部制作全层皮肤缺损创面,并根据不同的处理方式分为实验组（将负载毛囊干细胞的去细胞羊膜植入裸鼠全层皮肤缺损创面）、去细胞羊膜移植＋干细胞注射组（将去细胞羊膜覆盖创面后羊膜下注射5×106毛囊干细胞）和去细胞羊膜移植组（去细胞羊膜单独移植）,每组6只。分别于移植后第7、14、21、28天,测量各组创面面积并计算创面收缩率。移植后第28天,荧光显微镜观察创面组织绿色荧光蛋白（GFP）表达,HE染色光学显微镜观察新生皮肤结构特征。结果去细胞羊膜负载毛囊干细胞后第7天,HE染色光学显微镜观察发现,细胞连接成片状覆盖于去细胞羊膜表面。实验组和去细胞羊膜移植＋干细胞注射组移植后各时点的创面收缩率均显著小于去细胞羊膜移植组（P〈0.05）。移植后第28天,荧光显微镜观察显示实验组创面表皮层GFP表达阳性;HE染色光学显微镜观察发现实验组创面组织表皮层明显增厚,有类似毛囊样结构生成。结论去细胞羊膜负载毛囊干细胞可用于修复裸鼠全层皮肤缺损。