毛乳头细胞微囊诱导大鼠耳毛囊再生

目的:应用人头皮毛乳头细胞APA(alginate-polylysine-alginate,海藻酸钠-多聚赖氨酸-海藻酸钠)微囊诱导大鼠耳部毛囊再生。方法:以APA微囊包裹体外分离培养的人头皮毛乳头细胞;将毛乳头细胞微囊移植至大鼠耳部皮下,定时行组织学检查。结果:毛乳头细胞微囊移植3周后,组织学检查显示:移植部位皮下形成有密集的同心圆状毛囊结构形成,其数量、形态、分化程度等与正常鼠耳毛囊明显不同。结论:微囊化毛乳头细胞具备诱导大鼠耳部毛囊再生的功能。     

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。     

毛乳头细胞生长相关基因的筛选

研究表明 ,位于毛囊基底部的特殊间质成分———毛乳头在毛囊的生长发育、周期调控及维持毛发生长中起主导作用。毛乳头的功能障碍是导致毛囊周期失衡和脱发现象的主要起始因素。因此研究毛囊的生长发育、调控机制应不能脱离毛乳头细胞的功能状态。目前对毛乳头细胞的研究已发现一些活性蛋白及细胞因子对毛囊的生长有调节作用 ,但这些成分在毛囊生长调节中并不是关键因素 ,而是在生理 /病理发展过程中起辅助调节作用。在前期的研究中 ,我们证实了毛乳头在体外凝集性生长状态下较非凝集性生长状态生物学功能及诱导毛囊形成的能力均显著增强 ,…     

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，合成分泌细胞外基质的功能较非凝集生长的毛乳头细胞明显增强。     

培养基pH值对毛乳头细胞合成细胞外基质影响的组织化学研究

目的 研究不同pH值条件下毛乳头细胞的细胞外基质的合成情况及其与毛乳头细胞凝集性生长特性之间的关系.方法 通过用不同pH值的DMEM培养基培养低代毛乳头细胞12d,并对4种培养条件下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 阿新蓝-PAS联合染色后,pH6.8培养条件下的毛乳头细胞胞浆内以红色为主,仅含有少许蓝色.pH7.2、pH7.6培养条件下,大部分毛乳头细胞胞浆内既有红色又有蓝色,一部分细胞胞浆内则完全为蓝色,还有少部分细胞胞浆内完全为红色,而且毛乳头细胞在这两种培养条件下出现凝集性生长,毛乳头细胞凝集区域内红色和蓝色明显强于非凝集区.pH8.0培养条件下的毛乳头细胞胞浆内均为红色且未发现凝集性生长出现.结论 毛乳头细胞体外培养环境的最佳pH值为pH7.2～7.6,毛乳头细胞培养环境的酸碱度能够影响毛乳头细胞的细胞外基质的酸性黏多糖的合成.毛乳共细胞合成的酸性黏多糖与毛乳头细胞的凝集性生长有密切关系.     

毛囊细胞混合物移植诱导裸鼠毛囊样结构形成

目的 观察毛囊细胞复合物移植诱导裸鼠毛发再生和毛囊重建情况.方法 采用裸鼠移植技术,将混合分离出来的毛囊细胞,包括毛囊毛乳头细胞、毛囊外根鞘细胞、毛囊真皮鞘细胞和毛囊真皮成纤维细胞等,移植到裸鼠皮下,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 毛囊细胞混合物可以在体内诱导出毛囊样结构形成.     

活血补肾合剂对人头皮毛乳头细胞中血管内皮细胞生长因子表达的影响

目的:观察活血补肾合剂对人头皮毛乳头细胞增殖及其血管内皮细胞生长因子(VEGF)表达的影响。方法:将12只正常雄性SD大鼠随机分为3组,即活血补肾合剂组、养血生发胶囊组和生理盐水组,分别以不同药物灌饲大鼠。3d后采血收集血清,以三种药物血清分别培养人头皮毛乳头细胞;MTT法检测三种药物血清对毛乳头细胞增殖的影响;以RT-PCR方法检测三种药物血清对毛乳头细胞VEGF mRNA表达的影响;用免疫印迹法检测三种药物血清对毛乳头细胞VEGF蛋白表达的影响。结果:活血补肾合剂组和养血生发胶囊组毛乳头细胞数目显著多于生理盐水组(P0.05);活血补肾合剂组VEGF mRNA及其蛋白表达高于养血生发胶囊组和生理盐水组(P0.05)。结论:活血补肾合剂可能是通过上调人头皮毛乳头细胞VEGF表达以促进毛乳头细胞增殖和毛发生长。     

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.     

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.     

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.     

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.     

毛乳头在毛囊生长周期中的变化和作用

目的 了解人毛乳头结构在毛发生长周期中的变化及其对毛发生长的调控作用.方法 将整形手术后取下的头皮在无菌条件下分离成游离的毛囊,在Williams E培养基中培养;通过横断毛囊和完整毛囊的培养来观察毛囊的生长状况和处于不同生长周期的毛囊的毛乳头结构变化.结果 分离后的完整毛囊在Williams E培养基中平均可继续生长12 d,平均生长速度为每天0.2～0.3 mm;静止期或生长初期毛囊的毛乳头呈圆形贴在毛球底部,快速生长的毛囊毛乳头变长呈锥形,结构疏松,与毛根紧密相接,当毛囊进入衰退期直至停止生长后毛乳头萎缩变小,并与毛根逐渐分离.在低位横断毛囊,毛囊可再生毛球并继续生长,但生长速度减慢;在高位横断毛囊,毛囊不能继续生长.结论 毛乳头在毛发生长周期中不断变化,生长期毛乳头体积增大,与毛母质联系紧密,它在毛囊的生长和生长周期中起着不可缺少的调控作用.     

正常人毛乳头细胞和真皮鞘细胞的生长特性观察

目的 观察毛乳头细胞和真皮鞘细胞在体外培养条件下的生长特性。方法 采用胶原酶消化法培养正常人头皮毛囊的毛乳头细胞和真皮鞘细胞,在体外进行传代培养,测定毛乳头细胞不同时相点的细胞数,并观察表皮细胞生长因子、氢化可的松＋胰岛素、粒细胞巨噬细胞集落刺激因子对毛乳头细胞和真皮鞘细胞增殖的影响。结果 毛乳头细胞、真皮鞘成纤维细胞和真皮成纤维细胞的生长呈现３个时相,即停滞期、指数生长期和缓慢生长期。３种细胞都有４ｄ的停滞期,５～１１ｄ为指数生长期,之后呈现出缓慢生长。ＥＧＦ对３种细胞都有显的促进作用（Ｐ〈０．００１）,尤其是对真皮鞘成纤维细胞的作用更强。氢化可的松和胰岛素、ＧＭ－ＣＳＦ对３种细胞的作用不明显。毛乳头细胞、真皮鞘细胞和成纤维细胞的倍增时间分别为６０、５９．２、７６．８ｈ。结论 ＥＧＦ对毛乳头细胞、真皮鞘细胞和