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。     

毛囊真皮细胞的培养和组织化学研究

为了提高毛乳头细胞增减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０．２％胶原酶Ｄ直接消化产砂皮毛囊下部真皮鞘组织，分离出来的毛乳头予以浮培养。真皮鞘细胞和毛乳头细胞分别是用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，毛头头细胞阿新兰，甲苯胺Ｏ和ＰＡＳ染色阳性，有异染性，尤     

人毛囊外根鞘隆起、真皮鞘和毛乳头细胞高效同步分离培养的方法

目的 寻求一种快速高效同步分离培养人头皮毛囊外根鞘隆起(Bulge)细胞.真皮鞘细胞和毛乳头细胞的方法.方法 将人头皮标本剪成小块,中性蛋白酶中37℃孵育2h后镜下将带外根鞘的毛干从真皮鞘中拔出,组织块法培养外根鞘隆起(Bulge)细胞;从真皮一皮下组织交界处横断头皮,拔出含毛乳头的真皮鞘,胶原酶D 37℃孵育6～8h,多次低速离心,毛乳头沉淀重悬后培养,收集的真皮鞘细胞上清液经高速离心后重悬培养;培养的细胞分别用K19或α-平滑肌动蛋白组化鉴定.结果 同一标本可获得纯化的外根鞘Bulge细胞,真皮鞘细胞和毛乳头细胞.结论 将现有的分离方法改进和综合运用.可以从同一毛囊标本同步高效获取3种成分细胞.     

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     

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.     

毛囊细胞生物学和器官型培养的研究

毛囊细胞生物学和器官型培养的研究Ｓｔｕｄｙｏｎｔｈｅｃｅｌｌｂｉｏｌｉｇｙａｎｄｏｒｇａｎｏｔｙｐｉｃｃｕｌｔｕｒｅｏｆｈｕｍａｎｈａｉｒｆｏｌｌｉｃｌｅｓ伍津津，刘荣卿（第三军医大学附属西南医院皮肤科）重庆，６３００３８关键词毛囊；毛乳头细胞；器官...     

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.     

两种人毛囊外根鞘细胞培养方法的比较

目的　建立外根鞘细胞的培养方法 ,并比较不同方法的优缺点 ,以进一步探索毛囊干细胞的体外培养条件。方法　分别采用组织法和消化法进行外根鞘细胞的培养。结果　两种方法均能培养出大量的外根鞘细胞并进行传代 ,组织法操作简单 ,但细胞生长比率低 ,细胞不易融合 ,消化法培养细胞分布均匀 ,细胞克隆形成能力强 ,增殖迅速。结论　消化法适合于进行毛囊外根鞘细胞的体外培养。     

以表皮细胞及脱细胞真皮构建组织工程皮肤

目的 体外扩增培养表皮细胞，复合脱细胞真皮构建组织工程皮肤，以促进其进一步临床应用。方法 将人表皮细胞进行体外培养扩增后，以生长良好的表皮细胞接种于制备好的异种（猪）无细胞真皮支架上，行气液界面联合体外培养，构建组织工程皮肤。观察细胞生长情况及复合皮结构。结果 培养的表皮细胞扩增良好，脱细胞真皮支架去细胞完全，表皮细胞在脱细胞真皮上可以生长，可体外构建复合皮肤。结论 利用体外扩增的表皮细胞及制备的脱细胞真皮可体外联合构建具有复层结构的组织工程皮肤。     

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.     

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.     

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.     

尿激酶型纤溶酶原激活物在毛囊中的表达

目的 探讨尿激酶型纤溶酶原激活物（urokinase-type plasminogen activator,uPA)在毛囊发育过程中的原位表达及在体外培养的毛囊细胞中的表达。方法 用免疫细胞化学（immunocytochemistry,ICC)和原位杂交（in situ hybridization,ISH)技术研究了人及小鼠在毛囊生长周期的不同时期uPA的表达；体外培养小鼠毛囊外根鞘（outer root sheath,ORS)细胞并用ICC方法研究了ORS细胞uPA的表达。结果 在毛囊生长期的早期（即毛囊形成期），毛囊角质形成细胞（keratinocyte,Kc)表达uPA，而其它时期均不表达；体外培养的ORS有uPA表达。结论 uPA与Kc的快速增殖和迁移相关，在毛囊的发育中起重要作用。     

大鼠皮肤及毛囊骨桥蛋白研究

目的 对出生和出生后大鼠皮肤及毛囊的骨桥蛋白（ｏｓｔｅｏｐｏｎｔｉｎ）的ｍＲＮＡ进行研究。方法 确定妊齿１４～１８ｄ和出生后１～６１ｄ Ｗｉｓｔａｒ大鼠毛周期,用放性同位素标记的原位杂交法牟大鼠皮肤和毛囊骨桥蛋白ｍＲＮＡ的分布和含量进行研究。结果 骨桥蛋白ｍＲＮＡ在大鼠出生后第１８天首次在真皮乳头（ｄｅｒｍａｌｐａｐｉｌｌａ,ＤＰ）细胞中出现,在各毛发周期中骨桥蛋白ｍＲＮＡ信号均出现一毛囊退化期Ｄ