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.     

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。     

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。     

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。     

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.     

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     

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.     

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.     

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.     

影响毛囊器官型培养中胶原凝胶收缩的几个因素分析

目的 :观察鼠尾胶原、毛乳头细胞和毛囊上皮细胞的浓度对毛囊器官型培养模型中胶原凝胶收缩的影响。方法 :用不同浓度的鼠尾胶原、毛乳头细胞和毛囊上皮细胞制备成毛囊器官型培养模型 ,体外培养 10 d,隔日测量培养凝胶的直径 ,观察其对胶原凝胶收缩的影响。结果 :鼠尾胶原、毛乳头细胞和毛囊上皮细胞的浓度对毛囊器官型培养模型中胶原凝胶的收缩均有明显影响 (P<0 .0 1)。鼠尾胶原的浓度越高 ,胶原凝胶的收缩程度越小 ;而毛乳头细胞和毛囊上皮细胞的浓度对胶原凝胶收缩的影响则恰好相反 ,浓度越高 ,胶原凝胶的收缩程度越大。结论 :鼠尾胶原、毛乳头细胞和毛囊上皮细胞的浓度是影响毛囊器官型培养模型中胶原凝胶收缩的主要因素。     

毛囊球部细胞体外在胶原/壳聚糖多孔支架上形成皮肤样结构的研究

目的:观察毛囊球部细胞种植到胶原/壳聚糖多孔支架上皮肤重建情况.方法:采用细胞培养技术和细胞支架种植技术,将培养的毛囊球部细胞种植到胶原/壳聚糖多孔支架上,HE染色观察皮肤样结构形成情况,并作免疫组织化学鉴定,以证明细胞的来源属性.结果:毛囊球部细胞种植到胶原/壳聚糖多孔支架上可见皮肤样结构形成,表皮部分细胞分化良好,排列复层整齐,有角化现象,高分子与低分子角蛋白表达阳性.结论:低传代培养的毛囊球部细胞在体外胶原/壳聚糖多孔支架上可诱导皮肤样结构形成.