IGF-1、FGF7与毛乳头细胞生物学特性的相关性及复方甘草酸苷、地塞米松对毛乳头细胞活性影响的研究
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摘要
目的:作为皮肤的附属器官,毛发具有重要的生理学与社会学功能。毛发缺失或分布异常给患者带来很大的心理负担。目前对毛发生长机制的研究主要集中于毛囊。毛乳头细胞(Dermal papilla cell,DPC)是位于毛囊基底部的一群真皮源性细胞,它们是一簇特化的成纤维细胞(specialized fibroblast),其显著的生物学特性为体内外表现为凝集性的生长方式及体内外具有诱导毛囊形成的能力,并在毛囊的形态学发生及其周期性生长调控中处于中心环节。而在多次传代后,这种特性就会消失。毛乳头细胞的生物学作用是通过一系列信号分子通路、多种细胞生长因子完成的。胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)结构与胰岛素类似,能刺激黑素细胞、上皮细胞及毛囊的生长。成纤维细胞生长因子7(fibroblast growth factor7,FGF7)又称角质形成细胞生长因子,可刺激角质形成细胞和其它上皮细胞的增殖。
毛乳头细胞不仅对诱导和维持毛囊的周期性循环必不可少,毛乳头的大小也决定了毛囊的大小和毛囊生长期的长短。甘草是传统中药养发生发产品中使用频率较高的药物和添加成分,其主要成分为甘草酸,甘草提取物可促进体外培养的鼠触须毛囊生长。复方甘草酸苷(商品名:美能)的主要成分为甘草酸苷即β-甘草酸,被广泛用于皮肤病和肝病的治疗。临床实验发现复方甘草酸苷对斑秃治疗有效,其诱导毛发生长的机制尚不明确,其对体外培养的人毛乳头细胞的作用还未见报道。糖皮质激素应用于斑秃治疗迄今已有60余年的历史,是目前治疗斑秃最为常用、可靠及有效的方法之一。其主要机制可能与抗炎及免疫抑制有关,但对人毛乳头细胞的作用还未见报道。
为观察体外毛乳头细胞的生物学特性,及其与IGF-1和FGF7的关系,复方甘草酸苷和地塞米松对毛乳头细胞的影响,我们对人头皮毛乳头进行分离培养,并检测两种生长因子分别在凝集性、非凝集性生长的毛乳头细胞中的表达,以及IGF-1和复方甘草酸苷、地塞米松对毛乳头细胞增殖的影响,为临床筛选刺激毛囊及毛发生长的药物提供实验基础。
方法:
1毛乳头细胞的分离培养
采用“二步酶消化法”分离培养正常人毛乳头,观察体外培养毛乳头细胞形态和生长情况,并行α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)免疫组化染色鉴定。
2采用免疫组化法分别检测第3代和第9代培养的毛乳头细胞IGF-1、FGF7蛋白的表达水平。
3采用流式细胞仪分别检测第3代和第9代培养的毛乳头细胞IGF-1、FGF7蛋白的表达水平,及细胞周期变化和凋亡情况。
4 MTT比色分析法检测不同浓度IGF-1(2.5～100ng/ml)对人毛乳头细胞增殖的影响。
5 MTT比色分析法检测不同浓度复方甘草酸苷(0.5～100μg/ml)对人毛乳头细胞增殖的影响。
6 MTT比色分析法检测不同浓度地塞米松(0.5～100μg/ml)对人毛乳头细胞增殖的影响。
7运用统计软件SPSS13.0对数据进行统计处理,采用t检验、单因素方差分析,以α=0.05为差异显著性标准。
结果:
1人毛乳头细胞的分离、培养与鉴定
1.1人毛乳头细胞的分离、培养
经“二步酶消化法”得到的毛乳头,在倒置显微镜下观察呈圆形或椭圆形,第二天即可见贴壁,贴壁1天后可观察到细胞迁出,向四周呈放射状生长。2～3周后细胞大部分生长融合,低倍镜下可见细胞呈漩涡状生长,有多层凝集趋势,细胞呈长梭形。细胞消化传代后第2天可见细胞贴壁展开,随着细胞数目增多,又融合形成多层凝集性生长。细胞传代后增殖速度也加快,尤以3～5代细胞最为显著,且均可见凝集性生长。随毛乳头细胞传代次数增加,凝集性逐渐消失,细胞变为多极、梭形或细长的成纤维细胞样,生长速度变慢,传代周期明显延长。
1.2人毛乳头细胞的鉴定
人毛乳头细胞α-SMA免疫组化染色呈阳性。
2免疫组织化学S-p法分别检测3代、9代细胞IGF-1和FGF7的表达
IGF-1与FGF7均表达于细胞胞浆,着色由黄色到褐色不等。3代和9代毛乳头细胞免疫组化染色后IGF-1表达分别为+～++和+;FGF7表达分别为+～++和±～+。
3流式细胞仪分别检测3代、9代毛乳头细胞IGF-1、FGF7的表达
流式细胞技术检测结果显示,3代、9代毛乳头细胞IGF-1的均道值分别为270.98±26.84、232.14±14.80,前者大于后者,但统计分析表明两者相比无显著性差异(P>0.05)。3代、9代毛乳头细胞FGF7的均道值分别为373.51±24.97、270.91±32.83,前者大于后者,统计学分析表明两者有显著性差异(P<0.05)。
4流式细胞仪检测人毛乳头细胞凋亡及细胞周期分布
3代和9代毛乳头细胞处于G1期的细胞比率分别为55.53±3.34%、57.53±6.84%;处于S期的细胞比率分别为34.87±3.18%、34.10±6.36%;处于G2期的细胞比率分别为7.82±3.37%、8.40±3.19%。各期细胞两者比较均无明显差异(P>0.05)。两组细胞的凋亡率分别为1.36±0.24%、1.28±0.63%,无统计学差异(P>0.05)。
5 IGF-1对人毛乳头细胞增殖的影响
处于对数生长期的第4代毛乳头细胞加入不同浓度的重组人IGF-1培养72h后,MTT结果显示与对照组相比,各浓度组均有明显促进毛乳头细胞增殖的作用(P<0.05),其中以2.5ng/ml最为显著,其余各组间无明显差异,浓度再增加,促增殖作用并不增强。
6复方甘草酸苷对人毛乳头细胞增殖的影响
处于对数生长期的第4代毛乳头细胞加入不同浓度的复方甘草酸苷培养72h后,MTT结果显示在0.5～20μg/ml浓度内,促增殖作用呈剂量依赖性,以20μg/ml组刺激作用最强,有统计学意义(P<0.05),浓度再增加,其促增殖作用反而减弱。
7地塞米松对人毛乳头细胞增殖的影响
在处于对数生长期的第4代毛乳头细胞中加入不同浓度的地塞米松培养72h后,MTT结果显示在0.5～2.5μg/ml浓度内,促增殖作用呈剂量依赖性,以2.5μg/ml组刺激作用最强,有统计学意义(P<0.05),浓度再增加,其促增殖作用有所减弱。
结论:
1二步酶消化法可简便、快捷、有效地分离获得毛乳头。体外培养的毛乳头细胞在6代之前呈明显的凝集性生长特性,继续传代该特性逐渐消失。
2凝集性与非凝集性生长的毛乳头细胞各细胞周期与凋亡均无明显差异,表明体外培养的毛乳头细胞维持在比较稳定的增殖状态。
3 FGF7在凝集性生长的毛乳头细胞中表达明显高于非凝集性生长的毛乳头细胞,推测凝集性生长的毛乳头细胞促进毛母质细胞增殖角化可能与FGF7有关。
4 IGF-1可明显刺激毛乳头细胞增殖,同时受毛乳头细胞表面的IGF结合蛋白调节。IGF-1在凝集性生长的毛乳头细胞中表达高于非凝集性生长的毛乳头细胞,但无统计学差异,IGF-1促进毛囊生长的机制有待进一步探讨。
5复方甘草酸苷和地塞米松在抗炎、免疫调节及抑制的同时还可能通过调节毛乳头细胞的活性,诱导毛发生长。
Objective:As a subsidiary organ of skin, hair has an important function of physiology and sociology. Missing or abnormal distribution of hair brings great psychological burden to patients. The current research of hair growth mechanism has focused on hair follicles. Dermal papilla cells (DPCs) are a group of dermis-derived cells located at the bottom of follicle, which are specialized fibroblasts. The salient biological characteristics of DPCs are manifested aggregation of growth pattern and the ability to induce hair follicle formation in vivo and in vitro. DPCs occur at the central link in the morphology of the hair follicle and its cyclical growth regulation. This feature will disappear after the cells were passed on several times. A number of studies have shown that the biological functions of DPCs have a lot to the aggregation property. The biological functions are completed through a series of signaling molecules pathway and a variety of cell growth factors. Insulin-like growth factor-1(IGF-1) is similar with insulin that can stimulate the melanocytes, epithelial cells and hair follicle growth. Fibroblast growth factor 7 (FGF7) also known as keratinocyte growth factor, can stimulate keratinocytes and other epithelial cell proliferation.
DPCs are not only essential for the induction and maintenance of the cyclical nature of hair follicle cycle, but also for the size and anagen length of hair follicles. Licorice is frequently used in traditional Chinese medicine to promote hair growth. It is demonstrated that licorice can promote hair follicles growth in vitro. Stroner Neo-MinophagenC (SNMC), the essential component of which is glycyrrhizin compounded with glycine and cysteine, is widely used in the treatment of skin diseases and liver disease. Clinical trials found that SNMC is effective for alopecia areata. The mechanism is not clear, its effect on cultured human DPCs has not been reported yet. Glucocorticoid applied for treatment of alopecia areata have been more than 60 years so far, which is one of the most commonly used, reliable and effective methods. Foreign doctors often take the corticosteroid injection as the first treatment for adults with limitated alopecia areata. The mechanism may be related with anti-inflammatory and immune suppression, but the role to human DPCs has not been reported.
To observe the biological characteristics of DPCs in vitro as well as its correlation with IGF-1 and FGF7, and the effects of SNMC and dexameth on the activity of DPCs, we have managed to separate and culture dermal papilla, and detect two factors in the aggregative, non-aggregative growth of DPCs, and evaluate the proliferative effect of IGF-1, SNMC and dexameth on DPCs.
Methods:
1 Isolation and culture of dermal papilla cells
Dermal papillas were isolated from human scalp hair follicles by digestion with dispase and collagenase D and cultured. The morphology and growth of DPCs were observed in vitro.α-smooth muscle actin (α-SMA) was detected by immunohistochemical staining to identify DPCs.
2 The expression of IGF-1,FGF7 protein in different passages of cultured DPCs were detected by immunohistochemical method.
3 The expression level changes of IGF-1, FGF7 in different passages of cultured DPCs were detected by FCM.
4 The FCM was also used to detect the changes of the apoptosis rate and cell cycle in different passages.
5 Effect of different concentration of IGF-1(2.5～100ng/ml) on the cell proliferation of DPCs were measured by MTT.
6 Effect of different concentration of SNMC and dexameth (0.5～ 100ng/ml) on the cell proliferation of DPCs were measured by MTT.
7 Statistic analysis: data were analyzed by statistical software Spss13.0 for windows, using t-test, one-way ANOVA analysis, with significance of difference standardα=0.05.
Results:
1 Isolation, cultivation and identification of human DPCs.
1.1 Isolation and cultivation of human DPCs
The dermal papillas obtained by "two-step enzymatic digestion" were round or oval in the inverted microscope observation. They were adherent next day, and then DPCs could be observed move out to the surrounding in the radiative form. The majority cells were confluent after 2～3 weeks. Observed under low power lens, the cells were swirling growth, with aggregative growth tendency. The cells elongated spindle. Cells were adherent the next day after digestion, aggregative growth patten was formed again with the increase of cells number. Cell proliferation also accelerated after passage, especially in 3rd to 5th generations, and aggregative growth also could be observed. Continue passed on, the cells become multi-level, spindle or elongated fibroblast-like, the aggregative growth gradually disappeared. The cells growed more slowly, passaged longer.
1.2 Identification of human dermal papilla cells
α-SMA was expressed in DPCs by immunohistochemistry method.
2 The expression of IGF-1, FGF7 protein in 3rd and 9th generations of cultured DPCs by immunohistochemical method
IGF-1 and FGF7 were expressed in the cytoplasm, coloring ranging from yellow to brown. The expression of IGF-1 in 3rd, 9th generation DPCs after immunohistochemical staining were +～++, +; FGF7 were +～++,±～+。
3 The expression level changes of IGF-1, FGF7 in different passages of cultured DPCs by FCM
The results showed that fluorescence intensity of IGF-1 in 3rd and 9th generations were 270.98±26.84, 232.14±14.80 respectively, the former exceeded the latter, but the statistical analysis showed that there was no significant differences (P>0.05). The fluorescence intensity of FGF7 were 373.51±24.97, 270.91±32.83 respectively, the former exceeded the latter, statistical analysis showed a significant difference between them (P <0.05).
4 Apoptosis and cell cycle distribution of human DPCs were detected by FCM
The G1-phase ratio of 3rd and 9th generation cells were 55.53±3.34%, 57.53±6.84% respectively; the S-phase ratio of cells were 34.87±3.18%, 34.10±6.36%; the G2-phase ratio were 7.82±3.37%, 8.40±3.19%. The apoptosis of the two generations were 1.36±0.24, 1.28±0.36 respectively. There were no significant differences between the two passages in each phase (P> 0.05).
5 Effect of different concentration of IGF-1(2.5～100ng/ml) on the cell proliferation of DPCs
The DPCs of logarithmic 4th passage which cultured with different concentrations of recombinant human IGF-1 after 72h were observed under light microscope, no obvious morphological difference among them. The results of MTT showed that, compared with the control group, each experimental group could significantly promote the proliferation of DPCs (P <0.05). 2.5ng/ml was the most significant and there were no significant difference among the rest four groups (P> 0.05).
6 Effect of different concentration of SNMC (0.5～100μg/ml) on the cell proliferation of DPCs.
The DPCs of logarithmic 4th passage which cultured with different concentrations of recombinant human IGF-1 after 72h were observed under light microscope, no obvious morphological difference among them. The results of MTT showed that, the effect of the proliferation was dose-dependent with concentration of 0.5～20μg/ml. 20μg/ml could significantly promote the proliferation of DPCs (P <0.05).The effect was weakened when the concentration further increased, and there were no significant difference among the rest groups (P> 0.05).
7 Effect of different concentration of dexameth (0.5～100μg/ml) on the cell proliferation of DPCs
The DPCs of logarithmic 4th passage which cultured with different concentrations of dexameth after 72h were observed under light microscope, no obvious morphological difference among them. The results of MTT showed that, the effect of the proliferation was dose-dependent with concentration of 0.5～2.5μg/ml, of which 2.5μg/ml could significantly promote the proliferation of DPCs (P<0.05). The effect was weakened when the concentration further increased, and there were no significant difference among the rest groups (P> 0.05).
Conclusions:
1“Two-step digestive treatment with dispase and collagenase D”was an efficient and rapid method to isolate and culture human hair dermal papillas.
2 Aggregative growth property was visible in cultured DPCs before 6th generation, this feature gradually disappeared with DPCs were passed on.
3 The cell cycle and apoptosis of DPCs with aggregative and non-aggregative growth were no significant differences, indicating that cultured DPCs maintained at a stable proliferation of the state.
4 FGF7 in DPCs with aggregative growth expressed significantly higher than cells with non-aggregative growth, suggesting that DPCs promote hair matrix cell proliferation and cornification may be related to FGF7.
5 IGF-1 can significantly stimulate the proliferation of DPCs, but also regulated by IGF bingding protein. IGF-1 in the DPCs with aggregation growth expressed higher than that of non-aggregation growth, but no statistically significant difference. It remains to be further explored that the mechanism of IGF-1 promoting the growth of hair follicle.
6 The mechanism of SNMC and dexameth may also induce hair growth by regulating the activity of DPCs in addition to anti-inflammatory, immune regulation and suppression.
毛乳头细胞不仅对诱导和维持毛囊的周期性循环必不可少,毛乳头的大小也决定了毛囊的大小和毛囊生长期的长短。甘草是传统中药养发生发产品中使用频率较高的药物和添加成分,其主要成分为甘草酸,甘草提取物可促进体外培养的鼠触须毛囊生长。复方甘草酸苷(商品名:美能)的主要成分为甘草酸苷即β-甘草酸,被广泛用于皮肤病和肝病的治疗。临床实验发现复方甘草酸苷对斑秃治疗有效,其诱导毛发生长的机制尚不明确,其对体外培养的人毛乳头细胞的作用还未见报道。糖皮质激素应用于斑秃治疗迄今已有60余年的历史,是目前治疗斑秃最为常用、可靠及有效的方法之一。其主要机制可能与抗炎及免疫抑制有关,但对人毛乳头细胞的作用还未见报道。
为观察体外毛乳头细胞的生物学特性,及其与IGF-1和FGF7的关系,复方甘草酸苷和地塞米松对毛乳头细胞的影响,我们对人头皮毛乳头进行分离培养,并检测两种生长因子分别在凝集性、非凝集性生长的毛乳头细胞中的表达,以及IGF-1和复方甘草酸苷、地塞米松对毛乳头细胞增殖的影响,为临床筛选刺激毛囊及毛发生长的药物提供实验基础。
方法:
1毛乳头细胞的分离培养
采用“二步酶消化法”分离培养正常人毛乳头,观察体外培养毛乳头细胞形态和生长情况,并行α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)免疫组化染色鉴定。
2采用免疫组化法分别检测第3代和第9代培养的毛乳头细胞IGF-1、FGF7蛋白的表达水平。
3采用流式细胞仪分别检测第3代和第9代培养的毛乳头细胞IGF-1、FGF7蛋白的表达水平,及细胞周期变化和凋亡情况。
4 MTT比色分析法检测不同浓度IGF-1(2.5～100ng/ml)对人毛乳头细胞增殖的影响。
5 MTT比色分析法检测不同浓度复方甘草酸苷(0.5～100μg/ml)对人毛乳头细胞增殖的影响。
6 MTT比色分析法检测不同浓度地塞米松(0.5～100μg/ml)对人毛乳头细胞增殖的影响。
7运用统计软件SPSS13.0对数据进行统计处理,采用t检验、单因素方差分析,以α=0.05为差异显著性标准。
结果:
1人毛乳头细胞的分离、培养与鉴定
1.1人毛乳头细胞的分离、培养
经“二步酶消化法”得到的毛乳头,在倒置显微镜下观察呈圆形或椭圆形,第二天即可见贴壁,贴壁1天后可观察到细胞迁出,向四周呈放射状生长。2～3周后细胞大部分生长融合,低倍镜下可见细胞呈漩涡状生长,有多层凝集趋势,细胞呈长梭形。细胞消化传代后第2天可见细胞贴壁展开,随着细胞数目增多,又融合形成多层凝集性生长。细胞传代后增殖速度也加快,尤以3～5代细胞最为显著,且均可见凝集性生长。随毛乳头细胞传代次数增加,凝集性逐渐消失,细胞变为多极、梭形或细长的成纤维细胞样,生长速度变慢,传代周期明显延长。
1.2人毛乳头细胞的鉴定
人毛乳头细胞α-SMA免疫组化染色呈阳性。
2免疫组织化学S-p法分别检测3代、9代细胞IGF-1和FGF7的表达
IGF-1与FGF7均表达于细胞胞浆,着色由黄色到褐色不等。3代和9代毛乳头细胞免疫组化染色后IGF-1表达分别为+～++和+;FGF7表达分别为+～++和±～+。
3流式细胞仪分别检测3代、9代毛乳头细胞IGF-1、FGF7的表达
流式细胞技术检测结果显示,3代、9代毛乳头细胞IGF-1的均道值分别为270.98±26.84、232.14±14.80,前者大于后者,但统计分析表明两者相比无显著性差异(P>0.05)。3代、9代毛乳头细胞FGF7的均道值分别为373.51±24.97、270.91±32.83,前者大于后者,统计学分析表明两者有显著性差异(P<0.05)。
4流式细胞仪检测人毛乳头细胞凋亡及细胞周期分布
3代和9代毛乳头细胞处于G1期的细胞比率分别为55.53±3.34%、57.53±6.84%;处于S期的细胞比率分别为34.87±3.18%、34.10±6.36%;处于G2期的细胞比率分别为7.82±3.37%、8.40±3.19%。各期细胞两者比较均无明显差异(P>0.05)。两组细胞的凋亡率分别为1.36±0.24%、1.28±0.63%,无统计学差异(P>0.05)。
5 IGF-1对人毛乳头细胞增殖的影响
处于对数生长期的第4代毛乳头细胞加入不同浓度的重组人IGF-1培养72h后,MTT结果显示与对照组相比,各浓度组均有明显促进毛乳头细胞增殖的作用(P<0.05),其中以2.5ng/ml最为显著,其余各组间无明显差异,浓度再增加,促增殖作用并不增强。
6复方甘草酸苷对人毛乳头细胞增殖的影响
处于对数生长期的第4代毛乳头细胞加入不同浓度的复方甘草酸苷培养72h后,MTT结果显示在0.5～20μg/ml浓度内,促增殖作用呈剂量依赖性,以20μg/ml组刺激作用最强,有统计学意义(P<0.05),浓度再增加,其促增殖作用反而减弱。
7地塞米松对人毛乳头细胞增殖的影响
在处于对数生长期的第4代毛乳头细胞中加入不同浓度的地塞米松培养72h后,MTT结果显示在0.5～2.5μg/ml浓度内,促增殖作用呈剂量依赖性,以2.5μg/ml组刺激作用最强,有统计学意义(P<0.05),浓度再增加,其促增殖作用有所减弱。
结论:
1二步酶消化法可简便、快捷、有效地分离获得毛乳头。体外培养的毛乳头细胞在6代之前呈明显的凝集性生长特性,继续传代该特性逐渐消失。
2凝集性与非凝集性生长的毛乳头细胞各细胞周期与凋亡均无明显差异,表明体外培养的毛乳头细胞维持在比较稳定的增殖状态。
3 FGF7在凝集性生长的毛乳头细胞中表达明显高于非凝集性生长的毛乳头细胞,推测凝集性生长的毛乳头细胞促进毛母质细胞增殖角化可能与FGF7有关。
4 IGF-1可明显刺激毛乳头细胞增殖,同时受毛乳头细胞表面的IGF结合蛋白调节。IGF-1在凝集性生长的毛乳头细胞中表达高于非凝集性生长的毛乳头细胞,但无统计学差异,IGF-1促进毛囊生长的机制有待进一步探讨。
5复方甘草酸苷和地塞米松在抗炎、免疫调节及抑制的同时还可能通过调节毛乳头细胞的活性,诱导毛发生长。
Objective:As a subsidiary organ of skin, hair has an important function of physiology and sociology. Missing or abnormal distribution of hair brings great psychological burden to patients. The current research of hair growth mechanism has focused on hair follicles. Dermal papilla cells (DPCs) are a group of dermis-derived cells located at the bottom of follicle, which are specialized fibroblasts. The salient biological characteristics of DPCs are manifested aggregation of growth pattern and the ability to induce hair follicle formation in vivo and in vitro. DPCs occur at the central link in the morphology of the hair follicle and its cyclical growth regulation. This feature will disappear after the cells were passed on several times. A number of studies have shown that the biological functions of DPCs have a lot to the aggregation property. The biological functions are completed through a series of signaling molecules pathway and a variety of cell growth factors. Insulin-like growth factor-1(IGF-1) is similar with insulin that can stimulate the melanocytes, epithelial cells and hair follicle growth. Fibroblast growth factor 7 (FGF7) also known as keratinocyte growth factor, can stimulate keratinocytes and other epithelial cell proliferation.
DPCs are not only essential for the induction and maintenance of the cyclical nature of hair follicle cycle, but also for the size and anagen length of hair follicles. Licorice is frequently used in traditional Chinese medicine to promote hair growth. It is demonstrated that licorice can promote hair follicles growth in vitro. Stroner Neo-MinophagenC (SNMC), the essential component of which is glycyrrhizin compounded with glycine and cysteine, is widely used in the treatment of skin diseases and liver disease. Clinical trials found that SNMC is effective for alopecia areata. The mechanism is not clear, its effect on cultured human DPCs has not been reported yet. Glucocorticoid applied for treatment of alopecia areata have been more than 60 years so far, which is one of the most commonly used, reliable and effective methods. Foreign doctors often take the corticosteroid injection as the first treatment for adults with limitated alopecia areata. The mechanism may be related with anti-inflammatory and immune suppression, but the role to human DPCs has not been reported.
To observe the biological characteristics of DPCs in vitro as well as its correlation with IGF-1 and FGF7, and the effects of SNMC and dexameth on the activity of DPCs, we have managed to separate and culture dermal papilla, and detect two factors in the aggregative, non-aggregative growth of DPCs, and evaluate the proliferative effect of IGF-1, SNMC and dexameth on DPCs.
Methods:
1 Isolation and culture of dermal papilla cells
Dermal papillas were isolated from human scalp hair follicles by digestion with dispase and collagenase D and cultured. The morphology and growth of DPCs were observed in vitro.α-smooth muscle actin (α-SMA) was detected by immunohistochemical staining to identify DPCs.
2 The expression of IGF-1,FGF7 protein in different passages of cultured DPCs were detected by immunohistochemical method.
3 The expression level changes of IGF-1, FGF7 in different passages of cultured DPCs were detected by FCM.
4 The FCM was also used to detect the changes of the apoptosis rate and cell cycle in different passages.
5 Effect of different concentration of IGF-1(2.5～100ng/ml) on the cell proliferation of DPCs were measured by MTT.
6 Effect of different concentration of SNMC and dexameth (0.5～ 100ng/ml) on the cell proliferation of DPCs were measured by MTT.
7 Statistic analysis: data were analyzed by statistical software Spss13.0 for windows, using t-test, one-way ANOVA analysis, with significance of difference standardα=0.05.
Results:
1 Isolation, cultivation and identification of human DPCs.
1.1 Isolation and cultivation of human DPCs
The dermal papillas obtained by "two-step enzymatic digestion" were round or oval in the inverted microscope observation. They were adherent next day, and then DPCs could be observed move out to the surrounding in the radiative form. The majority cells were confluent after 2～3 weeks. Observed under low power lens, the cells were swirling growth, with aggregative growth tendency. The cells elongated spindle. Cells were adherent the next day after digestion, aggregative growth patten was formed again with the increase of cells number. Cell proliferation also accelerated after passage, especially in 3rd to 5th generations, and aggregative growth also could be observed. Continue passed on, the cells become multi-level, spindle or elongated fibroblast-like, the aggregative growth gradually disappeared. The cells growed more slowly, passaged longer.
1.2 Identification of human dermal papilla cells
α-SMA was expressed in DPCs by immunohistochemistry method.
2 The expression of IGF-1, FGF7 protein in 3rd and 9th generations of cultured DPCs by immunohistochemical method
IGF-1 and FGF7 were expressed in the cytoplasm, coloring ranging from yellow to brown. The expression of IGF-1 in 3rd, 9th generation DPCs after immunohistochemical staining were +～++, +; FGF7 were +～++,±～+。
3 The expression level changes of IGF-1, FGF7 in different passages of cultured DPCs by FCM
The results showed that fluorescence intensity of IGF-1 in 3rd and 9th generations were 270.98±26.84, 232.14±14.80 respectively, the former exceeded the latter, but the statistical analysis showed that there was no significant differences (P>0.05). The fluorescence intensity of FGF7 were 373.51±24.97, 270.91±32.83 respectively, the former exceeded the latter, statistical analysis showed a significant difference between them (P <0.05).
4 Apoptosis and cell cycle distribution of human DPCs were detected by FCM
The G1-phase ratio of 3rd and 9th generation cells were 55.53±3.34%, 57.53±6.84% respectively; the S-phase ratio of cells were 34.87±3.18%, 34.10±6.36%; the G2-phase ratio were 7.82±3.37%, 8.40±3.19%. The apoptosis of the two generations were 1.36±0.24, 1.28±0.36 respectively. There were no significant differences between the two passages in each phase (P> 0.05).
5 Effect of different concentration of IGF-1(2.5～100ng/ml) on the cell proliferation of DPCs
The DPCs of logarithmic 4th passage which cultured with different concentrations of recombinant human IGF-1 after 72h were observed under light microscope, no obvious morphological difference among them. The results of MTT showed that, compared with the control group, each experimental group could significantly promote the proliferation of DPCs (P <0.05). 2.5ng/ml was the most significant and there were no significant difference among the rest four groups (P> 0.05).
6 Effect of different concentration of SNMC (0.5～100μg/ml) on the cell proliferation of DPCs.
The DPCs of logarithmic 4th passage which cultured with different concentrations of recombinant human IGF-1 after 72h were observed under light microscope, no obvious morphological difference among them. The results of MTT showed that, the effect of the proliferation was dose-dependent with concentration of 0.5～20μg/ml. 20μg/ml could significantly promote the proliferation of DPCs (P <0.05).The effect was weakened when the concentration further increased, and there were no significant difference among the rest groups (P> 0.05).
7 Effect of different concentration of dexameth (0.5～100μg/ml) on the cell proliferation of DPCs
The DPCs of logarithmic 4th passage which cultured with different concentrations of dexameth after 72h were observed under light microscope, no obvious morphological difference among them. The results of MTT showed that, the effect of the proliferation was dose-dependent with concentration of 0.5～2.5μg/ml, of which 2.5μg/ml could significantly promote the proliferation of DPCs (P<0.05). The effect was weakened when the concentration further increased, and there were no significant difference among the rest groups (P> 0.05).
Conclusions:
1“Two-step digestive treatment with dispase and collagenase D”was an efficient and rapid method to isolate and culture human hair dermal papillas.
2 Aggregative growth property was visible in cultured DPCs before 6th generation, this feature gradually disappeared with DPCs were passed on.
3 The cell cycle and apoptosis of DPCs with aggregative and non-aggregative growth were no significant differences, indicating that cultured DPCs maintained at a stable proliferation of the state.
4 FGF7 in DPCs with aggregative growth expressed significantly higher than cells with non-aggregative growth, suggesting that DPCs promote hair matrix cell proliferation and cornification may be related to FGF7.
5 IGF-1 can significantly stimulate the proliferation of DPCs, but also regulated by IGF bingding protein. IGF-1 in the DPCs with aggregation growth expressed higher than that of non-aggregation growth, but no statistically significant difference. It remains to be further explored that the mechanism of IGF-1 promoting the growth of hair follicle.
6 The mechanism of SNMC and dexameth may also induce hair growth by regulating the activity of DPCs in addition to anti-inflammatory, immune regulation and suppression.
引文
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47 ReeK. Reduction of Langerhans cells in human epidermis during PUVA therapy: a morphometric study. Invest dermatol 1982,78(6): 488-492
48 Happle R. Topical immunotherapy in alopecia areata. Invest dermatol 1991,96(5):71S-72S
49 Taylor CR,Hawk JL. PUVA treatment of alopecia areata partialis, totalis and universalis: audit of 10 years’experience at St John’s Institute of Dermatology. Br J Dermatol,1995,133(6):914-918
50 Healy E,Rogers S. PUVA treatment for alopecia areata—does it work? A retrospective review of 102 cases. Br J Dermatol,1993,129(1): 42-44
51 Claudy AL,Gagnaire D. PUVA treatment of alopecia areata. Arch Dermatol,1983,119: 975-978
52 Madani S,Shapiro J. Alopecia areata update.J Am Acad Dermatol, 2000,42(4): 549-566
53 Schmoeckel C,Weissmann I, Plewig G, et al. Treatment of alopecia areata by anthralin-induced dermatitis. Arch Dermatol,1979,115(10): 1254-1255
54 Fielder-Weiss VC, Buys CM. Evaluation of anthralin in the treatment of alopecia areata. Arch Dermatol,1987,123(11): 1491-1493
55 Fiedler VC,Wendrow A,Szpunar GJ,et al. Treatment-resistant alopecia areata. Response to therapy with minoxidil plus anthralin. Arch Dermatol 1990,126(6): 756-769
56 Ross EK,Shapiro J. Management of hair loss. Dermatol Clin,2005, 23(2): 227-243
57 Schreiber SL,Crabtree GR. The mechanism of action of cyclosporin A and FK506. ImmunolToday,1992,13(4): 136-142
58 Yamamoto S,Jiang H,Kato R: Stimulation of hair growth by topical application of FK506,a potent immunosuppressive agent. Invest Dermatol,1994,102(2): 160-164
59 McElwee KJ,Tobin DJ,Bystryn JC,et al. Alopecia areata: An autoimmune disease? Exp Dermatol,1999,8(5): 371-379
60 Freyschmidt-Paul P,Seiter S,Zoller M,et al. Treatment with an anti-CD44v10-specific antibody inhibits the onset of alopecia areata in C3H/HeJ mice. Invest Dermatol,2000,115(4): 653-657
61 Ferrando J,Grimalt R. Partial response of severe alopecia areata tocyclosporine A. Dermatology,1999,199(1): 67-69
62 Shapiro J,Lui H,Tron V,et al. Systemic cyclosporine and low-dose prednisone in the treatment of chronic severeb alopecia areata: a clinical and immunopathologic evaluation. Am Acad Dermatol, 1997,36(1): 114-117
63 Taylor M,Ashcroft AT,Messenger AG. Cyclosporin A prolongs human hair growth in vitro. Invest Dermatol,1993,100(3):237-239.
64 Gilhar A,Winterstein F,Krueger GG Hair growth in human split-thickness skin grafts transplanted onto nude rats:The role of cyclosporine[Abstract]. Berlin : Seventh World Congress of Dermatology,1987,426
65 Gold R. Mechanism of action of glucocorticosteroid hormones: Possible implications for therapy of neuroimmunological disorders. Neurommunol,2001,117 (1): 1-8
66周静,杨勤萍,徐峰,等.复方甘草酸苷治疗斑秃10例临床疗效观察.中国皮肤性病学杂志,2008,22(7): 446-447
67 McMichael AJ. The new biologics in psoriasis: possible treatments for alopecia areata. Invest Dermatol,2003,8(2):217-218
68 Strober B,Siu K,Alexis A,et al. Etanercept does not effectively treat moderate to severe alopecia areata: an open-label study. Am Acad Dermatol,2005,52(6): 1082-1084
69 Posten W,Swan J. Recurrence of alopecia areata in a patient receiving etanercept injections. Arch Dermatol 2005,141(6): 759-760
70 Ettefagh L,Neodorost S,Mirmirani P. Alopecia areta in a patient using infliximab: new insights into the role of tumor necrosis factor on human hair follicles. Arch Dermatol,2004,140(8): 1012
71 Kolde G, Meffert H,Rowe E. Successful treatment of alopecia areata with efalizumab. Eur Acad Dermatol Venereol,2008,22(3): 468-473
72 Posten W,Swan J. Recurrence of alopecia areata in a patient receiving etanercept injections. Arch Dermatol,2004,141(6): 759-760
73 Pelivani N,Hassan A,Braathen L,et al. Alopecia areata universaliselicited during treatment with adalimumab. Dermatol,2008,216(4): 320-323
74 Chaves Y,Duarte G,Ben-Said B,et al. Alopecia areata universalis during treatment of rheumatoid arthritis with anti-TNF-a antibody (adalimumab). Dermatol,2008,217(4):380
75 Gundogan C,Greve B,Raulin C. Treatment of alopecia areata with the 308-nm xenon chloride excimer laser : case report of two successful treatments with the excimer laser. Lasers Surg Med. 2004, 34(2): 86-90
76 Zakaria W,Passeron T,Ostovari N,et a1.308 nm excimer laser therapy in alopecia areata. Am Acad Dermatol,2004,51(5): 837-838
77 Raulin C,Gundogan C,Greve B,et a1. Excimer laser therapy of alopecia areata side-by-side evaluation of a representative area. Dtsch Dermatol Ges 2005,3(7): 524-526
78 Jahoda CAB,Home KA,Oliver RF. Induction of hair growth by imp1antation of cultured dermal papilla cells,Nature,1984, 311(5986): 560-562
79李宇,李国强,蔡湘娜,等.微囊化人头皮毛乳头细胞移植诱导毛发形成.中华医学美容杂质,2006,12(6): 350-353
80 Madani S,Shapiro J. Alopecia areata update. J Am Acad Dermatol, 2000, 42(4): 549-566
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1 McDonagh AJG, Messenger AG. The pathogenesis of alopecia areata. Dermatol Clin,1996,14(4): 661-670
2 Seema Garg and Andrew G. Messenger. Alopecia Areata: Evidence-Based Treatments. Semin Cutan Med Surg,2009,28: 15-18
3 de Andrade M,Jackow CM,Danm N,et al.Alopecia areata in families: association with the HLA locus. Investig Dermatol Symp proc, 1999,4(3): 220-223
4 Tan E,Tay YK,Giam YCh. A clinical study of childhood alopecia areata in Singapore. Pediatric Dermatol,2002,19: 298-301
5 Tan E,Tay Y-K,Goh Ch-L,et al.The pattern of alopecia areata in Singapore-a study of 219 Asians. Dermatol,2002,41(11): 748-753
6 Jahoda CA,Horne KA,Oliver RF. Induction of hair growth by implantation of cultured dermal papilla cells.Nature,1984,11-17; 311(5986): 560-562
7 Madani S,Shapiro J. Alopecia areata update. Am Acad Dermatol 2000;42(4): 549-566
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21 Madani S,Shapiro J. Alopecia areata update. J Am Acad Dermatol, 2000,42(4):549-566
22 Drake LA,Ceilley RI,Cornllison RL. Guidelines of care for alopeeia areata. Am Acad Dermatol,1992,26: 247-250
23 Dillaha CJ,Rothman S. Therapeutic experiments in alopecia areata with orally administered cortisone. AmMed Ass,1952,150(6): 546-550
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40 Ranchoff RE,Bergfeld WF,Steck WD,et al. Extensive alopecia areata. Results of treatment with 3% topical minoxidil. Clin J Med 1989,56(2): 149-154
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42 White SI, Friedmann PS. Topical minoxidil lacks efficacy in alopecia areata. Arch Dermatol,1985,121(5): 591
43 Frentz G. Topical minoxidil for extended areate alopecia. Acta Derm Venereol,1985,65(2): 172-175
44 Maitland JM,Alridge RD,Main RA,et al. Topical minoxidil in the treatment of alopecia areata. Br Med J,1984,288(6419): 794
45 Vestey JP,Savin JA. A trial of 1% minoxidil used topically for severe alopecia areata. Acta DermVenereol (Stockh),1986,66(2):179-180
46 Fransway AF,Muller SA. 3 percent topical minoxidil compared with placebo for the treatment of chronic severe alopecia areata. Cutis, 1988,41(6):431-435
47 ReeK. Reduction of Langerhans cells in human epidermis during PUVA therapy: a morphometric study. Invest dermatol 1982,78(6): 488-492
48 Happle R. Topical immunotherapy in alopecia areata. Invest dermatol 1991,96(5):71S-72S
49 Taylor CR,Hawk JL. PUVA treatment of alopecia areata partialis, totalis and universalis: audit of 10 years’experience at St John’s Institute of Dermatology. Br J Dermatol,1995,133(6):914-918
50 Healy E,Rogers S. PUVA treatment for alopecia areata—does it work? A retrospective review of 102 cases. Br J Dermatol,1993,129(1): 42-44
51 Claudy AL,Gagnaire D. PUVA treatment of alopecia areata. Arch Dermatol,1983,119: 975-978
52 Madani S,Shapiro J. Alopecia areata update.J Am Acad Dermatol, 2000,42(4): 549-566
53 Schmoeckel C,Weissmann I, Plewig G, et al. Treatment of alopecia areata by anthralin-induced dermatitis. Arch Dermatol,1979,115(10): 1254-1255
54 Fielder-Weiss VC, Buys CM. Evaluation of anthralin in the treatment of alopecia areata. Arch Dermatol,1987,123(11): 1491-1493
55 Fiedler VC,Wendrow A,Szpunar GJ,et al. Treatment-resistant alopecia areata. Response to therapy with minoxidil plus anthralin. Arch Dermatol 1990,126(6): 756-769
56 Ross EK,Shapiro J. Management of hair loss. Dermatol Clin,2005, 23(2): 227-243
57 Schreiber SL,Crabtree GR. The mechanism of action of cyclosporin A and FK506. ImmunolToday,1992,13(4): 136-142
58 Yamamoto S,Jiang H,Kato R: Stimulation of hair growth by topical application of FK506,a potent immunosuppressive agent. Invest Dermatol,1994,102(2): 160-164
59 McElwee KJ,Tobin DJ,Bystryn JC,et al. Alopecia areata: An autoimmune disease? Exp Dermatol,1999,8(5): 371-379
60 Freyschmidt-Paul P,Seiter S,Zoller M,et al. Treatment with an anti-CD44v10-specific antibody inhibits the onset of alopecia areata in C3H/HeJ mice. Invest Dermatol,2000,115(4): 653-657
61 Ferrando J,Grimalt R. Partial response of severe alopecia areata tocyclosporine A. Dermatology,1999,199(1): 67-69
62 Shapiro J,Lui H,Tron V,et al. Systemic cyclosporine and low-dose prednisone in the treatment of chronic severeb alopecia areata: a clinical and immunopathologic evaluation. Am Acad Dermatol, 1997,36(1): 114-117
63 Taylor M,Ashcroft AT,Messenger AG. Cyclosporin A prolongs human hair growth in vitro. Invest Dermatol,1993,100(3):237-239.
64 Gilhar A,Winterstein F,Krueger GG Hair growth in human split-thickness skin grafts transplanted onto nude rats:The role of cyclosporine[Abstract]. Berlin : Seventh World Congress of Dermatology,1987,426
65 Gold R. Mechanism of action of glucocorticosteroid hormones: Possible implications for therapy of neuroimmunological disorders. Neurommunol,2001,117 (1): 1-8
66周静,杨勤萍,徐峰,等.复方甘草酸苷治疗斑秃10例临床疗效观察.中国皮肤性病学杂志,2008,22(7): 446-447
67 McMichael AJ. The new biologics in psoriasis: possible treatments for alopecia areata. Invest Dermatol,2003,8(2):217-218
68 Strober B,Siu K,Alexis A,et al. Etanercept does not effectively treat moderate to severe alopecia areata: an open-label study. Am Acad Dermatol,2005,52(6): 1082-1084
69 Posten W,Swan J. Recurrence of alopecia areata in a patient receiving etanercept injections. Arch Dermatol 2005,141(6): 759-760
70 Ettefagh L,Neodorost S,Mirmirani P. Alopecia areta in a patient using infliximab: new insights into the role of tumor necrosis factor on human hair follicles. Arch Dermatol,2004,140(8): 1012
71 Kolde G, Meffert H,Rowe E. Successful treatment of alopecia areata with efalizumab. Eur Acad Dermatol Venereol,2008,22(3): 468-473
72 Posten W,Swan J. Recurrence of alopecia areata in a patient receiving etanercept injections. Arch Dermatol,2004,141(6): 759-760
73 Pelivani N,Hassan A,Braathen L,et al. Alopecia areata universaliselicited during treatment with adalimumab. Dermatol,2008,216(4): 320-323
74 Chaves Y,Duarte G,Ben-Said B,et al. Alopecia areata universalis during treatment of rheumatoid arthritis with anti-TNF-a antibody (adalimumab). Dermatol,2008,217(4):380
75 Gundogan C,Greve B,Raulin C. Treatment of alopecia areata with the 308-nm xenon chloride excimer laser : case report of two successful treatments with the excimer laser. Lasers Surg Med. 2004, 34(2): 86-90
76 Zakaria W,Passeron T,Ostovari N,et a1.308 nm excimer laser therapy in alopecia areata. Am Acad Dermatol,2004,51(5): 837-838
77 Raulin C,Gundogan C,Greve B,et a1. Excimer laser therapy of alopecia areata side-by-side evaluation of a representative area. Dtsch Dermatol Ges 2005,3(7): 524-526
78 Jahoda CAB,Home KA,Oliver RF. Induction of hair growth by imp1antation of cultured dermal papilla cells,Nature,1984, 311(5986): 560-562
79李宇,李国强,蔡湘娜,等.微囊化人头皮毛乳头细胞移植诱导毛发形成.中华医学美容杂质,2006,12(6): 350-353
80 Madani S,Shapiro J. Alopecia areata update. J Am Acad Dermatol, 2000, 42(4): 549-566