Wnt/β-catenin信号通路活化对人表皮细胞表型变化的影响

目的 探讨Wnt/β-catenin信号通路活化对人表皮细胞表型变化的影响.方法 差速贴壁法分离人成熟表皮细胞.并将细胞分为对照组和诱导组,诱导组义分为氯化锂(LiCI,20 mmol/L)诱导组和GSK-3β抑制子(15μmol/L)诱导组,诱导6d后观察细胞的形态变化,并采用Western blot检测衷皮细胞内β-catenin的表达水平,免疫组织化学法检测表皮细胞表面标志性蛋白CK10、CK14、CK19和B1整合素的表达变化.结果 人表皮细胞内β-catenin的表达在被Licl(2.15±0.54)和GSK-3β(2.58±0.65)抑制子诱导后分别比对照组(0.71±0.23)增加了2倍和2.5倍(P<0.01).而且诱导后细胞体积变小变圆,细胞核、细胞质比例变大.免疫组织化学检测结果表明,诱导前表皮细胞高表达CK10,部分细胞表达CK14,CK19和β1整合素表达阴性;诱导后已无CK10阳性细胞存在,有部分CK14阳性细胞,而且CK19和β1整合素的表达呈强阳性.结论 Wnt/β-catenin通路活化可引起人成熟表皮细胞的去分化,即Wnt/β-catenin通路可能是调控人表皮细胞去分化的关键环节.     

Analysis of differentially expressed genes in fetal skin of scarless and scar-forming periods of gestational rats

Objective: To study the differences of gene expression between earlier gestational skin and later gestational skin of rats with the aids of single primer amplification (SPA) and high-density oligonucleotide DNA array to understand the molecular mechanism of scarless healing. Methods: Total RNAs were isolated from fetal rat skin of the scarless (E15) and scar-forming (E18) periods of gestation (term =21.5 days). The RNAs from earlier gestational skin (EGS) and later gestational skin (LGS) were both reversely transcribed to cDNAs, then labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes by SPA method. The mixed probes were then hybridized to the oligonucleotide DNA arrays which contained 5 705 probes representing 5 705 rat genes. After highly stringent washing, these DNA arrays were scanned for fluorescent signals to display the differentially expressed genes between the 2 groups of skin. Results: Among 5 705 rat genes, there were 53 genes (0.93%) with differentially expressed levels between EGS and LGS groups, 27 genes, including fibroblast growth factor 2 ( FGF2 ) and follistatin were up-regulated (0.47%) and 26 genes were down-regulated (0.46%) in fetal skin during scarless period versus scar-forming period. Higher expressions of FGF2 and follistatin in EGS than those in LGS were also revealed by RT-PCR method. Conclusions: High-density oligonucleotide DNA array provided a powerful tool for investigating differential gene expression in earlier and later gestational fetal skins. This technology validates that the mechanism of fetal scarless healing is very complicate and the change of many gene expressions is associated with fetal scarless healing.     

细胞外信号调节激酶1/2传导通路在不同发育阶段皮肤组织内的变化

为研究磷酸化细胞外信号调节激酶1/2（p-ERK1/2）、Ras和C-fos在不同发育阶段皮肤中的表达特征及其可能的生物学意义,用免疫组化技术检测这3种蛋白在8例胎儿皮肤和8例成人皮肤中的表达变化规律。结果发现,在胎儿发育初期,p-ERK1/2、Ras和C-fos蛋白表达较低,随着胎儿生长发育,这3种蛋白的阳性细胞率逐渐增大,在妊娠后期胎儿和成人皮肤组织中,3种蛋白的阳性细胞率明显高于妊娠早期胎儿皮肤（P<0.01）。p-ERK1/2、Ras和C-fos蛋白在不同发育阶段皮肤组织内都有表达,显示它们可能对皮肤的发生、结构和功能的维持以及损伤后修复十分重要。     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

正常皮肤和瘢痕组织表皮干细胞定位与增殖分化特征的比较性研究

目的采用特殊染色法研究少儿与成年人大面积重度烧伤后瘢痕组织表皮干细胞分布与表达β1整合素和角蛋白19、14、10(K19,K14,K10)的特征与规律,在此基础上确定表皮干细胞及短暂扩充细胞分布、数量的差异以及这些改变与瘢痕愈合关系.方法分别取4～12岁少儿及35～53岁成年人2组健康皮肤及大面积深度烧伤后瘢痕组织.采用免疫组织化学SP法检测表皮干细胞、短暂扩充细胞特异表达的β1整合素和K19以及分化表皮细胞表达的K14和K10.结果瘢痕组织表皮基底层表达β1整合素与K19的阳性细胞数较健康皮肤明显减少,阳性强度降低.瘢痕组织表皮中表达K14的阳性细胞仅位于表皮底部2～3层,明显少于健康皮肤,而K10表达阳性细胞则较健康皮肤分布广泛.结论瘢痕组织表皮基底层具有增殖能力的表皮干细胞和短暂扩充细胞明显少于健康皮肤,且瘢痕组织表皮干细胞的分化过程与健康皮肤不同,处于有丝分裂后分化阶段的细胞比例降低,而终末分化细胞的比例明显增高.提示瘢痕组织表皮的增殖能力下降,细胞的分化行为紊乱,这可能是瘢痕组织表皮结构与功能改变、愈合能力下降的原因之一.     

皮肤扩张过程中p63和细胞角蛋白的表达特征及意义

目的:初步观察皮肤扩张术对表皮细胞增殖分化的影响并探讨其机制。方法:用iWstar大鼠制作扩张动物模型,将72个扩张器埋于36只wistar鼠背部浅筋膜下,利用表皮干细胞特异表达p63和角蛋白19及短暂扩充细胞表达角蛋白14的特点,采用免疫组织化学Powervision M二步法检测扩张过程中表皮干细胞和短暂扩充细胞的分布与数量差异。结果:扩张皮肤表皮层增厚,表皮干细胞和短暂扩充细胞的数量明显增多,在棘层和颗粒层可见表皮干细胞和短暂扩充细胞的阳性表达。结论:皮肤扩张术能诱导表皮干细胞增殖分化,表皮干细胞在扩张过程中对机械应力和创伤修复的响应机制可能是其主要的生物学基础。     

不同发育阶段人皮肤表皮干细胞增殖分化特征及其与创面修复结局关系的研究

目的从细胞分化角度研究人胎儿期、少儿期、成年人期皮肤中表皮干细胞增殖分化特征,以及这些特征与创面修复结局的关系.方法分别取因创伤等原因致流产的22～24周龄胎儿和4～12周岁少儿、35～53岁成年人3组全层皮肤.采用免疫组化方法检测表皮干细胞特异表达的β1整合素和细胞角蛋白19(K19).结果胎儿期皮肤表皮基底层细胞β1整合素和K19染色均为强阳性.少儿期表皮基底层细胞中60%～80%的细胞表达β1整合素和K19.成年人表皮基底层中表达β1整合素和K19的细胞较少儿组进一步减少,且染色强度较弱.结论本实验结果提示,胎儿期表皮基底层增殖细胞均为表皮干细胞和短暂扩充细胞,而少儿期表皮基底层中部分细胞为于细胞和短暂扩充细胞,成年人期干细胞与短暂扩充细胞所占的比例则进一步降低.这种干细胞增殖分化的差异可能与三种不同发育阶段皮肤损伤的完全与不完全修复有关.     

增生性瘢痕中丝裂素活化蛋白激酶基因表达的变化

增生性瘢痕的形成受多种信号通路的调控 ,其中丝裂素活化蛋白激酶(MAPKs)可能起着十分重要的作用。MAPKs家族成员主要包括细胞外信号调节蛋白激酶 (erk1/2 ) ,应急活化蛋白激酶 (sapk)和蛋白激酶 p3 8MAPK[1] 。目前 ,关于增生性瘢痕内这 4种基因表达变化的研究还少有报道 ,对这一规律的阐明 ,将有助于揭示增生性瘢痕发生的机制。一、材料与方法1.标本来源及制备 :取住院进行整形手术患者的增生性瘢痕 8例 ,其中男 5例 ,女 3例 ,年龄 3～ 5 2岁 ,瘢痕增生时间 1年以内 4例 ,1～ 2年 2例 ,2年以上2例。其对应的正常皮肤 8例 ,取自增生性…     

胎儿和出生后机体皮肤内TGF-β1,TGF-β3及其受体基因表达变化的比较性研究

目的:探讨转化生长因子-β1(TGF-β1),转化生长因子-β3(TGF-β3)及其Ⅰ-型受体(TBRⅠ)和Ⅱ-型受体(TBRⅡ)在不同胎龄胎儿的皮肤和出生后机体皮肤组织中表达变化特征及其可能的生物学意义.方法:用病理学技术检测不同发育时期皮肤的结构特征后,提取18例不同胎龄(13～32周)的胎儿皮肤和6例出生后机体皮肤组织的总RNA后,分离mRNA,用RT-PCR方法检测这4种基因在不同组织中的表达变化规律.结果:TGF-β1,TBRⅠ和TBRⅡ基因在不同妊娠时期的胎儿皮肤和出生后机体皮肤组织中都有表达.在早期妊娠胎儿的皮肤组织中,这3种基因表达较弱,随着胎龄的增加,皮肤组织内这3种基因表达逐渐增强.在出生后机体的皮肤组织中,这些基因的表达量分别为早期妊娠胎儿皮肤的1.3倍、1.3倍和1.2倍,基因表达显著升高(P＜0.05).TGF-β3基因表达变化规律恰恰相反,在早期妊娠胎儿的皮肤组织中,该基因表达较强,而在出生后机体的皮肤组织中,该基因表达产物的密度值为0.158±0.095,与早期妊娠胎儿皮肤相比显著下降(P＜0.01).结论:TGF-β1,TGF-β3及其受体基因在不同发育阶段人皮肤组织内都有表达,显示这些细胞因子与其受体结合后引起的信号通路可能对皮肤的发生、结构功能的维持以及伤后修复十分重要.在早期妊娠胎儿皮肤中TGF-β1,TBRⅠ和TBRⅡ基因低表达,TGF-β3基因的高表达可能与胎儿皮肤创面无瘢痕修复密切相关.