人体皮肤上皮细胞异常增殖和分化特征及其与创（烧）伤不同修复结局关系的研究
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摘要
人体皮肤上皮细胞异常增殖和分化特征及其与创(烧)伤不同修复结局关系的研究
【目的】皮肤创(烧)伤后创面自然愈合会产生不同的修复结局，包括正常瘢痕、增生性瘢痕(hypertrophic scar，HS)、瘢痕疙瘩(keloid，Ke)、慢性溃疡和假性上皮瘤样增生(pseudoepitheliomatous hyperplasia，PEH)病变等。这些病理性结局的产生固然与自身免疫状态、局部损伤深度、感染和炎症反应等综合因素有关，但作为深度损伤的修复主体-皮肤附件(SAs)与表皮(干)细胞对创面再上皮化和病变形成发挥至关重要的作用。因此，本研究将从胚胎表皮和附件形态发生、深Ⅱ度烧伤创面汗腺上皮细胞复层上皮化，以及HS、Ke和PEH形成过程中表皮和皮肤附件(SAs)变化等方面入手，探讨生理和病理条件下上皮细胞增殖和分化(转分化)对组织发生和不同修复结局的影响，为认识创伤修复规律和提高创面愈合质量提供理论依据。
【方法】收集不同时间点人胎儿、成人正常皮肤(NS)标本、皮肤深Ⅱ度(DBW)和浅Ⅱ度烧伤创面(SBW)标本以及不同愈合结局(HS、Ke和PEH)组织标本，在组织形态学观察的基础上，采用免疫组化和免疫荧光(双)标记、mRNA原位杂交、流式细胞分析和计算机辅助图象扫描分析技术等，调查人胚胎发育阶段表皮细胞及其SAs形态发生、临床DBW汗腺复层上皮化以及病理性愈合结局SAs破坏过程中，上皮细胞组织表型与分化表型之间的关系。具体包括：(1)不同角蛋白分子表达与上皮增殖和分化的关系，如增殖性蛋白标记-细胞角蛋白(CK)5&6、CK14和CK17，(胚胎)表皮干细胞标记-CK8&18和CK19，以及终末分化标记-CK10，腺上皮细胞功能性标记-分泌成分(SC)等；(2)上皮细胞表达金属蛋白酶(MMP-2，-3，-9)和金属蛋白酶抑制物(TIMP-1，-2)对Ⅳ型胶原(ColIV)、层粘连蛋白(laminin，LN)基底膜结构成分形成的影响：(3)细胞间黏附分子-1(ICAM-1)、上皮细胞钙粘附蛋白(E-cadherin，E-Cad)和β-链蛋白(β-catenin，β-Cat)表达水平，对上皮细胞异常分化和迁移的影响；(4)组织中干细胞因子(stem cell factor，SCF)及其受体
博士后名丹究工作J晨告
(c一Kit/CDx17)和白介素门L)一6等表达水平对分化杭原簇一14(cluste:of
difterentiation，eol4)、en6s和肥大细胞类胰蛋白酶(l刀ast eell tryptase，MCT)
阳性细胞分布、细胞生长和分化的诱导作用;(5)上皮细胞表达广谱CK〔cKp)、
转化生长因子一田(TGF一pl)及其受体TGFpRI表达水平与波形蛋白(vim)和a一sMA
表达之间的关系，探讨 TGF一印/TG FpRI信号系统对成体上皮一间质细胞转化
‘巨州~二一epithelia卜mesenellylllal trans、，·:二ati。。，
EMT)和胚胎间质一上皮细胞转化(mesenchyma一epstllelia一transfor，nation，MET)
的诱导作用及其生物学意义;(6)细胞增殖性核抗原(PcNA)和凋亡相关蛋白分子
(Bcl一2和Bax)表达水平与表皮、皮肤附件上皮细胞增殖和分化以及结构异常的
关系;(7)成纤维细胞生长因子(FGF) 10(KGFZ)及其受体(FGFRZb/Bek)信号
系统对胚胎表皮细胞增殖、分化和皮肤附件发生的诱导作用。
此外，在建立新生大鼠表皮角肮细胞分离、培养和纯化方法和技术的基础上，
对培养第3一5代角阮细胞采用重组人TGF一p】(rh TGF一日!)和/或重组人表皮生长因
子(rhEGF)进行离体刺激实验，并对Dispase和胰蛋白酶分离的早期人增生性疲
痕表皮细胞，采用进行免疫组化、免疫荧光和流式细胞分析等方法动态观察EMT
的组织表型和分子表型的改变。
具体分以下五个部分进行总结:
1.胚胎期表皮细胞及其皮肤附件发生
【结果】在人胚胎胎龄(EAG)6一sw期间，外胚层细胞Vi:n+一a一sMA一向cKS&!8十-
CK19十表皮干细胞转变，并检测到中等强度(+十)TGF口R工信号，但TGF口!信
号十分微弱;至EA GI ow以后呈现表皮细胞典型的组织形态学特征。在EAG 1 lw，
表皮细胞开始表达CK一9和eKS&6，在表皮下过度表达FGF一。、PCNA和 Bel·2的
间质细胞开始成团聚集;在EAG I3w时，分层的表皮细胞浅层出现CKIO，周皮细
胞开始脱落;在基底层细胞CK14、PcNA、Bek和p一Cat信号明显增强的区域，细
胞局灶性增殖形成毛囊原基(胚芽)，在其向真皮内侵入生长的同时，上皮细胞
E一cad表达水平与周围间质细胞表达FGF10呈现相反的表达趋势:在EAG16w皮
脂腺发生的同时，表皮一真皮依次重复上述信号并开始汗腺形态发生。
[结论l(1) EAG6一sw是人胚胎皮肤表皮细胞经由MET发生的重要时期，TGF
博士后对究互‘乍报告
.‘亩亩‘菌‘‘亩亩.亩‘亩‘‘‘亩‘亩亩‘亩‘‘.‘亩亩‘‘亩‘‘亩‘亩~‘
口RI信号通路可能参与了MET调变过程，但其配体一TGF日】起源及其作用机制有
待研究。(2)间质细胞源性FGF10以旁分泌方式作用于上皮细胞Bek，并通过合
理调配p一cat和E一cad信号，诱导表皮干细胞增殖分化和SAs形态发生·
2.汗睐上皮细胳表皮细胞转化机制与DBW愈合的关系
l结果1 NS汗腺分泌部基底层的肌上皮细胞表达卜Cat、Bcl一2、CK19、P63和
eK14:浅51度烧伤创面(superfieial 110 bLlrn wounds，sBW)愈合期间深层汗腺分
泌部与NS组相似。在深11度烧伤创面(deep 110 bL!rn woL一
Study on the characteristics of abnormal
proliferation and differentiation of epithelial cells and different relations to outcomes of wound (burn) repair in human skin
[Objective] Different patterns of repair, including normal scars, liypertrophic scars (HS), keloids (Ke), chronic ulcers and pseudoepitheliomatous hyperplasia (PEH), could result from healing of a wound. These pathological healing processes were not only associated with autoimmunity status, depth of local lesion, infection and inflammatory reaction, but also related to the main original mass of cells in the wound, such as epithelial cells and skin appendages (SAs). In this study, the effect of proliferation and differentiation of epithelial cells under physiological and pathological conditions on histogenesis and different repairing outcomes of repair was explored from those aspects including morphogenesis of embryonic epidermis and SAs, stratified epithelization of glandular epithelial cells in deep II burn wound (DBW), and the changes in epidermis and skin appendages during the course of HS, Ke and PEH formation, in order to elucidate the rule of wound healing and to provide a theoretical basis of promotin
g improvement in quality of wound healing.
[Methods] Fetal skin of different embryonic ages, adult normal skin, wound tissues of DBW and SBW, tissues of healed skin wound, including HS, Ke and PEH. were collected from our hospital. The use of these samples was approved by the Committee of Scientific and Technology of 304th Hospital, Beijing, and signed consent of the involved patients. After morphological changes in epithelial tissue was observed with light and electron microscopy, the morphologic development of epidermal cells and skin appendages during human embryonic developmental stage, the relationship of the development of epithelial cells and differentiation during stratified epithelization of sweat gland in deep II degree burned wounds, and destruction of cells of appendages after pathological healing of wound, were explored with immunohistochemical method, immunofluorescent double labeling technology, mRNA in situ hybridization, flow cytometry analysis, and computer ancillary image scan and analysis. (1)The contents were as following: the re
lationship of proliferation and differentiation of epidermal cells with protein expression of various cytokeratins (CKs), such as labeling proteins of proliferative
cells (CK5&6, CK14 and CK17), labeling molecules of epidermal stem cells (CK8&18 and CK19) and labeling proteins of terminal-differentiated cells (CK10), marking molecules of glandular epithelial cells (stem cell factor); (2) Effect of protein expression of matrix metalloproteinase (MMP)-2,-3,-9, Tissue inhibitor of matrix metalloproteinase (TIMP)-1,-2, IV-type collagen and laminin (LN) in epithelial cells on component formation of basement membrane. (3) Influence of expression levels of focal adhesion kinase (FAK), (CAM-1, E-cadherin (E-Cad) and B -catenin ( B -Cat) on abnormal differentiation and migration of epithelial cells.(4)The effect of the levels of expression of stem cell factor (SCF), its receptor (c-Kit/CD1 17 ) and interleukin (IL ) -6 on distribution, growth and differentiation of cells expressive CD14, CD68 and mast cell tryptase (MCT). (5) Correlationship of the expression of pan-cytokeratin (CKp), TGF-B1 and its receptor with the expression of Vim and a -SMA in epithelial cells underlying the effect of signal transduction mediated by transforming growth factor (TGF)-B1 / TGFBR I on epithelial- mesenchyrnal transformation epithelial-mesenchymal transformation (EMT) and mesenchymal-epithelial transformation (MET). (6)The relationship of protein expression and distribution of PCNA and apoptosis-related proteins (Bcl-2 and Bax) with proliferation and differentiation of epidermis, and epithelial cells in skin appendages.(7) Effect of signal transduction mediated by KGF2 and its receptor (FGFR2b/Bek) on proliferation and differentiation of embryonic epidermal cells and induction roles of skin appendage formation.
Furtherm
【目的】皮肤创(烧)伤后创面自然愈合会产生不同的修复结局，包括正常瘢痕、增生性瘢痕(hypertrophic scar，HS)、瘢痕疙瘩(keloid，Ke)、慢性溃疡和假性上皮瘤样增生(pseudoepitheliomatous hyperplasia，PEH)病变等。这些病理性结局的产生固然与自身免疫状态、局部损伤深度、感染和炎症反应等综合因素有关，但作为深度损伤的修复主体-皮肤附件(SAs)与表皮(干)细胞对创面再上皮化和病变形成发挥至关重要的作用。因此，本研究将从胚胎表皮和附件形态发生、深Ⅱ度烧伤创面汗腺上皮细胞复层上皮化，以及HS、Ke和PEH形成过程中表皮和皮肤附件(SAs)变化等方面入手，探讨生理和病理条件下上皮细胞增殖和分化(转分化)对组织发生和不同修复结局的影响，为认识创伤修复规律和提高创面愈合质量提供理论依据。
【方法】收集不同时间点人胎儿、成人正常皮肤(NS)标本、皮肤深Ⅱ度(DBW)和浅Ⅱ度烧伤创面(SBW)标本以及不同愈合结局(HS、Ke和PEH)组织标本，在组织形态学观察的基础上，采用免疫组化和免疫荧光(双)标记、mRNA原位杂交、流式细胞分析和计算机辅助图象扫描分析技术等，调查人胚胎发育阶段表皮细胞及其SAs形态发生、临床DBW汗腺复层上皮化以及病理性愈合结局SAs破坏过程中，上皮细胞组织表型与分化表型之间的关系。具体包括：(1)不同角蛋白分子表达与上皮增殖和分化的关系，如增殖性蛋白标记-细胞角蛋白(CK)5&6、CK14和CK17，(胚胎)表皮干细胞标记-CK8&18和CK19，以及终末分化标记-CK10，腺上皮细胞功能性标记-分泌成分(SC)等；(2)上皮细胞表达金属蛋白酶(MMP-2，-3，-9)和金属蛋白酶抑制物(TIMP-1，-2)对Ⅳ型胶原(ColIV)、层粘连蛋白(laminin，LN)基底膜结构成分形成的影响：(3)细胞间黏附分子-1(ICAM-1)、上皮细胞钙粘附蛋白(E-cadherin，E-Cad)和β-链蛋白(β-catenin，β-Cat)表达水平，对上皮细胞异常分化和迁移的影响；(4)组织中干细胞因子(stem cell factor，SCF)及其受体
博士后名丹究工作J晨告
(c一Kit/CDx17)和白介素门L)一6等表达水平对分化杭原簇一14(cluste:of
difterentiation，eol4)、en6s和肥大细胞类胰蛋白酶(l刀ast eell tryptase，MCT)
阳性细胞分布、细胞生长和分化的诱导作用;(5)上皮细胞表达广谱CK〔cKp)、
转化生长因子一田(TGF一pl)及其受体TGFpRI表达水平与波形蛋白(vim)和a一sMA
表达之间的关系，探讨 TGF一印/TG FpRI信号系统对成体上皮一间质细胞转化
‘巨州~二一epithelia卜mesenellylllal trans、，·:二ati。。，
EMT)和胚胎间质一上皮细胞转化(mesenchyma一epstllelia一transfor，nation，MET)
的诱导作用及其生物学意义;(6)细胞增殖性核抗原(PcNA)和凋亡相关蛋白分子
(Bcl一2和Bax)表达水平与表皮、皮肤附件上皮细胞增殖和分化以及结构异常的
关系;(7)成纤维细胞生长因子(FGF) 10(KGFZ)及其受体(FGFRZb/Bek)信号
系统对胚胎表皮细胞增殖、分化和皮肤附件发生的诱导作用。
此外，在建立新生大鼠表皮角肮细胞分离、培养和纯化方法和技术的基础上，
对培养第3一5代角阮细胞采用重组人TGF一p】(rh TGF一日!)和/或重组人表皮生长因
子(rhEGF)进行离体刺激实验，并对Dispase和胰蛋白酶分离的早期人增生性疲
痕表皮细胞，采用进行免疫组化、免疫荧光和流式细胞分析等方法动态观察EMT
的组织表型和分子表型的改变。
具体分以下五个部分进行总结:
1.胚胎期表皮细胞及其皮肤附件发生
【结果】在人胚胎胎龄(EAG)6一sw期间，外胚层细胞Vi:n+一a一sMA一向cKS&!8十-
CK19十表皮干细胞转变，并检测到中等强度(+十)TGF口R工信号，但TGF口!信
号十分微弱;至EA GI ow以后呈现表皮细胞典型的组织形态学特征。在EAG 1 lw，
表皮细胞开始表达CK一9和eKS&6，在表皮下过度表达FGF一。、PCNA和 Bel·2的
间质细胞开始成团聚集;在EAG I3w时，分层的表皮细胞浅层出现CKIO，周皮细
胞开始脱落;在基底层细胞CK14、PcNA、Bek和p一Cat信号明显增强的区域，细
胞局灶性增殖形成毛囊原基(胚芽)，在其向真皮内侵入生长的同时，上皮细胞
E一cad表达水平与周围间质细胞表达FGF10呈现相反的表达趋势:在EAG16w皮
脂腺发生的同时，表皮一真皮依次重复上述信号并开始汗腺形态发生。
[结论l(1) EAG6一sw是人胚胎皮肤表皮细胞经由MET发生的重要时期，TGF
博士后对究互‘乍报告
.‘亩亩‘菌‘‘亩亩.亩‘亩‘‘‘亩‘亩亩‘亩‘‘.‘亩亩‘‘亩‘‘亩‘亩~‘
口RI信号通路可能参与了MET调变过程，但其配体一TGF日】起源及其作用机制有
待研究。(2)间质细胞源性FGF10以旁分泌方式作用于上皮细胞Bek，并通过合
理调配p一cat和E一cad信号，诱导表皮干细胞增殖分化和SAs形态发生·
2.汗睐上皮细胳表皮细胞转化机制与DBW愈合的关系
l结果1 NS汗腺分泌部基底层的肌上皮细胞表达卜Cat、Bcl一2、CK19、P63和
eK14:浅51度烧伤创面(superfieial 110 bLlrn wounds，sBW)愈合期间深层汗腺分
泌部与NS组相似。在深11度烧伤创面(deep 110 bL!rn woL一
Study on the characteristics of abnormal
proliferation and differentiation of epithelial cells and different relations to outcomes of wound (burn) repair in human skin
[Objective] Different patterns of repair, including normal scars, liypertrophic scars (HS), keloids (Ke), chronic ulcers and pseudoepitheliomatous hyperplasia (PEH), could result from healing of a wound. These pathological healing processes were not only associated with autoimmunity status, depth of local lesion, infection and inflammatory reaction, but also related to the main original mass of cells in the wound, such as epithelial cells and skin appendages (SAs). In this study, the effect of proliferation and differentiation of epithelial cells under physiological and pathological conditions on histogenesis and different repairing outcomes of repair was explored from those aspects including morphogenesis of embryonic epidermis and SAs, stratified epithelization of glandular epithelial cells in deep II burn wound (DBW), and the changes in epidermis and skin appendages during the course of HS, Ke and PEH formation, in order to elucidate the rule of wound healing and to provide a theoretical basis of promotin
g improvement in quality of wound healing.
[Methods] Fetal skin of different embryonic ages, adult normal skin, wound tissues of DBW and SBW, tissues of healed skin wound, including HS, Ke and PEH. were collected from our hospital. The use of these samples was approved by the Committee of Scientific and Technology of 304th Hospital, Beijing, and signed consent of the involved patients. After morphological changes in epithelial tissue was observed with light and electron microscopy, the morphologic development of epidermal cells and skin appendages during human embryonic developmental stage, the relationship of the development of epithelial cells and differentiation during stratified epithelization of sweat gland in deep II degree burned wounds, and destruction of cells of appendages after pathological healing of wound, were explored with immunohistochemical method, immunofluorescent double labeling technology, mRNA in situ hybridization, flow cytometry analysis, and computer ancillary image scan and analysis. (1)The contents were as following: the re
lationship of proliferation and differentiation of epidermal cells with protein expression of various cytokeratins (CKs), such as labeling proteins of proliferative
cells (CK5&6, CK14 and CK17), labeling molecules of epidermal stem cells (CK8&18 and CK19) and labeling proteins of terminal-differentiated cells (CK10), marking molecules of glandular epithelial cells (stem cell factor); (2) Effect of protein expression of matrix metalloproteinase (MMP)-2,-3,-9, Tissue inhibitor of matrix metalloproteinase (TIMP)-1,-2, IV-type collagen and laminin (LN) in epithelial cells on component formation of basement membrane. (3) Influence of expression levels of focal adhesion kinase (FAK), (CAM-1, E-cadherin (E-Cad) and B -catenin ( B -Cat) on abnormal differentiation and migration of epithelial cells.(4)The effect of the levels of expression of stem cell factor (SCF), its receptor (c-Kit/CD1 17 ) and interleukin (IL ) -6 on distribution, growth and differentiation of cells expressive CD14, CD68 and mast cell tryptase (MCT). (5) Correlationship of the expression of pan-cytokeratin (CKp), TGF-B1 and its receptor with the expression of Vim and a -SMA in epithelial cells underlying the effect of signal transduction mediated by transforming growth factor (TGF)-B1 / TGFBR I on epithelial- mesenchyrnal transformation epithelial-mesenchymal transformation (EMT) and mesenchymal-epithelial transformation (MET). (6)The relationship of protein expression and distribution of PCNA and apoptosis-related proteins (Bcl-2 and Bax) with proliferation and differentiation of epidermis, and epithelial cells in skin appendages.(7) Effect of signal transduction mediated by KGF2 and its receptor (FGFR2b/Bek) on proliferation and differentiation of embryonic epidermal cells and induction roles of skin appendage formation.
Furtherm
引文
[1] 易梅生．表皮及其衍生物的发育[M]．见：桂建芳，易梅生 主编．发育生物学．北京：科学出版社(笫1版)2002，149-150
[2] Hay ED. An overview of epithelio-mesenchymal transformation[J]. Acta Anat, 1995,154: 8-20.
[3] 赵志力，付小兵．不同发育阶段细胞的分化与调控机制[J]，中国临床康复，2002，6(22)：3412-3413
[4] 周健．波形蛋白与晶状体纤维细胞分化及白内障形成[J]．国外医学眼科学分册，2000，24(2)：112-115．
[5] Toma JG, Akhavan M, Femandes KJL,et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin[J].Nat Cell Biol,2001,3:778-784.
[6] Okochi H. Stem cells in the skin and regenerative medicine[J].Jpn J Dermatol. 2003,113(3):247-251.
[7] Okada H, Danoff TM, Kalluri R, et al. Early role of FSPI in epithelial-mesenehynal transformation[J]. Am J Physiol, 1997, 273:F563-F574.
[8] Lan HY. Tubular epithelial-myofibroblast transdifferentiation mechanisms in proximal tubule ceils[J]. Cure Optn Nephrol Hypertens,2003.12(1):25-29.
[9] Funderburgh .IL, Funderburgh ML, Mann MM, et al. Proteoglycan expression during transforming growth factor beta -induced keratocyte-myofibroblast transdifferentiation[J]. J Biol Chem, 2001, 276 (47):44173-44178,
[2] Hay ED. An overview of epithelio-mesenchymal transformation[J]. Acta Anat, 1995,154: 8-20.
[3] 赵志力，付小兵．不同发育阶段细胞的分化与调控机制[J]，中国临床康复，2002，6(22)：3412-3413
[4] 周健．波形蛋白与晶状体纤维细胞分化及白内障形成[J]．国外医学眼科学分册，2000，24(2)：112-115．
[5] Toma JG, Akhavan M, Femandes KJL,et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin[J].Nat Cell Biol,2001,3:778-784.
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