瘢痕疙瘩周围皮肤成纤维细胞周期分析及P53基因突变检测

目的：探讨瘢痕疙瘩周围皮肤中是否有生物学活性异常的成纤维细胞， 以期进一步了解瘢痕疙瘩的发展机制。方法：对所取新鲜组织标本进行细胞培养，通过碘化丙啶（PI）染色，激光流式细胞仪分析细胞周期，比较各组成纤维细胞处于增殖期细胞的百分比。采用PCR技术对P53基因外显子4、5、6进行扩增并测序。结果：瘢痕疙瘩周围皮肤增殖期成纤维细胞百分比与瘢痕疙瘩中央部相似（P＞0．05），介于瘢痕疙瘩边缘部与正常皮肤之间，与两者的差异均有显著性意义（P＜0．05）。所有体外培养瘢痕疙瘩周围皮肤成纤维细胞（6／6）均存在P53外显子4的点突变，3例（2／6）P53外显子5存在移码突变，均与同病人瘢痕疙瘩成纤维细胞基因突变一致，而两组正常皮肤成纤维细胞则未发现有任何形式的基因突变，结论：瘢痕疙瘩周围0.5cm皮肤内存在生物学活性异常成纤维细胞，这可能为瘢痕疙瘩浸润性生长的结果及其治疗后易复发的原因之一。     

成纤维细胞体外培养、冻存及复苏的实验研究

目的：探讨正常皮肤和瘢痕疙瘩组织成纤维细胞在体外培养情况下其生物学特性的差异，以及与细胞冻存和复苏的方法及其应用价值。方法：对8例正常皮肤和8例瘢痕疙瘩组织标本分别进行体外成纤维细胞的原代培养、传代培养，观察培养的成纤维细胞形态，并作生长曲线比较细胞增殖情况。选取处于对数生长期的培养细胞梯度降温后置入液氮（-196℃）中保存4—6个月，进行细胞复苏培养，以2％台盼蓝确定细胞活力，并观察复苏的成纤维细胞形态学状况。结果：体外培养的正常皮肤和瘢痕疙瘩组织成纤维细胞形态和生长曲线基本相同，但瘢痕疙瘩原代成纤维细胞萌出生长较正常皮肤快，生长融合后成纤维细胞排列紊乱，极性消失，有明显的交叉重叠现象；体外培养的成纤维细胞，经冷冻保存4～6个月后，复苏率超过80％，细胞形态无明显改变。结论：体外培养的正常皮肤和瘢痕疙瘩组织成纤维细胞形态与生长特性基本相同。瘢痕疙瘩成纤维细胞可成功冻存和复苏。     

正常皮肤,增生性瘢痕和瘢痕疙瘩成纤维细胞培养,生物 …

目的 探讨正常皮肤、增生性瘢痕和瘢痕疙瘩成纤维细胞的体外培养、生物学特征及超微结构、阐明其应用价值。方法 采用成纤维细胞体外培养技术，从正常皮肤成纤维细胞与增生性瘢痕，瘢痕疙瘩成纤维细胞在细胞增殖，细胞形态，细胞遗传学特征及细胞超微结构方面进行比较研究。结果 体外培养的正常皮肤成纤维细胞与增生性瘢痕、瘢痕疙瘩成纤维生长率和形态学相同，细胞遗传学特征和超微结构相似。结论 据此结果和其他学者的观点，认     

正常皮肤、增生性瘢痕和瘢痕疙瘩成纤维细胞培养、生物学特征及超微结构的比较研究

目的探讨正常皮肤、增生性瘢痕和瘢痕疙瘩成纤维细胞的体外培养、生物学特征及超微结构，阐明其应用价值。方法采用成纤维细胞体外培养技术，从正常皮肤成纤维细胞与增生性瘢痕、瘢痕疙瘩成纤维细胞在细胞增殖、细胞形态、细胞遗传学特征及细胞超微结构方面进行比较研究。结果体外培养的正常皮肤成纤维细胞与增生性瘢痕、瘢痕疙瘩成纤维细胞生长率和形态学相同，细胞遗传学特征和超微结构相似。结论据此结果和其他学者的观点，认为建立体外培养的正常皮肤成纤维细胞的细胞实验模型，可用于瘢痕防治的研究。     

病理性瘢痕中c—fos原癌基因的表达

目的：病理性瘢痕是创伤过度愈合的结果，以成纤维细胞的异常增殖，合成及分泌大量胶原和细胞外基质为特征，其形成机理仍不清楚，探讨原癌基因c-fos的表达与病理性瘢痕形成的相关性，方法：应用免疫组化SP法，检测c-fos蛋白在增生性产痕，产痕疙瘩及正常皮肤组织中的表达和分布，并用图像定量分析比较其差异。结果；在增生性瘢痕和瘢痕疙瘩的成纤维细胞中c-fos呈强阳性表达，两组间无明显差异，而与正常皮肤对照组均有显著性差异。结论：增生性瘢痕与瘢痕疙瘩中c-fos蛋白表达升高，存在c-fos癌基因的激活，可能参与了成纤维细胞的分化增殖，胶原合成与降解以及对细胞因子的调控，并导致瘢痕增生。     

正常皮肤、增生性瘢痕和瘢痕疙瘩成纤维细胞培养、生物学特征及超微结构的比较研究

目的探讨正常皮肤、增生性瘢痕和瘢痕疙瘩成纤维细胞的体外培养、生物学特征及超微结构,阐明其应用价值。方法采用成纤维细胞体外培养技术,从正常皮肤成纤维细胞与增生性瘢痕、瘢痕疙瘩成纤维细胞在细胞增殖、细胞形态、细胞遗传学特征及细胞超微结构方面进行比较研究。结果体外培养的正常皮肤成纤维细胞与增生性瘢痕、瘢痕疙瘩成纤维细胞生长率和形态学相同,细胞遗传学特征和超微结构相似。结论据此结果和其他学者的观点,认为建立体外培养的正常皮肤成纤维细胞的细胞实验模型,可用于瘢痕防治的研究。     

瘢痕疙瘩成纤维细胞低密度培养的克隆能力分析

目的：比较瘢痕疙瘩成纤维细胞（KFs）和正常真皮成纤维细胞（NFs）间克隆形成能力的差异,探讨瘢痕疙瘩中病理性干细胞是否存在,及其对瘢痕疙瘩发生发展的影响。方法利用酶消化法获得瘢痕疙瘩和正常皮肤组织的原代细胞,以4000个/皿的密度接种,进行低密度培养,2周后观察细胞克隆的形成及形态变化。结果低密度培养条件下的瘢痕疙瘩成纤维细胞和正常皮肤成纤维细胞都可形成克隆,但KFs可形成明显的克隆集落,NFs形成的克隆不明显且松散。KFs克隆形成率高于NFs,为（0.80±0.21）%,而NFs为（0.18±0.06）%,两者间差异显著（P〈0.05）。结论低密度培养条件下,瘢痕疙瘩成纤维细胞的克隆形成能力高于正常皮肤成纤维细胞,可能与瘢痕疙瘩组织中存在病理性瘢痕疙瘩干细胞有关。     

病理性瘢痕中原癌基因c—myc和c—fos的表达

目的：探讨原癌基因的表达与病理性瘢痕形成的相关性。方法：应用免疫组化SP法。检测c-myc和c-fos蛋白在增生性瘢痕，瘢痕疙瘩和正常皮肤组织中的表达和分布，并用图像定量分析比较其差异。结果：在增生性瘢痕和瘢痕疙瘩的成纤维细胞中c-myc,c-fos呈强阳性表达，两组间无明显差异，而与正常皮肤对照组均有显著性差异，结论：增生性瘢痕与瘢痕疙瘩中c-myc,c-fos蛋白表达升高，存在c-myc和c-fos原癌基因的激活，可能参与了成纤维细胞的分化增殖或表型转化，胶原合成与降解以及对细胞因子的调控，并导致瘢痕增生。     

细胞周期蛋白E在瘢痕疙瘩不同区域中的表达及意义

目的探讨细胞周期蛋白E在瘢痕疙瘩形成过程中的作用及意义。方法应用免疫组织化学链霉亲和素-过氧化物酶（SP）法检测瘢痕疙瘩不同区域和正常皮肤组织成纤维细胞（fibro-blast,FB）中细胞周期蛋白E的表达。结果在瘢痕疙瘩周边部FB中存在细胞周期蛋白E的高表达;而在瘢痕疙瘩中央部阳性表达的FB数量少,8例标本FB中细胞周期蛋白E表达阴性。正常皮肤FB中细胞周期蛋白E表达阴性。经统计学处理,周边部与正常皮肤组及中央部比较,差异有统计学意义（P〈0.01及P〈0.05）;中央部与正常皮肤（NS）组比较差异无统计学意义（P〉0.05）。结论细胞周期蛋白E在瘢痕疙瘩周边部FB中的高表达,不仅可能促进了瘢痕疙瘩的形成,并可能是瘢痕疙瘩侵犯周围正常组织,呈浸润性生长的原因之一。     

病理性瘢痕中c-fos原癌基因的表达

目的　病理性瘢痕是创伤过度愈合的结果 ,以成纤维细胞的异常增殖、合成及分泌大量胶原和细胞外基质为特征 ,其形成机理仍不清楚。探讨原癌基因c -fos的表达与病理性瘢痕形成的相关性。方法　应用免疫组化SP法 ,检测c -fos蛋白在增生性瘢痕、瘢痕疙瘩及正常皮肤组织中的表达和分布 ,并用图像定量分析比较其差异。结果　在增生性瘢痕和瘢痕疙瘩的成纤维细胞中c-fos呈强阳性表达 ,两组间无明显差异 ,而与正常皮肤对照组均有显著性差异。结论　增生性瘢痕与瘢痕疙瘩中c -fos蛋白表达升高 ,存在c -fos癌基因的激活 ,可能参与了成纤维细胞的分化增殖、胶原合成与降解以及对细胞因子的调控 ,并导致瘢痕增生     

Fas介导瘢痕疙瘩周围皮肤成纤维细胞凋亡及其基因检测研究

目的　探讨瘢痕疙瘩周围皮肤中是否有生物学活性异常成纤维细胞 ,以期进一步了解瘢痕疙瘩的发展机制。方法　所取新鲜组织标本进行细胞培养 ,通过碘化丙啶 (PI)染色、激光流式细胞仪比较不同浓度Fas单克隆抗体 (FasMcAb ,0～ 10 0 0 μg/L)作用后各组成纤维细胞凋亡率。采用聚合酶链反应 (PCR)技术对Fas基因外显子 8进行DNA扩增并测序。结果　FasMcAb作用2 4h后 ,瘢痕疙瘩成纤维细胞在各浓度段均不能凋亡 ,瘢痕疙瘩周围皮肤成纤维细胞随FasMcAb作用浓度的增加凋亡率虽有所增加 ,但总体比较与瘢痕疙瘩差异无显著性 (P >0 .0 5 ) ,而与正常皮肤差异有显著性 (P <0 .0 1)。瘢痕疙瘩周围皮肤 6例标本中有 4例 (4 /6)Fas基因外显子 8及其下游序列存在点突变及移码突变 ,均与同患者瘢痕疙瘩细胞基因序列分析结果一致 ,而两组正常皮肤成纤维细胞则未发现有任何形式的基因突变。结论　瘢痕疙瘩周围 0 .5cm皮肤内存在生物学活性异常细胞 ,这可能为瘢痕疙瘩浸润性生长的结果及其治疗后易复发的原因之一。     

增殖瘢痕成纤维细胞接触后增殖及生物合成特性对异常瘢痕形成的机理

目的 为明确不同异常瘢痕成纤维细胞在体外完全接触后其增殖活性及生物全成功能的特性。方法 以瘢痕疙瘩、增生性瘢痕和正常皮肤（各6例）为材料，通过细胞培养、免疫组织化学及分子生物学等方法，对不同成纤维细胞在细胞接触及未接触时通过检测增殖细胞核内抗原、P16、Ⅰ、Ⅲ型胶原蛋白及前胶原基因表达对成纤维细胞的增殖、抑制及生物合成进行了研究。结果 瘢痕疙瘩成纤维细胞接触表现为细胞交叉重叠及较高的增殖活性及旺盛的生物合成功能，提示其失去了接触性抑制及密度抑制。皮肤成纤维细胞接触后则增殖及生物合成功能明显下降。增生性瘢痕成纤维细胞接触后表现为旺盛的生物合成功能，但其增殖活性处于瘢痕疙瘩和正常皮肤成纤维细胞之间。结论 不同瘢痕成纤维细胞接触后增殖及生物合成的特性可能是形成不同瘢痕的机理之一。     

瘢痕成纤维细胞中cyclin D1、p16的表达及关系研究

目的:了解细胞周期蛋白D1、p16在病理性瘢痕中的表达及它们之间的相互关系,以探讨他们在瘢痕形成过程中的作用。方法:采用免疫组化(SP法)对8例成熟瘢痕、11例增生性瘢痕、11例瘢痕疙瘩及8例正常皮肤组织进行染色,观察CyclinD1和p16在不同组织中的表达。结果:正常皮肤及普通瘢痕成纤维细胞中CyclinD1、p16均为阴性;增生性瘢痕与瘢痕疙瘩成纤维细胞中CyclinD1、p16与正常皮肤相比均有极显著性差异(P<0.05);瘢痕疙瘩成纤维细胞CyclinD1表达高于增生性瘢痕,且有显著性差异(P<0.05);p16在瘢痕疙瘩成纤维细胞的表达比增生性瘢痕为高,但两者之间没有统计学差异.结论:CyclinD1、p16在病理性瘢痕的发生及发展中起重要的作用。在瘢痕疙瘩里p16的细胞抑制作用可能无法与CyclinD1的促细胞增殖作用相拮抗,所以细胞呈现持续增殖状态;而在增生性瘢痕里CyclinD1与p16可能处于相对的平衡状态,所以其生长具有一定的自限性。     

Fibronectin gene expression differs in normal and abnormal human wound healing

The overproduction of fibronectin and type I collagen in keloids and hypertrophic scars implicates altered regulation of extracellular matrix components as an important aspect of these wound healing pathologies. However, little is known about the similarities and differences in extracellular matrix gene expression during normal and abnormal wound healing. This study compared the content of fibronectin messenger RNA and rates of fibronectin protein biosynthesis in fibroblasts derived from normal skin, normal scar, keloid, and hypertrophic scar. Fibronectin expression was enhanced in cells from both normal and abnormal wounds relative to cells from quiescent normal skin. Matched pairs of normal and keloid fibroblasts from the same individuals were also compared, and three of the four pairs showed higher fibronectin expression by the keloid cells at the levels of messenger RNA and protein synthesis. This was consistent with previous studies showing elevated steady state content of fibronectin in keloid cells relative to normal cells from the same individual. Fibronectin messenger RNA and protein content in the tissues from which these cells were derived was examined by in situ hybridization and immunohistochemistry. These studies revealed that in vivo, the steady state content of fibronectin messenger RNA and protein was highest in abnormal wounds, less in most normal scars, and lowest in normal skin. Thus, fibroblasts from keloids and hypertrophic scars overexpressed fibronectin in vivo relative to normal skin and normal scar and retain this characteristic in vitro relative to normal skin. Although normal scars contained little fibronectin protein and messenger RNA, cultured fibroblasts derived from these scars had contents of fibronectin messenger RNA and rates of biosynthesis in vitro similar to those of keloid fibroblasts. This indicates that the fibronectin regulatory pathway in scar fibroblasts is influenced by the tissue environment. These results are discussed with respect to the relationship of fibronectin expression in keloids, hypertrophic scars, and normal wounds in human beings.     

瘢痕疙瘩和正常皮肤成纤维细胞对白介素—1β和白介素—6的…

OBJECTIVE: The purpose of this study was to explore the responses of fibroblasts from keloids and normal skin to interleukin-1 beta and interleukin-6. METHODS: Six samples of keloids and 6 samples of normal skin were collected as the experimental and control group respectively. The means of cell culture was used to investigate the responses of fibroblasts from three different parts of keloids and normal skin to interleukin-1 beta (200 U/ml) and interleukin-6 (100 U/ml). RESULTS: Interleukin-1 beta could inhibit the growth of fibroblasts from the proliferative part of keloids but stimulate growth of those from normal skin, while it did not affect the growth of those from other parts of keloids. Fibroblasts from different parts of keloids and normal skin were all inhibited by interleukin-6. CONCLUSION: The responses of fibroblasts from three parts of keloids and normal skin to interleukin-1 beta and interleukin-6 were not much similar.     

Decreased expression of inhibitory SMAD6 and SMAD7 in keloid scarring.

Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. They are characterised by an abnormal deposition of extracellular matrix components, in particular collagen. There is evidence that transforming growth factor-beta (TGFbeta) is involved in keloid formation. SMAD proteins play a crucial role in TGFbeta signaling and in terminating the TGFbeta signal by a negative feedback loop through SMAD6 and 7. It is unclear how TGFbeta signaling is connected to the pathogenesis of keloids. Therefore, we investigated the expression of SMAD mRNA and proteins in keloids, in normal skin and in normal scars. Dermal fibroblasts were obtained from punch-biopsies of keloids, normal scars and normal skin. Cells were stimulated with TGFbeta1 and the expression of SMAD2, 3, 4, 6 and 7 mRNA was analysed by real time RT-PCR. Protein expression was determined by Western blot analysis. Our data demonstrate a decreased mRNA expression of the inhibitory SMAD6 and 7 in keloid fibroblasts as compared to normal scar (p<0.01) and normal skin fibroblasts (p<0.05). SMAD3 mRNA was found to be lower in keloids (p<0.01) and in normal scar fibroblasts (p<0.001) compared to normal skin fibroblasts. Our data showed for the first time a decreased expression of the inhibitory SMAD6 and SMAD7 in keloid fibroblasts. This could explain why TGFbeta signaling is not terminated in keloids leading to overexpression of extracellularmatrix in keloids. These data support a possible role of SMAD6 and 7 in the pathogenesis of keloids.     

病理性瘢痕和正常皮肤成纤维细胞缝隙连接介导的细胞间通讯

目的 研究和比较不同来源的成纤维细胞缝隙连接介导的细胞间通讯的差异。方法 取手术切除的正常皮肤、增生性瘢痕、瘢痕疙瘩组织各6例,通过细胞培养6-8代后,应用粘附式细胞仪检测及比较各种来源的细胞缝隙连接介导的细胞间通讯。结果 正常皮肤成纤维细胞的细胞间通讯正常,增生性瘢痕成纤维细胞的细胞间通讯受到抑制,瘢痕疙瘩成纤维细胞的细胞间通讯被阻断。结论 细胞间通讯被证明与细胞生长的接触抑制及细胞的浸润性生长密切相关。细胞间通讯被阻断是瘢痕疙瘩呈“蟹足样”浸润性生长的细胞生物学机理之一。     

增殖细胞核抗原在瘢痕疙瘩成纤维细胞中的表达及意义

探讨瘢痕和正常皮肤成纤维细胞原位增殖情况。方法　对 2 4例瘢痕疙瘩组织和周围正常皮肤行增殖细胞核抗原免疫组织化学染色 ,比较瘢痕疙瘩和正常皮肤增殖指数 (Proliferatingindex ,PI)。结果　 2 4例瘢痕疙瘩PI平均值为 1.44,两者经配对t检验 ,P <0 .0 0 1,两者具有显著性差异 ,表明瘢痕疙瘩成纤维细胞增殖活跃。结论　成纤维细胞异常增殖在瘢痕疙瘩发生、发展中起重要作用。     

A novel model of humanised keloid scarring in mice

Treatments for keloid scarring are a major challenge to scientists and physicians for their unknown aetiology. Although several models, including monolayer cell culture to tissue‐engineered models, were developed, further research on keloid has more or less been hindered by the lack of appropriate animal models. Because these aberrant scars are specific to humans, we obtained human normal and keloid skin tissues and isolated dermal fibroblasts from them. Cell morphology, growth and immunohistochemical staining of myofibroblastmarker α‐SMA were examined, and the cell medium of 2‐hour culture and 24‐hour culture was implanted on the back of nude mice. The cell medium of 2‐hour culture and 24‐hour culture was also analysed by a protein array for the detection of distinction in inflammatory factors. We showed that keloid fibroblasts had similar morphology and growth compared to normal skin fibroblasts, but the α‐SMA expression was obviously up‐regulated. After 6 weeks, mice of the 2‐hour keloid‐derived culture medium group exhibited keloid‐like hypertrophic nodules macroscopically, while mice of 24‐hour keloid‐derived culture medium group were similar to normal skin. Histological findings confirmed that the reconstituted skin tissues had the typical features of human keloids. The protein array data revealed that RANTES were involved in humanised fibrotic occurrence in mice, also suggesting they were important modulators of this inflammatory event. This novel model might help to understand the key events that result in the formation of these abnormal scars and provide new therapeutic options.