皮肤肿瘤

20043072　几丁糖对瘢痕疙瘩成纤维细胞形态及增殖的作用/张敬德(二军大长海医院整形外科)…//临床皮肤科杂志.-2004,33(4).-199～201用组织块法进行瘢痕疙瘩、正常皮肤成纤维细胞的体外培养,用光学显微镜和电子显微镜观察细胞的形态结构,用四甲基偶氮唑蓝(MTT)比色法和流式细胞仪检测不同浓度几丁糖对不同来源成纤维细胞生长增殖和细胞周期影响。结果显示,随着几丁糖浓度升高,瘢痕疙瘩来源的成纤维细胞细胞器萎缩,A值减小,G0 G1期的细胞百分比增多,S期、G2 M期的细胞百分比减少。说明几丁糖对正常皮肤和瘢痕疙瘩来源的成纤维细胞的生长…     

积雪草甙和苦参碱对皮肤瘢痕成纤维细胞生长及胶原合成影响的研究

目的 探讨积雪草甙、苦参碱对皮肤瘢痕成纤维细胞生长及胶原合成的影响，并对两者的抗瘢痕作用强度进行比较。方法 体外培养瘢痕疙瘩成纤维细胞，分别加入不同浓度的积雪草甙、苦参碱，通过倒置显微镜观察细胞形态、MTT法测定细胞活力、流式细胞仪检测细胞周期改变、氯胺T法测定羟脯氨酸含量、乳酸脱氢酶检测细胞毒作用，分别观测两种中药对瘢痕成纤维细胞生长及胶原合成的影响。结果 与空白对照组相比，实验组中积雪草甙为0．5g／L、苦参碱为1．00g／L后成纤维细胞数量均明显减少；羟脯氨酸含量降低（P〈0．05）；细胞处于G2-M期比例增加（P〈0．05）；当积雪草甙浓度低于5g/L时乳酸脱氢酶含量在正常范围内。结论 在一定浓度下，积雪草甙和苦参碱均能抑制皮肤瘢痕成纤维细胞生长及胶原合成，积雪草甙的抗瘢痕作用强于苦参碱。     

正常皮肤、增生性瘢痕与瘢痕疙瘩成纤维细胞培养及生物学特征的研究

目的:分析正常皮肤、增生性瘢痕和瘢痕疙瘩成纤维细胞在形态学、生长动力学及生物学的特性.方法:采用组织块培养法培养细胞;MTT法检测细胞在血清饥饿状态下的活性;免疫组织化学法检测3种成纤维细胞中结缔组织生长因子(CTGF)和胸苷磷酸化酶(TP)的表达.结果:正常皮肤、增生性瘢痕和瘢痕疙瘩成纤维细胞在形态与生长动力学上基本相似,血清饥饿对不同来源的成纤维细胞生长的影响并没有显著的差别.CTGF、TP在增生性瘢痕和瘢痕疙瘩成纤维细胞中均呈强阳性表达,而在正常皮肤成纤维细胞中的表达却很微弱,且有显著差别.结论:正常皮肤、增生性瘢痕与瘢痕疙瘩3类成纤维细胞在体外培养状态下,其形态、生长特性以及对血清饥饿的影响无显著差异.3类成纤维细胞对细胞因子具有不同的反应特性.     

五没食子酰基葡萄糖对瘢痕疙瘩成纤维细胞增殖与凋亡的影响

目的：确定五没食子酰基葡萄糖对瘢痕疙瘩细胞增殖与凋亡的影响。方法：用细胞生长实验。细胞周期分析,细胞姬姆萨染色比较五没食子酰基葡萄糖、五倍子单宁酸和秋水仙碱对瘢痕组织成纤维细胞．Hacat细胞和肿瘤细胞在增殖与凋亡方面的影响。结果：五没食子酰基葡萄糖对瘢痕疙瘩成纤维细胞和黑素瘤A375细胞的增殖有较强的抑制作用,对成纤维细胞的作用呈时间和剂量依赖性,对HaCat细胞的增殖呈双相作用,低浓度为促进作用,高浓度为抑制作用,可使增殖受抑制细胞的细胞周期发生改变,表现为S期阻滞。凋亡细胞显著增加。结论：五没食子酰基葡萄糖对瘢痕疙瘩成纤维细胞有抑制增殖和促进凋亡的作用。对治疗瘢痕疙瘩可能是一个具有开发潜力的活性物质。     

槲皮素对瘢痕疙瘩成纤维细胞生长的影响

目的探讨槲皮素对人体瘢痕疙瘩成纤维细胞在生长增殖、细胞凋亡方面的作用,为临床瘢痕疙瘩的治疗提供实验基础。方法MTT法测定瘢痕疙瘩成纤维细胞干预前后的生长曲线;光学显微镜观察干预后细胞的形态学变化;流式细胞仪分析成纤维细胞在干预前后的细胞周期变化;原位细胞凋亡检测成纤维细胞干预后的细胞凋亡率;免疫细胞化学和蛋白印迹实验检测成纤维细胞干预前后p53、bcl-2、bax蛋白表达水平的变化。结果MTT法显示槲皮素对体外培养的瘢痕疙瘩成纤维细胞有抑制作用,抑制效应呈剂量时间依赖关系;槲皮素作用后48h瘢痕疙瘩成纤维细胞发生G2/M期阻滞,并且出现细胞凋亡,光镜可见细胞形态学上出现细胞皱缩,染色质浓集;流式细胞仪结果显示有凋亡峰出现;原位细胞凋亡检测显示凋亡率为16.5%±2.7%。槲皮素作用后细胞p53蛋白和bax蛋白表达升高,bcl-2蛋白表达降低。结论槲皮素通过抑制细胞增殖和促进细胞凋亡来抑制瘢痕疙瘩成纤维细胞的生长。     

肥大细胞在瘢痕疙瘩发病机理中作用的研究

为探讨肥大细胞在瘢痕疙瘩发病机理中的作用，用透射电镜观察活跃增生瘢痕疙瘩的超微结构。用组胺分别处理体外培养正常皮肤与瘢痕疙瘩成张纤维细胞，采用^3H-脯氨酸掺入、胃蛋白酶消化法检测其胶原合成量。结果：瘢痕疙瘩中有大量含有高度发达粗面内质网的成纤维细胞，还可见有脱颗粒的肥大细胞。瘢痕疙瘩成纤维细胞在胶原合成量已有增加的基础上，经一定浓度组胺作用，其胶原合成量仍有显著增加。结果示肥大细胞可有助于瘢痕疙瘩成纤维细胞胶原合成功能的活化及其活化表型的维持。     

己酮可可碱对瘢痕疙瘩成纤维细胞增殖、胶原合成及转化生长因子-β1表达的影响北大核心CSCD

目的探讨己酮可可碱对瘢痕疙瘩成纤维细胞增殖、胶原合成及转化生长因子（TGF）-β1表达的影响。方法取人瘢痕疙瘩及正常皮肤组织培养的第5～8代成纤维细胞,在含有0．1—3g／L己酮可可碱的环境中培养。应用噻唑蓝（M1Tr）法检测成纤维细胞增殖,双抗体夹心-ELISA法测定TGF—β1表达,RT—PCR检测I、Ⅲ型前胶原mRNA表达。结果0．1～2g／L己酮可可碱能明显抑制瘢痕疙瘩和正常皮肤成纤维细胞的增殖,呈明显的量效一时效关系,抑制作用在浓度2g／L时达到最高。浓度为0．5～2g／L时己酮可可碱能降低瘢痕疙瘩和正常皮肤成纤维细胞TGF—B1表达,1或2g／L时己酮可可碱能降低瘢痕疙瘩和正常皮肤成纤维细胞I、Ⅲ型前胶原mRNA表达。结论己酮可可碱对瘢痕疙瘩和正常皮肤成纤维细胞的增殖、TGF—β1以及I、Ⅲ型前胶原表达均有明显的抑制作用。     

曲尼司特对人正常皮肤和瘢痕疙瘩成纤维细胞部分细胞因子表达的影响

目的 ：研究曲尼司特对正常皮肤和瘢痕疙瘩成纤维细胞转化生长因子-β1（TGF-β1）、碱性成纤维细胞生长因子（bFGF）和白介素-6（IL-6）表达的影响。方法：在体外无血清培养的人正常皮肤成纤维细胞和瘢痕疙瘩成纤维细胞中，分别加入0、10、25、50和250μg／mL曲尼司特孵育24、72、96h，用双抗体夹心酶联免疫吸附试验（ABC—ELISA）法测定其上清液中TGF-β1、bFGF和IL-6的表达水平。结果：与对照组相比。25、50和250μg／mL曲尼司特能抑制瘢痕疙瘩成纤维细胞TGF-β1的表达；50μg／mL和250μg／mL曲尼司特能增加瘢痕疙瘩成纤维细胞bFGF的表达；10～250μg／mL曲尼司特可降低IL-6的表达，上述改变在一定时段差异有统计学意义（P〈0．05）。不同浓度曲尼司特对正常皮肤成纤维细胞的影响相似。结论：曲尼司特能降低瘢痕疙瘩成纤维细胞TGF-β1的产生。增加bFGF的合成，减少IL-6的表达，这或许可解释其在抑制异常瘢痕形成中的作用。     

Gravin在瘢痕疙瘩中的表达

目的 探讨Gravin在瘢痕疙瘩形成中的可能作用机制。方法 荧光定量PCR法检测并比较Gravin在瘢痕疙瘩和正常皮肤中的表达情况,免疫荧光双标法分析Gravin在正常皮肤和瘢痕疙瘩中的定位。 结果 荧光定量PCR结果显示：Gravin在瘢痕疙瘩中的表达量明显降低（0.0953 ± 0.0664）,与正常皮肤相比（0.4565 ± 0.1728）差异有统计学差异（P < 0.01）。免疫荧光双标结果显示：正常皮肤组织中,Gravin主要定位于成纤维细胞;在瘢痕疙瘩中,Gravin定位于巨噬细胞和成纤维细胞,主要位于巨噬细胞。结论 瘢痕疙瘩中Gravin表达量明显下降且主要定位于巨噬细胞。这种表达和定位的改变可能通过影响瘢痕疙瘩中成纤维细胞和炎症细胞的增殖及活化,参与瘢痕疙瘩的形成。     

雷公藤多甙片对皮肤瘢痕成纤维细胞生长及胶原合成影响的研究

目的探讨雷公藤多甙片对瘢痕疙瘩成纤维细胞生长及胶原合成的影响。方法体外培养瘢痕疙瘩成纤维细胞,分别加入不同浓度的雷公藤多甙,通过倒置显微镜观察、MTT法测定细胞活力、流式细胞仪检测细胞周期改变、羟脯氨酸含量测量、乳酸脱氢酶检测等方法观测雷公藤多甙对瘢痕疙瘩成纤维细胞生长及胶原合成的影响。结果与空白对照组相比,加入雷公藤多甙5μg/mL后成纤维细胞数量明显减少;A值降低,最大抑制率为59.60%;羟脯氨酸含量降低(P<0.05);细胞处于G2-M期比例增加(P<0.05);当药物浓度低于500μg/mL时乳酸脱氢酶含量与空白对照组相比没有明显差异(P>0.05)。结论有效浓度下,雷公藤多甙能抑制皮肤瘢痕成纤维细胞生长及胶原合成。     

Differences in collagen production between normal and keloid-derived fibroblasts in serum-media co-culture with keloid-derived keratinocytes

Keloids are characterized by the deposition of excessive extracellular-matrix collagen by abnormal fibroblasts in response to cutaneous injury. Studies to date have largely concentrated on the role of the keloid fibroblast in the pathogenesis of this lesion. Recent studies have highlighted the important concept of epithelial-mesenchymal interactions in normal skin biology. Extrapolating this to keloids in two recent serum-free in vitro studies, we demonstrated increased growth and proliferation, as well as induction of keloid-like collagen secretory characteristics in normal fibroblasts co-cultured with keloid-derived keratinocytes. Most fibroblast culture work to date has been performed in nutrient and growth factor-rich serum media. To investigate how a serum co-culture system might influence epithelial-mesenchymal interactions, [3H] proline incorporation was examined in normal and keloid fibroblasts co-cultured in serum with keratinocytes derived either from normal skin or keloid tissue. Results showed increased [3H] proline incorporation when normal fibroblasts were co-cultured with keloid keratinocytes, which was significantly increased when keloid fibroblasts were co-cultured with keloid keratinocytes. Taken with previous results, this study demonstrates a good correlation between both serum and serum-free co-culture systems, and supports the significance of epithelial-mesenchymal interactions in keloid pathogenesis.     

2-Methoxyestradiol inhibits the proliferation level in keloid fibroblasts through p38 in the MAPK/Erk signaling pathway

Background

The MAPK/Erk signaling pathway is a classic pathway in cell proliferation. Our former study showed that keloid tissue revealed a higher proliferation level than physiological scars and normal skin. As a natural metabolite of estradiol, 2-methoxyestradiol (2ME2) showed an inhibition proliferation effect on tumor cells.

Aim

In this study, the treatment effect of 2ME2 and its potential mechanisms are explored.

Methods

Six keloid patients and six non-keloid patients were randomly selected from the Department of Plastic Surgery at our hospital during June 2021 to December 2021. Six groups were established: normal skin fibroblasts (N); keloid fibroblasts (K); keloid fibroblasts treated with 2ME2 (K + 2ME2); keloid fibroblasts treated with dimethyl sulfoxide (DMSO) (K + DMSO); keloid fibroblasts treated with doramapimod (K + IN); keloid fibroblasts treated with doramapimod (p38 inhibitor) and 2ME2 (K + IN+2ME2). The fibroblast activity and key factor expression of the MAPK/Erk signaling pathway were measured.

Results

In the results, 2ME2 significantly inhibited keloid fibroblast activity and key factor expression (except STAT1).

Conclusion

The proliferation levels were reduced by both the p38 inhibitor and 2ME2, indicating 2ME2 may achieve an antiproliferation effect by targeting p38 in keloid fibroblasts.     

DHMEQ, a novel NF-kappaB inhibitor, suppresses growth and type I collagen accumulation in keloid fibroblasts

BACKGROUND: Keloid is a benign dermal tumor characterized by proliferation of dermal fibroblasts and overproduction of extracellular matrix (ECM). Nuclear factor kappaB (NF-kappaB) plays an important role in regulation of inflammation, immune response and cell proliferation. Activation of the NF-kappaB pathway is thought to be closely linked to abnormal cell proliferation and ECM production in keloid fibroblasts. OBJECTIVE: This study was set out to investigate the effects of a novel selective NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on keloid fibroblasts. METHODS: Primary normal and keloid dermal fibroblasts were used for this study. NF-kappaB activity was assessed by DNA-binding assay and immunohistochemistry. The effect of DHMEQ was evaluated by cell viability, cell growth and type I collagen accumulation. RESULTS: Basal NF-kappaB activity was constitutively elevated in keloid fibroblasts, indicating that this pathway is involved in keloid pathogenesis. DHMEQ markedly reduced cell proliferation and type I collagen accumulation in keloid fibroblasts. CONCLUSION: The inhibition of NF-kappaB by DHMEQ may be an attractive therapeutic approach for keloids.     

SB-431542 inhibits TGF-beta-induced contraction of collagen gel by normal and keloid fibroblasts

BACKGROUND: Transforming growth factor (TGF)-beta induces fibroblast contraction, which is implicated in wound healing and keloid formation. SB-431542 is a novel specific inhibitor of TGF-beta type I receptor kinase activity. OBJECTIVE: We sought to determine whether SB-431542 inhibited TGF-beta-induced fibroblast contraction. METHODS: We used an in vitro type I collagen gel contraction assay with normal or keloid dermal fibroblasts incorporated. RESULTS: TGF-beta induced contraction of collagen gels with normal dermal fibroblasts incorporated, which was efficiently suppressed by SB-431542. Keloid fibroblasts showed higher basal contraction of collagen gels in the absence of TGF-beta than normal fibroblasts, which was enhanced by addition of TGF-beta. SB-431542 suppressed both the basal and TGF-beta-enhanced contraction of collagen gels by keloid fibroblasts. These inhibitory effects of SB-431542 were associated with suppression of TGF-beta-induced alpha-smooth muscle actin (alpha-SMA) expression and phosphorylation of Smad2 in normal and keloid fibroblasts. CONCLUSION: SB-431542 can suppress TGF-beta-induced contraction of collagen gel by normal and keloid dermal fibroblasts. Importantly, SB-431542 can inhibit basal contraction of collagen gel by keloid fibroblasts. These results suggest that an inhibitor of TGF-beta type I receptor kinase activity may have therapeutic potential for excessive skin contraction as observed in keloid.     

雷公藤甲素对瘢痕疙瘩成纤维细胞增殖和胶原合成的影响

目的探讨雷公藤甲素对瘢痕疙瘩成纤维细胞增殖和胶原合成的影响。方法取手术切除的瘢痕疙瘩组织作成纤维细胞的原代培养,传代培养后加入不同浓度的雷公藤甲素作用,观察细胞的形态学变化,应用MTT法检测细胞的活性,应用免疫组化和Western-blot法检测雷公藤甲素对瘢痕疙瘩成纤维细胞Ⅰ型、Ⅲ型前胶原合成的影响。结果雷公藤甲素能显著抑制瘢痕疙瘩成纤维细胞的增殖,且存在一定的浓度依赖性;雷公藤甲素作用后瘢痕疙瘩成纤维细胞Ⅰ型、Ⅲ型前胶原表达减弱。结论雷公藤甲素能有效抑制瘢痕疙瘩成纤维细胞的增殖和Ⅰ型、Ⅲ型前胶原的合成。     

Studies of transforming growth factors beta 1-3 and their receptors I and II in fibroblast of keloids and hypertrophic scars

Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. They are characterized by an abnormal deposition of extracellular matrix components, particularly collagen. There is uncertain evidence that transforming growth factor-beta (TGFss) is involved in keloid formation. Therefore we investigated the expression of TGFss1, 2 and 3 and their receptors in keloids, hypertrophic scars and normal skin. Dermal fibroblasts were obtained from punch biopsies of patients with keloids and hypertrophic scars and from normal skin of healthy individuals. Total RNA was isolated and the expression of TGFss1, 2 and 3 and of TGFss receptors I and II (TGFssRI and II) was analysed by real-time PCR using the Lightcycler technique. Our data demonstrate significantly lower TGFss2 mRNA expression in hypertrophic scar fibroblasts as compared with fibroblasts derived from keloids and normal skin (p<0.05). In contrast, TGFss3 mRNA expression was significantly lower in keloid fibroblasts in comparison with fibroblasts derived from hypertrophic scar and normal skin (p<0.01). TGFssRI mRNA expression was significantly decreased in hypertrophic scar fibroblasts (p<0.01) and TGFssRII mRNA expression was decreased in keloids compared with hypertrophic scar fibroblasts (p<0.001). The ratio of TGFssRI/TGFssRII expression was increased in keloids compared with hypertrophic scar and normal skin fibroblasts. As recently supposed, an increased TGFssRI/TGFssRII ratio could promote fibrosis. Therefore our data support a possible role of TGFssRI and TGFssRII in combination with a certain TGFss expression pattern as fibrosis-inducing factors in keloids.     

Keloid fibroblasts resist ceramide-induced apoptosis by overexpression of insulin-like growth factor I receptor

Keloids are benign dermal tumors, characterized by overgrowth of lesions, invasiveness beyond the original boundary of the insult, and recurrence of lesions. The exact etiology is unknown, however. Our hypothesis is that keloids are acquired as a result of an abnormal or prolonged wound healing process, with persistent proliferation and extracellular matrix production of fibroblasts that should otherwise discontinue in normal wound healing. In this study, we examined the response of keloid fibroblasts to proapoptotic signaling. Cell-permeable ceramide, N-acetyl-D-sphingosine, induced apoptosis of dermal fibroblasts in a dose- and time-dependent manner, which was detected by phase contrast microscopy, fluorescent microscopy, the TUNEL method, flow cytometric analysis, and WST-1 assay. In contrast, keloid fibroblasts resisted apoptosis induced by N-acetyl-D-sphingosine (percent survival with 40 mM ceramide treatment for 12 h, normal versus keloid: 9.6% +/- 6.6% vs 66.8% +/- 5.5%). Western blotting analysis showed insulin-like growth factor I receptor overexpression in keloid fibroblasts, but not in normal fibroblasts. Exogenously added insulin-like growth factor I enhanced the resistance of keloid fibroblasts to ceramide-induced apoptosis. Wort- mannin, a phosphatidylinositol 3 kinase inhibitor, suppressed the antiapoptotic action of insulin-like growth factor I in keloid fibroblasts. Our results suggest that keloid fibroblasts overexpressing insulin-like growth factor I receptor are resistant to apoptosis, thus allowing persistent proliferation and production of excessive extracellular matrix. J Invest Dermatol 115:1065-1071 2000