转化生长因子β1对培养的瘢痕成纤维细胞增殖的调控作用

目的探讨TGF-β1对瘢痕成纤维细胞增殖的调控作用。方法采用细胞培养、MTT法、流式细胞仪和免疫组化方法观察TGF-β1在不同作用剂量和时间对增生性瘢痕来源的成纤维细胞的增殖影响。结果在20%胎牛血清条件下TGF-β1可刺激瘢痕成纤维细胞增殖,其作用在10ng-50ng/ml之间呈剂量效应关系;50ng/ml TGF-β1作用12h开始增殖显著,并可持续到72h。细胞周期分析表明:在TGF-β1作用下,G0/G1%呈降低趋势,而S%呈升高趋势,反映细胞增殖活力的PI值也呈升高趋势。免疫组化结果也显示增殖细胞核内抗原(PCNA)随用药浓度增加而逐渐增加。说明TGF-β1对体外培养的瘢痕成纤维细胞具有促增殖作用。结论TGF-β1可能通过调控成纤维细胞的增殖而在增生性瘢痕的形成过程中起重要作用。     

可调脉宽倍频Nd:YAG激光干预兔耳增生性瘢痕的实验研究

目的:探讨可调脉宽倍频Nd:YAG激光对兔耳增生性瘢痕的治疗作用.方法:制作兔耳增生性瘢痕模型,术后30天对兔耳增生性瘢痕进行可调脉宽倍频Nd:YAG激光干预,观察兔耳瘢痕大体形态和组织病理改变,并采用免疫组化的方法检测兔耳瘢痕治疗前后血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达情况.结果:可调脉宽倍频Nd:YAG激光干预后,兔耳增生性瘢痕出现明显的萎缩,免疫组化检查VEGF表达减弱.结论:可调脉宽倍频Nd:YAG激光可能通过损伤瘢痕组织内的微血管促进兔耳增生性瘢痕萎缩,对早期增生性瘢痕具有治疗作用.     

咪喹莫特调节Th1、Th2细胞相关趋化因子表达抑制兔耳瘢痕增生

目的:免疫因素在增生性瘢痕的发生中起重要作用,本实验研究免疫抑制剂咪喹莫特对兔耳增生性瘢痕组织中辅助性T淋巴(Th)细胞亚群Th1、Th2细胞相关趋化因子CXCL10、CXCL12、CCL2、CCL3、CCL5、CCL7、CCL13表达的影响,探讨咪喹莫特抑制兔耳瘢痕增生的作用机制。方法:选取16只新西兰大耳白兔,雌雄不限。建立兔耳增生性瘢痕模型,每只兔耳腹侧做四个直径为1 cm的圆形创面,每个相距1.5 cm,双侧对称,右耳为咪喹莫特组涂抹5%咪喹莫特软膏,左耳为空白对照组涂抹等量凡士林软膏,待术后14天上皮化均完全后开始涂抹,一日一次,持续一个月。分别于术后第21、28、35、42、49、56、63天同一时间空气栓塞法随机处死2只兔子,收集所有瘢痕标本。另外处死2只健康兔并采集兔耳正常皮肤组织。所有标本行HE染色及Masson三色法染色,观察形态学差异;测量并计算瘢痕增生指数(Scar elevation index,SEI);行Real-time PCR检测CXCL10、CXCL12、CCL2、CCL3、CCL5、CCL7、CCL13的表达。结果:HE及Masson染色可见咪喹莫特组胶原沉积较空白对照组明显减少,SEI显示空白对照组于术后第28天增生程度达到高峰,其增生程度明显高于咪喹莫特组(P0.05);Real-time PCR结果可见咪喹莫特组Th2细胞相关趋化因子CCL2、CCL3、CCL5、CCL7及CCL13表达在各时间点较空白对照组明显降低,Th1细胞相关趋化因子CXCL10、CXCL12的表达在各时间点较空白对照组明显增高(P0.05)。结论:咪喹莫特可通过调节Th1、Th2细胞相关趋化因子的表达来发挥抑制兔耳瘢痕增生的作用。     

PI3K/AKt/mTOR信号通路介导黄芩苷抑制增生性瘢痕组织成纤维细胞的增殖

黄芩苷作为一种黄酮类成分可通过抑制细胞增殖、促进凋亡发挥抗肿瘤作用,但它是否对异常增生的瘢痕具有抑制增生的作用尚不清楚.本研究探讨黄芩苷抑制人增生性瘢痕组织成纤维细胞增殖的分子机制. 采用MTT比色法检测不同浓度的黄芩苷（2.24×10-2 ～ 2.24×102 mmol/L）对体外培养的增生性瘢痕组织成纤维细胞增殖的抑制作用.发现浓度为2.24×100～2.24×102 mmol/L黄芩苷处理组明显抑制增生性瘢痕组织成纤维细胞的增殖（P<0.05）.转染后的荧光素酶报告基因活性检测、RT-PCR及Western印迹分析技术检测其mRNA水平及细胞的帽状依赖翻译的表达.2.24×102 mmol/L黄芩苷处理后,黄芩苷作用组的mRNA水平并无明显差异（P>0.05）;增生性瘢痕成纤维细胞的帽状依赖结构的翻译明显被黄芩苷所抑制.采用Western印迹分析检测被黄芩苷干预的增生性瘢痕组织成纤维细胞的增殖相关的蛋白的表达;m7GTP琼脂糖珠沉淀结合蛋白4E-BP1与eIF4E的变化.发现增殖相关的蛋白mTOR、p70S6K、S6、4EBP1、eIF4E及其上游的AKT表达明显下调（P<0.05）,而PTEN表达明显上调.p-AKT(Ser473)、p-mTOR(Ser2448)、p-S6(Ser235/236)、p-4EBP1(Thr37/ 46)、p-PTEN（T380/S382/383）磷酸化水平下降（P<0.05）.在黄芩苷作用下的增生性成纤维细胞中的游离的4E-BP1明显减少（P<0.05）,而与eIF4E结合的4E-BP1明显增加（P<0.05）黄芩苷诱导游离的4E-BP1与eIF4E结合,从而抑制帽状依赖蛋白翻译.以上结果说明,黄芩苷可通过抑制PI3K/AKT/mTOR信号通路抑制人增生性瘢痕组织成纤维细胞的增殖.     

咪喹莫特抑制兔耳瘢痕增生的机制研究

目的:研究咪喹莫特抑制兔耳瘢痕增生的作用及可能机制.方法:选取10只新西兰大耳白兔,雌雄不限,根据本实验室的改良方法建立兔耳瘢痕模型,每只兔耳腹侧做六个直径为1cm的圆形创面,每个相距1.5cm,双侧对称,左耳为实验组涂抹5％咪喹莫特软,右耳为对照组涂抹等量凡士林软膏,待术后14天上皮化均完全后开始涂抹,一日一次.分别于术后14天、21天、28天、35天及42天同一时间点空气栓塞随机处死2只兔子,后沿每个瘢痕边缘外周的0.5cm处环形切下,经瘢痕最凸点直径一切为二,一半固定后行苏木精-伊红(HE)染色及Masson三色法染色,观察形态学差异,测量并计算瘢痕增生指数(Scar elevation index,SEI):另一半用于提取RNA,反转录后行Real-time PCR检测细胞外基质I型胶原(Collagen-I,Col-I),细胞因子IFN-γ及IL-4的表达.结果:HE及Masson染色可见实验组胶原沉积较对照组明显减少,SEI显示空白对照组于术后第21天增生程度达到高峰,其增生程度明显高于实验组;Real-time PCR结果可见实验组Col-I及IL-4的表达在各时间点明显降低,且IFN-γ的表达较对照组增高.结论:咪喹莫特可能通过调节IFN-γ及IL-4的表达来发挥抑制瘢痕增生的作用.     

Nd：YAG激光对牙龈成纤维细胞增殖及超微结构的影响

这探讨Ｎｄ：ＹＡＧ激光照射对牙龈成纤维细胞增殖状况及超微结构的影响,采用体外细胞培养技术,经一定能量密度（２０ｍｊ／ｃｍ＾２－１２０ｍｊ／ｃｍ＾２）的脉冲Ｎｄ：ＹＡＧ激肖照射后,观察牙龈成纤维细胞的增殖和细胞超微结构的变化。观察到２０ｍｊ／ｃｍ＾２－１２０ｍｊ／ｃｍ＾２能量密度的Ｎｄ：ＹＡＧ激光均能使细胞的增殖受到抑制,透射电镜下可见,８０ｍｊ／ｃｍ＾２－１２０ｍｊ／ｃｍ＾２组细胞内结构明显受损。     

Wnt5a在增生性瘢痕中的表达及其临床意义

目的：探讨wnt5a在增生性瘢痕中的表达及其临床意义。方法：选择12例增生性瘢痕患者,术中取成熟期增生性瘢痕6份,增殖期增生性瘢痕6份,正常皮肤组织6份。光镜下观察其形态学的差异,通过免疫组化技术检测和比较其Wnt5a阳性表达的细胞面积率。结果：与正常皮肤相比,增殖期增生性瘢痕中有大量的成纤维细胞,胶原纤维含量丰富,且排列紊乱,其间有大量的炎性细胞,成熟期增生性瘢痕也含有丰富的成纤维细胞和胶原,但炎性细胞很少。增殖期增生性瘢痕和成熟期增生性瘢痕组织中真皮浅层和真皮深层Wnt5a阳性表达的细胞面积率均显著高于正常皮肤组织（P〈0．05）,且增殖期增生性瘢痕组织中wnt5a阳性表达的细胞面积率显著高于成熟期增生性瘢痕（P〈0．05）。但正常皮肤组织、成熟期增生性瘢痕、增殖期增生性瘢痕各组间真皮浅层与真皮深层Wnt5a阳性表达的细胞面积率比较均无显著性差异（P〉0．05）。结论：Wnt5a的表达上调可能在增生性瘢痕的形成中起重要作用,并可能与增生性瘢痕的增殖活性有关。     

丝裂霉素C对瘢痕疙瘩成纤维细胞Cyclin D1、CDK4及P27基因表达的影响

目的探讨丝裂霉素C（Mitomycin C,MMC）对瘢痕疙瘩成纤维中Cyclin D1、CDK4及P27基因表达的影响,以阐明MMC对瘢痕疙瘩的作用机制,为瘢痕疙瘩的治疗提供理论依据。方法用不同浓度的MMC作用于体外培养的瘢痕疙瘩成纤维细胞,MTT法测量细胞增殖情况,利用半定量RT-PCR、免疫组化分析用药前后Cydin D1、CDK4及P27的mRNA和蛋白水平表达的变化。结果MMC可明显抑制体外培养的瘢痕疙瘩成纤维细胞的增殖,并呈时间和剂量依赖性抑制成纤维细胞增殖;MMC作用后,使Cyclin D1和CDK4的mRNA及蛋白表达减少,P27基因的表达增加。结论MMC可能是通过调节cyclin D1／CDK4／P27细胞周期调控通路,抑制瘢痕疙瘩成纤维细胞增殖而发挥治疗瘢痕疙瘩的作用。     

脂氧素A4对瘢痕成纤维细胞生物学活性的影响及机制研究

最新文献表明,脂氧素A4(lipoxin A4,LXA4)对组织纤维化及相关疾病有防治作用。为了观察脂氧素A4对瘢痕成纤维细胞增殖、凋亡和胶原合成的影响并探讨其抗瘢痕形成的机理,该文首先通过RT-PCR和Western blot法检测瘢痕成纤维细胞是否表达脂氧素受体ALX;然后将不同浓度脂氧素A4加入瘢痕成纤维细胞培养液中分别作用相应时间后,MTT法检测细胞的增殖程度,流式细胞仪检测细胞的凋亡情况,羟脯氨酸测试盒检测细胞培养液中羟脯氨酸含量,ELISA法检测细胞培养上清中TGF-β水平。结果发现,瘢痕成纤维细胞表达ALX,脂氧素A4抑制瘢痕成纤维细胞增殖、羟脯氨酸释放及TGF-β分泌,同时还诱导细胞凋亡。综上所述,脂氧素A4抑制瘢痕成纤维细胞增殖和胶原合成并诱导其凋亡,可能是防治瘢痕形成的重要潜在药物。     

658 nm低能量激光照射对人牙周膜细胞生物学效应的影响

目的:探讨658 nm低能量激光照射对人牙周膜细胞增殖、碱性磷酸酶活性及纤维连接蛋白合成的影响.方法:改良组织块法体外培养人牙周膜细胞.通过658 nm激光照射人牙周膜细胞,观察能量密度为1.86 J/cm2和3.72 J/cm2激光照射后不同时间点细胞增殖效应、碱性磷酸酶活性和纤维连接蛋白的变化.结果:1.86 J/cm2和3.72 J/cm2能量密度的激光照射人牙周膜细胞,可显著促进细胞增殖效应.3.72 J/cm2能量密度的激光照射可提高人牙周膜细胞碱性磷酸酶活性;能量密度为1.86 J/cm2的激光照射人牙周膜细胞72 h后,细胞中纤维连接蛋白分泌量增加.结论:658 nm低剂量激光照射可促进人牙周膜细胞增殖;适量的低剂量激光照射人牙周膜细胞可促进其碱性磷酸酶活性及纤维连接蛋白的分泌.     

Therapeutic effects of liposome-enveloped Ligusticum chuanxiong essential oil on hypertrophic scars in the rabbit ear model

Hypertrophic scarring, a common proliferative disorder of dermal fibroblasts, results from an overproduction of fibroblasts and excessive deposition of collagen. Although treatment with surgical excision or steroid hormones can modify the symptoms, numerous treatment-related complications have been described. In view of this, we investigated the therapeutic effects of essential oil (EO) from rhizomes of Ligusticum chuanxiong Hort. (Umbelliferae) on formed hypertrophic scars in a rabbit ear model. EO was prepared as a liposomal formulation (liposome-enveloped essential oil, LEO) and a rabbit ear model with hypertrophic scars was established. LEO (2.5, 5, and 10%) was applied once daily to the scars for 28 days. On postoperative day 56, the scar tissue was excised for masson's trichrome staining, detection of fibroblast apoptosis, assays of the levels of collagens I and III, and analysis of the mRNA expression of matrix metalloproteinase-1 (MMP-1), caspase-3 and -9, and transforming growth factor beta 1 (TGF-β(1)). In addition, the scar elevation index (SEI) was also determined. As a result, LEO treatment significantly alleviated formed hypertrophic scars on rabbit ears. The levels of TGF-β(1), MMP-1, collagen I, and collagen III were evidently decreased, and caspase -3 and -9 levels and apoptosis cells were markedly increased in the scar tissue. SEI was also significantly reduced. Histological findings exhibited significant amelioration of the collagen tissue. These results suggest that LEO possesses the favorable therapeutic effects on formed hypertrophic scars in the rabbit ear model and may be an effective cure for human hypertrophic scars.     

Effect of Hematoporphyrin Monomethyl Ether-Sonodynamic Therapy (HMME-SDT) on Hypertrophic Scarring

Objective

The aim of the present study was to explore the potential for hematoporphyrin monomethyl ether-Sonodynamic Therapy (HMME-SDT) treatment of hypertrophic scars within rabbit ears.

Methods

60 white rabbits were randomly divided into five groups: 1) untreated controls, 2) lesioned, 3) lesioned + HMME, 4) lesioned + US (Ultrasound), and 5) lesioned +HMME-SDT. After induction of a lesion upon the ears of the rabbits, hypertrophic scars were assessed at 14, 28, 42 and 56 days post-lesion +/− treatment. Assessments consisted of visual inspection in the change of the skin, scar formation pathological morphology by hematoxylin and eosin (HE) staining technique with optical microscopy, calculation of a hypertrophic index, fibroblastic density measures, and observation of collagen changes in the scar tissue by Van Gieson''s (VG)Stain along with calculation of collagen area density.

Results

With continued HMME-SDT treatment there was a gradual improvement in all parameters over the duration of the experiment. The lesion-induced scars of rabbits receiving HMME-SDT treatment were soft, the size was reduced, hyperplasia was flat and the color pale. The fibroblasts and collagens were reduced and the collagens were light red, sparse and orderly. The hypertrophic index was reduced, since the fibroblastic density was lowered and collagen area density was decreased.

Conclusion

HMME is an effective sonosensitizer and the combination of HMME-SDT treatment can exert significant benefits in reducing the formation of hypertrophic scars.     

Abrogation of transforming growth factor-beta signaling by SMAD7 inhibits collagen gel contraction of human dermal fibroblasts

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-beta in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-beta signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-beta functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-beta antagonists, e.g. soluble TGF-beta receptor, latency-associated peptide, and anti-TGF-beta1 antibodies, confirms autocrine TGF-beta stimulation of both cell populations. Further, Smad7 expression inhibited alpha1 (I) collagen and alpha-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-beta/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation.     

Effect of Mederma on hypertrophic scarring in the rabbit ear model

Currently accepted conservative treatments of hypertrophic scars are limited to steroid injections, radiation therapy, and silicone occlusive therapy. However, the use of Mederma for these problematic lesions has become quite prevalent in the clinical setting. Little scientific evidence exists to support the efficacy of this product in reducing hypertrophic scars. The aim of this study was to study the effects of Mederma on hypertrophic scars in the rabbit hypertrophic scar model, allowing the histologic quantification of scar elevation, dermal collagen organization, vascularity, and inflammation and the gross examination of scar erythema. Full-thickness wounds down to cartilage, four per ear, were created in four New Zealand White rabbits, for a total of 32 scars. Twenty-eight days after the initial wounding, the hypertrophic scars were photographed, and treatment of half of the scars on each ear was begun with Mederma three times per day for a total of 4 weeks. The untreated scars served as control scars and were left exposed to air. After 4 weeks of treatment, the scars were once again photographed. The rabbits were then killed, and the scars were analyzed histologically. The pretreatment and posttreatment photographs were compared by using computer quantification of magenta, yellow, and cyan expression within the scars.Histologic analysis demonstrated no significant reduction in scar hypertrophy or scar elevation index. However, a significant improvement in dermal collagen organization was noted on comparing Mederma-treated scars with untreated control scars (p < 0.05). No significant difference in dermal vascularity or inflammation was noted. Computer analysis of the scar photographs demonstrated no significant reduction in scar erythema with Mederma treatment. The active product in Mederma, allium cepa, has as its derivative quercetin, a bioflavonoid noted for its antiproliferative effects on both normal and malignant cells, and its antihistamine release effects. These properties could theoretically prove beneficial in reversing the inflammatory and proliferative responses noted in hypertrophic scars. Despite the authors' inability to demonstrate a reduction in scar hypertrophy, the improvement in collagen organization noted in the Mederma-treated scars suggests it may have an effect on the pathophysiology of hypertrophic scar formation.     

脉冲1 064 nm激光视网膜损伤动物模型的建立

目的:建立脉冲1 064 nm Nd:YAG激光致视网膜出血性损伤及非出血性损伤动物模型,为治疗药物评价提供技术基础.方法:应用自由振荡脉冲及调Q脉冲1 064 nm激光照射青紫蓝灰兔视网膜,通过在光路中加人透镜获得直径200μm眼底光斑,加入衰减片改变角膜入射激光能量.照射即刻对损伤应用检眼镜进行实时观察,并用眼底相...     

血管钠肽抑制低氧刺激心脏成纤维细胞增殖的机制研究

目的：研究血管钠肽（VNP）抑制低氧刺激的心脏成纤维细胞增殖的机制。方法：发离、培养乳鼠心脏成纤维细胞,随机分为四组：对照组、低氧组、低氧＋VNP组和低氧＋8－Bromo-cGMP组。以MTT法观察各组细胞的生长情况,分别采用放射免疫和免疫组化的方法研究了VNP对细胞内cGMP水平和增殖细胞核抗原（PCNA）表达的影响。结果：低氧24h可以使培养的乳鼠心脏成纤维细胞MTT A490nm值显著升高（P＜0．05vs对照组）,VNP（10^-7mol/L和8－Bromo-cGMP(10^-3mol/L)均可以显著降低低氧刺激的心脏成纤维细胞MTT A490nm值（P＜0．05vs低氧组）;对照组和低氧组细胞内cGMP水平无显著差异,而VNP（10^-7mol/L）能升高细胞内cGMP水平（P＜0．05vs对照组、低氧组）;低氧组PCNA的表达显著强于对照组（P＜0．05vs对照组）,VNP（10^-7mol/L可以使低氧刺激的心脏成纤维细胞PCNA表达减弱（P＜0．05vs低氧组）。结论：VNP抑制低氧刺激的心脏成纤维细胞增殖与升高细胞内cGMP水平、减弱PCNA的表达有关。