电子线对瘢痕疙瘩成纤维细胞生物学作用的影响

目的　研究电子线治疗瘢痕疙瘩的机制及其有效的安全剂量。方法　分别用 5 ,10 ,15 ,2 0Gy的电子线照射体外培养的瘢痕疙瘩成纤维细胞 ,然后观测其生物学效应。结果　 5Gy以上的放射剂量即能抑制成纤维细胞增殖及胶原合成 ,但剂量超过 2 0Gy ,成纤维细胞即被杀死。剂量在 10～ 15Gy之间 ,成纤维细胞Ⅲ型胶原的合成明显增加。结论　 10～ 15Gy的电子线应是安全有效的放射剂量     

电子线对瘢痕疙瘩成纤维细胞生物学作用的影响

目的研究电子线治疗瘢痕疙瘩的机制及其有效的安全剂量.方法分别用5,10,15,20Gy的电子线照射体外培养的瘢痕疙瘩成纤维细胞,然后观测其生物学效应.结果 5Gy以上的放射剂量即能抑制成纤维细胞增殖及胶原合成,但剂量超过20Gy,成纤维细胞即被杀死.剂量在10～15Gy之间,成纤维细胞Ⅲ型胶原的合成明显增加.结论 10～15Gy的电子线应是安全有效的放射剂量.     

重组CDglyTK双自杀基因腺病毒对瘢痕疙瘩成纤维细胞周期的影响

目的观察携带胞嘧啶脱氨酶（CD）基因和胸腺嘧啶核苷激酶（TK）基因两者融合基因（CDglyTK）的重组腺病毒对瘢痕疙瘩成纤维细胞周期的影响,以探讨CDglyTK双自杀基因作用的最佳条件和机制。方法采用改良的AdEasy系统构建携带CDglyTK双自杀基因的重组腺病毒,分别以10、20、40、80的感染复数（MOI）感染成纤维细胞,观察最佳感染效率的MOI,然后给予无毒的前体药物5-氟胞嘧啶（5-Fc）和无环鸟苷（GCV）,分别于给药后24、48、72h行MTT法检测,观察药物作用的最佳时间和最佳MOE。流式细胞仪检测成纤维细胞周期的变化情况。结果在荧光显微镜下观察,重组双自杀基因腺病毒在MOI为20时感染成纤维细胞的感染效率大于95%;给药后48、72hMTT检测其差异无统计学意义（P〉0.05）,给药后48h,转染双自杀基因组的成纤维细胞在G1期的数量与未转染的数量其差异有统计学意义（P〈0.05）,成纤维细胞被阻滞于G1期。结论重组CDglyTK双自杀基因腺病毒在MOI为20时能有效地感染瘢痕疙瘩成纤维细胞,给药后48h是最佳的作用时间,成纤维细胞被阻滞于G期是CDglyTK双自杀基因影响其生长的主要机制。     

电子线对瘢痕疙瘩成纤维细胞生物学作用的影响

目的 研究电子线治疗瘢痕疙瘩的机制及其有效的安全剂量。方法 分别用5，10，15，20Gy的电子线照射体外培养的瘢痕疙瘩成纤维细胞，然后观测其生物学效应。结果 5Gy以上的放射剂量即能抑制成纤维细胞增殖及胶原合成，但剂量超过20Gy，成纤维细胞即被杀死。剂量在10～15Gy之间，成纤维细胞Ⅲ型胶原的合成明显增加。结论 10～15Gy的电子线应是安全有效的放射剂量。     

DNA甲基转移酶1在人瘢痕疙瘩成纤维细胞中的表达及意义

目的 探讨DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)在瘢痕疙瘩的不同生长期和不同部位的成纤维细胞表达及其意义.方法 收集53例瘢痕疙瘩患者手术切除的瘢痕疙瘩标本,并按生长期分为A(＜6个月)、B(6 ～12个月)、C(1～2年)、D(＞2年)4组;免疫组织化学法检测各组标本正常皮肤、浸润部、增生部及老化部成纤维细胞DNMT1的表达并比较差异;RT-PCR法检测各组标本及不同部位成纤维细胞DNMT1mRNA的表达.结果 DNMT1在成纤维细胞中主要分布于细胞核内;瘢痕疙瘩成纤维细胞DNMT1蛋白阳性表达率(72％)及DNMT1mRNA表达均高于正常皮肤成纤维细胞(8％,P =0.001);瘢痕疙瘩生长期较短组成纤维细胞DNMT1阳性表达率(100％,评分:10.47±1.85)及DNMT1mRNA表达均高于生长期较长组(45％,评分:7.71 +1.80,P =0.007);瘢痕疙瘩浸润部(100％,评分:10.47±1.85)成纤维细胞DNMT1阳性表达率及DNMT1mRNA表达均高于增生部(78％,评分:6.63±1.75)与老化部(38％,评分:5.53 +1.55,P=0.001).结论 DNMTI在瘢痕疙瘩发病中具有重要作用;随着瘢痕疙瘩生长期的延长,DNMT1的表达有降低趋势;随着瘢痕疙瘩中央成纤维细胞老化,DNMT1表达降低.提示DNMT1在瘢痕疙瘩生长过程中起着重要的作用,并且与瘢痕疙瘩浸润生长密切相关.     

瘢痕疙瘩中成纤维细胞耐药基因mdr1的研究

目的 研究瘢痕疙瘩成纤维细胞(KF)与正常皮肤组织成纤维细胞(NF)耐药基因mdr1的表达差异。方法 利用RT-PCR、免疫组化及流式细胞分析等方法,检测瘢痕疙瘩与正常皮肤组织中成纤维细胞耐药基因mdr1及其蛋白ABCB1的表达;并对30例不同部位瘢痕疙瘩的ABCB1蛋白表达量进行流式细胞分析。结果 瘢痕疙瘩成纤维细胞中耐药基因mdr1及其蛋白ABCB1的表达明显高于正常皮肤;流式细胞分析显示,瘢痕疙瘩原代成纤维细胞中ABCB1表达率:胸部>背部、腹部,差异有统计学意义。结论 瘢痕疙瘩成纤维细胞表达耐药基因mdr1及其蛋白ABCB1,且较正常皮肤中的表达量增高。     

消瘢醑对瘢痕疙瘩成纤维细胞Ⅰ、Ⅲ型胶原蛋白的影响

目的　研究复方中药制剂消瘢醑对体外培养的瘢痕疙瘩成纤维细胞 (Keloidfibroblast,KFB)I、III型胶原蛋白的影响。　方法　 6例瘢痕疙瘩成纤维细胞为实验组 ,6例正常皮肤成纤维细胞 (Normalfibroblast,NFB)为对照组 ,采用成纤维细胞体外培养、ABC免疫细胞化学染色技术及积分光密度 (IOD)分析 ,观察在 10 μg ml消瘢醑浓度作用下 ,瘢痕疙瘩和正常皮肤成纤维细胞I、III型胶原蛋白的表达。　结果　瘢痕疙瘩成纤维细胞I、III型胶原阳性染色表达强度均显著高于正常皮肤成纤维细胞 (11113 1± 130 4 9vs 35 19 6± 2 36 0与 1115 7 7± 130 0 3vs 2 6 2 6 5± 4 2 6 3,t值分别为 14 4 2和 13 4 7,P值分别为 0 0 0 0 0 3和 0 0 0 0 0 4 )。 10 μg ml浓度的消瘢醑作用 4 8小时后 :1.瘢痕疙瘩成纤维细胞I、III型胶原阳性染色表达强度均显著高于正常皮肤成纤维细胞 (76 75 4± 82 5 5vs 2 30 5 2± 32 0 4与 10 5 95 2± 1311 5vs 2 4 34 8± 35 6 9,t值分别为 13 37和 12 6 6 ,P值分别为0 0 0 0 0 4和 0 0 0 0 0 5 ) ;2 .瘢痕疙瘩和正常皮肤成纤维细胞呈阳性表达的I、III型胶原染色强度明显下降 ,与未经药物处理的相应空白对照相比具有显著性差异 (76 75 4± 82 5 5vs 11113 1± 130 4 9与     

阻抑结缔组织生长因子表达对瘢痕疙瘩成纤维细胞的影响

目的 观察结缔组织生长因子(CTGF)RNA干扰对瘢痕疙瘩成纤维细胞(KF)增殖以及I型胶原合成的影响.方法 设计合成3条CTGF小干扰RNA(siRNA)序列并克隆到pRNAT-6.1/Neo载体中;实时定量逆转录-聚合酶链反应(qRT-PCR)检测KF中CTGF和I型胶原的表达,计算KF细胞数目.结果 合成的3个CTGF siRNA载体中CTGF siRNA3的干扰效果最强,将CTGF mRNA的相对表达量从3.13±0.17降至0.71±0.02,并能将KF中的I型胶原的mRNA水平从2.04±0.10降至0.60±0.04(P<0.01).KF对照组5 d的细胞数目为(10.50±0.25)×10~4,而CTGF siRNA3组5d的细胞数目为(6.83±0.29)×10~4(P<0.01).结论 CTGF RNA干扰能抑制KF的CTGF表达,产生抑制I型胶原合成和细胞增殖的生物学作用.     

缺氧对瘢痕疙瘩成纤维细胞影响的实验研究

目的 研究不同程度缺氧条件下对瘢痕疙瘩成纤维细胞的影响及其与HIF-1α基因表达的关系.方法 设立不同O2浓度组,在常氧(20%)、不同缺氧(10%、5%、1%)条件下,培养瘢痕疙瘩成纤维细胞.24 h后,应用MTT法、流式细胞仪技术和羟脯氨酸比色法,分别检测各组中成纤维细胞的活力、细胞周期和胶原合成水平.结果 不同程度缺氧各组中的瘢痕疙瘩成纤维细胞增殖均明显高于常氧对照组,且S期比例也增高;随着O2浓度的降低,成纤维细胞胶原合成水平逐渐增加.结论 缺氧条件下能增强瘢痕疙瘩成纤维细胞的增殖活力,提高胶原合成水平.这一结果 很可能是通过HIF-1α及其调控的缺氧通路产生作用.提示阻断瘢痕疙瘩内HIF-1α调控的缺氧通路有望成为预防及治疗瘢痕增生的新途径.     

野生型p16基因对人瘢痕疙瘩成纤维细胞生长及代谢影响的实验研究

目的　探讨野生型 p16基因对人瘢痕疙瘩成纤维细胞 (keloidfibroblasts,KFb)生长增殖及DNA合成代谢的影响。　方法　构建含p16cDNA片段的真核表达载体 pcDNA3 p16 ,采用脂质体介导的基因转染法 ,将其导入体外培养的KFb中 ,并用G4 18筛选阳性克隆。随后对已转染及未转染的KFb进行免疫细胞化学染色 ,观察p16蛋白的表达 ,并通过绘制细胞生长曲线及采用氚标记胸腺嘧啶脱氧核苷 (3 H TdR)掺入法 ,比较转染前后细胞在生长增殖及DNA合成代谢方面的变化。结果　经酶切鉴定证实 ,pcDNA3 p16构建成功。已转染的KFb经G4 18筛选 ,出现阳性克隆 ,并有 p16蛋白表达 ;与正常KFb比较 ,其生长增殖速度明显减缓 ,DNA合成代谢能力明显减弱 (P <0 .0 5 )。　结论　p16基因对KFb的生长增殖及DNA合成代谢有明显的抑制作用。     

The deubiquitinating enzyme USP37 promotes keloid fibroblasts proliferation and collagen production by regulating the c-Myc expression

Previous research testifies that c-Myc may promote keloid fibroblast proliferation and collagen accumulation. Ubiquitin-specific peptidase 37 (USP37)-mediated deubiquitination and stabilisation of c-Myc are vital for lung cancer proliferation, while the potential role of USP37 in keloid fibroblasts is not investigated. Elevated USP37, c-Myc, and Collagen I content were detected in keloid tissue with RT-PCR or ELISA assay. USP37 over-expression plasmids or USP37 short hairpin RNAs (shRNAs) were transfected into keloid fibroblasts with Lipofectamine 3000 to decipher the role of USP37 in keloid fibroblasts. USP37 overexpression could promote the proliferation of keloid fibroblasts with increased c-Myc and Collagen I expression. On the other hand, USP37 shRNAs inhibited the proliferation of keloid fibroblasts with diminished c-Myc and Collagen I expression. It was worth noting that C-Myc overexpression promoted the proliferation of keloid fibroblasts inhibited by USP37 shRNAs with increasing Collagen I expression. All of these results demonstrate that USP37 could regulate c-Myc to promote the proliferation and collagen deposit of keloid fibroblasts, and USP37 could be targeted in future keloid therapy.     

PI3K/AKT pathway promotes keloid fibroblasts proliferation by enhancing glycolysis under hypoxia

Our previous study demonstrated altered glucose metabolism and enhanced phosphorylation of the PI3K/AKT pathway in keloid fibroblasts (KFb) under hypoxic conditions. However, whether the PI3K/AKT pathway influences KFb cell function by regulating glucose metabolism under hypoxic conditions remains unclear. Here, we show that when PI3K/AKT pathway was inactivated with LY294002, the protein expression of glycolytic enzymes decreased, while the amount of mitochondria and mitochondrial membrane potential increased. The key parameters of extracellular acidification rate markedly diminished, and those of oxygen consumption rate significantly increased after inhibition of the PI3K/AKT pathway. When the PI3K/AKT pathway was suppressed, the levels of reactive oxygen species (ROS) and mitochondrial ROS (mitoROS) were significantly increased. Meanwhile, cell proliferation, migration and invasion were inhibited, and apoptosis was increased when the PI3K/AKT pathway was blocked. Additionally, cell proliferation was compromised when KFb were treated with both SC79 (an activator of the PI3K/AKT pathway) and 2-deoxy-d-glucose (an inhibitor of glycolysis), compared with the SC79 group. Moreover, a positive feedback mechanism was demonstrated between the PI3K/AKT pathway and hypoxia-inducible factor-1α (HIF-1α). Our data collectively demonstrated that the PI3K/AKT pathway promotes proliferation and inhibits apoptosis in KFb under hypoxia by regulating glycolysis, indicating that the PI3K/AKT signalling pathway could be a therapeutic target for keloids.     

CircSLC8A1 targets miR-181a-5p/HIF1AN pathway to inhibit the growth,migration and extracellular matrix deposition of human keloid fibroblasts

BackgroundCircular RNAs (circRNAs) are identified as important regulators in human diseases, including keloid. The purpose of this study is to reveal the role and molecular mechanism of circSLC8A1 in keloid formation.MethodsExpression of circSLC8A1, microRNA (miR)-181a-5p, and hypoxia inducible factor 1 alpha inhibitor (HIF1AN) were detected by quantitative real-time PCR. Protein expression of extracellular matrix (ECM) deposition markers and HIF1AN was detected by western blot analysis. Furthermore, the interaction between miR-181a-5p and circSLC8A1 or HIF1AN was confirmed by dual-luciferase reporter assay, RIP assay and RNA pull-down assay.ResultsExpression of circSLC8A1 was downregulated in keloid tissues and HKFs. Overexpression of circSLC8A1 suppressed HKFs proliferation, migration, ECM deposition, and promoted apoptosis. MiR-181a-5p is targeted by circSLC8A1, and its mimic reversed the effect of circSLC8A1 on the biological function of HKFs. HIF1AN was a target of miR-181a-5p, and it was positively regulated by circSLC8A1. Knockdown of HIF1AN also reversed the negatively regulation of circSLC8A1 on the biological functions of HKFs.ConclusionOur data showed that circSLC8A1 regulates the miR-181a-5p/HIF1AN axis to restrain HKFs biological functions, confirming that circSLC8A1 might serve as a novel therapeutic target for keloids.     

Keloid explant culture: a model for keloid fibroblasts isolation and cultivation based on the biological differences of its specific regions

In vitro studies with keloid fibroblasts frequently present contradictory results. This may occur because keloids present distinct genotypic and phenotypic characteristics in its different regions, such as the peripheral region in relation to the central region. We suggest an explant model for keloid fibroblasts harvesting, standardising the initial processing of keloid samples to obtain fragments from different regions, considering its biological differences, for primary cell culture. The different keloid regions were delimited and fragments were obtained using a 3‐mm diameter punch. To remove fragments from the periphery, the punch was placed in one longitudinal line extremity, respecting the lesion borders. For the central region, it was placed in the intersection of lines at the level of the largest longitudinal and transversal axes, the other fragments being removed centrifugally in relation to the first one. Primary fibroblast culture was carried out by explant. Flow cytometry analysis showed cell cycle differences between the groups, confirming its different origins and biological characteristics. In conclusion, our proposed model proved itself efficient for keloid fibroblast isolation from specific regions and cultivation. Its simplicity and ease of execution may turn it into an important tool for studying the characteristics of the different keloid‐derived fibroblasts in culture.     

MTS1基因在人BPH中突变缺失的初步研究

目的探讨ＭＴＳ１基因在人前列腺增生症（ＢＰＨ）发病机制中的作用和变异情况。方法用ＰＣＲ银染ＳＳＣＰ技术检测１８例正常前列腺和３８例ＢＰＨ中抑癌基因ＭＴＳ１各外显子的纯合性缺失和突变。结果１２例ＢＰＨ出现ＭＴＳ１基因缺失,总缺失率为３２％;正常前列腺组织中有１例出现ＭＴＳ１基因缺失,缺失率为６％,两者之间差异有显著性（Ｐ＜００５）;无１例有ＭＴＳ１基因的突变。结论ＢＰＨ的发病机制可能与ＭＴＳ１的纯合性缺失有关,未见有突变发生。应对ＭＴＳ１基因在人ＢＰＨ中的作用作更深入的研究     

反义结缔组织生长因子对人瘢痕疙瘩成纤维细胞的作用

目的：研究结缔组织生长因子（CTGF）反义寡核苷酸（ASODN）对人瘢痕疙瘩成纤维细胞CTGFmRNA的表达和细胞增殖及胶原合成的影响,探讨瘢痕疙瘩的基因治疗。方法：体外分离、培养人正常皮肤成纤维细胞（NSF）和瘢痕疙瘩成纤维细胞（KF）,以脂质体介导方法将CTGFASODN转染KF中,用逆转录-聚合酶链式反应（RT-PCR）方法检测细胞中CTGFmRNA的表达;采用MTT方法测细胞增殖3;H-脯氨酸掺入法检测细胞的胶原合成量。结果：CTGFmRNA在NSF中几乎无表达,在KF中表达增高,CTGFASODN可以抑制其增殖和胶原合成（P〈0.05）。结论：CTGFASODN能够抑制成纤维细胞CTGFmRNA表达和细胞增殖及胶原合成,表明阻断CTGF可能是延缓瘢痕纤维化的有效手段。     

姜黄素对瘢痕疙瘩成纤维细胞增殖和胶原合成的影响

目的:观察姜黄素对体外培养的人瘢痕疙瘩成纤维细胞增殖及胶原合成的作用,以寻找治疗人瘢痕疙瘩的有效药物。方法:体外培养人瘢痕疙瘩成纤维细胞,分别应用MTT比色法和羟脯氨酸比色法检测姜黄素作用后细胞增殖及胶原合成的变化。结果:和对照组相比,姜黄素能够显著抑制瘢痕疙瘩成纤维细胞的增殖及胶原合成(P<0.01)。结论:姜黄素具有体外抗瘢痕疙瘩的作用,可能成为治疗瘢痕疙瘩的有效药物。     

水飞蓟宾对体外培养瘢痕疙瘩成纤维细胞增殖的影响

目的 研究水飞蓟宾(SB)对体外培养的瘢痕疙瘩成纤维细胞增殖的影响,并探讨其作用机制.方法 采用CCK-8法观察不同浓度的SB对瘢痕疙瘩成纤维细胞增殖的影响;用免疫组化、RT-PCR和Western Blot检测SB 干预前后成纤维细胞中mTOR和HIF-1α的表达情况.结果 CCK-8检测体外培养的瘢痕疙瘩成纤维细胞的吸光度值,随着SB浓度的逐渐增大及干预时间的延长而减小,各组间差异有统计学意义(P<0.05);在浓度为200 μmoL/L的SB作用下,mTOR及HIF-1α基因和蛋白的表达在常氧和缺氧条件均降低,不同组之间比较,其差异有统计学意义(P<0.01).结论 SB通过抑制瘢痕疙瘩成纤维细胞中mTOR和HIF-1α的表达,来抑制瘢痕疙瘩成纤维细胞的增殖,从而抑制瘢痕疙瘩的发生.