SMAD3在瘢痕疙瘩成纤维细胞中的表达与分布

目的：研究SMAD3在瘢痕疙瘩成纤维细胞（Keloid fibroblast,KFB)和正常人皮肤成纤维细胞（Normal skin fibroblast,NFB)中的表达和分布。方法：采用Western蛋白印迹法和细胞内免疫荧光法测定SMAD3的表达和分布。结果：SMAD3在KFB和NFB两种细胞中的表达差别不大（P＞0．05），KFB核内的荧光强度强于NFB。结论：SMAD3在KF3细胞核内的分布高于NFB。     

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.     

Hint1基因抑制HepG2细胞AP-1转录因子活性的实验研究

目的 探索体外过表达Hint1基因对人肝肿瘤HepG2细胞AP-1转录因子活性的影响.方法 分子克隆获得Hint1基因序列,构建pHA-Hint1重组融合基因表达载体.阳离子脂质体介导,pHA-Hint1转导人肝母细胞瘤HepG2细胞,RT-PCR和Western blot检测HepG2细胞中外源HA-Hint1表达.pHA-Hint1质粒、启动子荧光素酶报告质粒AP-1/Luc及巨细胞病毒-β-半乳糖酶报告质粒(β-gel)共转导HepG2细胞.36 h后测定荧光素酶活性及β-gel活性.结果 重组质粒经EcoR Ⅰ和BamH Ⅰ双酶切,获约100 bp特异条带,测序证实成功构建pHA-Hint1表达载体.pHA-Hint1及空载体pcDNA3/HA转导HepG2细胞,半定量RT-PCR结果显示pHA-Hint1组Hint1mRNA表达高于空载体组(t=3.89,P<0.05);Western blot结果显示pHA-Hint1组HA标签蛋白表达高于空载体组(t=3.12,P<0.05).AP-1转录因子活性检测结果:pHA-Hint1终浓度为0 μg/ml,0.5 μg/ml,1.0 μg/ml,1.5 μg/ml,2.0 μg/ml时,AP-1转录因子相对活性(106)为:5.12±0025、4.24±0.74、3.43±0.31、2.62±0.48、2.09±0.21.随着pHA-Hint1浓度增加,荧光素酶活性呈明显下降,当pHA-Hint1浓度达到1.5 μg及2.0 μg时,差异有统计学意义(F=72.009,P<0.05).结论 过表达Hint1基因可抑制HepG2细胞AP-1转录因子活性.     

Resveratrol inhibits fibrogenesis and induces apoptosis in keloid fibroblasts

Keloids are benign dermal fibrotic tumors arising during the wound healing process. The mechanisms of keloid formation and development still remain unknown, and no effective treatment is available. Resveratrol, a dietary compound, has anticancer properties and, from recent studies, it has been suggested that resveratrol may have an antifibrogenic effect on organs such as the liver and kidney. Based on this idea, we investigated its effect on the regulation of extracellular matrix expression, proliferation, and apoptosis of keloid fibroblasts. Type I collagen, α‐smooth muscle actin, and heat shock protein 47 expression decreased in resveratrol‐treated keloid fibroblasts in a dose‐dependent manner. In addition, resveratrol diminished transforming growth factor‐β1 production by keloid fibroblasts. We also demonstrated that it suppressed their proliferation and induced apoptosis of the fibroblasts. Conversely, resveratrol did not decrease type I collagen, α‐smooth muscle actin, and heat shock protein 47 mRNA expression in normal skin fibroblasts and barely suppressed cell proliferation. Our data indicate that resveratrol may have an antifibrogenic effect on keloid fibroblasts without any adversely effects on normal skin fibroblasts, suggesting the potential application of resveratrol for the treatment of keloids.     

抑癌基因MTS1对人瘢痕疙瘩成纤维细胞的生物学影响

目的：探讨多肿瘤抑制基因1（MTS1）对人瘢痕疙瘩成纤维细胞在生长增殖、DNA合成代谢及胶原合成量等方面的影响。方法：将外源性MTS1基因导入人瘢痕疙瘩成纤维细胞后，通过MTT法测定细胞的生长曲线，通过^3H-胸腺嘧啶核苷（^3H-TdR）及^3H-脯氨酸掺入的方法测定细胞的DNA合成量及胶原合成量，来分别比较转染前后细胞的生物学变化。结果：在转染MTS1后瘢痕疙瘩成纤维细胞的生长增殖受到明显的抑制，同时其DNA合成量及胶原合成量也明显降低，而转染空载体组及正常组细胞均未出现上述变化。结论：外源性多肿瘤抑制基因1(MTS1）能明显抑制人瘢痕疙瘩成纤维细胞在生长增殖、DNA合成及胶原合成等方面生物学功能，为瘢痕疙瘩的临床治疗提供了新的思路。     

阻断转化生长因子-β受体信号转导降低瘢痕疙瘩细胞转化生长因子-β1自分泌的研究

目的 研究瘢痕疙瘩成纤维细胞转化生长因子－β1（TGF－β1）的自分泌现象和阻断TGF－β受体信号转导对TGF－β1自分泌的调控。方法 组织块法体外培养瘢痕疾疙瘩成纤维细胞，加入重组人源（rh）TGF－β1（5mg/L）或含缩短型TGF－βⅡ型体的重组腺病毒（50pfu/cell），并用Northern印迹观察它们对TGF－β1极其Ⅰ、Ⅱ型受体的基因调控。结果 rhTGF－β1能上调TGF－β1（30％－50％）和Ⅰ型受体（30％－90％）的基因表达，但不影响Ⅱ型受体的基因表达。短型TGF－βⅡ型受体在瘢痕疙瘩细胞的过度表达减少细胞TGF－β1（50％－65％）和Ⅰ型受体（21％－55％）的基因表达，但不影响Ⅱ型受体的基因表达。结论 瘢痕疙瘩细胞存在TGF－β1的自分泌现象，而缩短型TGF－βⅡ型受体的过度表达可以通过阻断TGF－β的信号转导来降低TGF－β1的自分泌。     

p53 and apoptosis alterations in keloids and keloid fibroblasts

Keloids are the result of a dysregulated wound-healing process and are characterized by formation of excess scar tissue that proliferates beyond the boundaries of the inciting wound. In this study, we investigated the expression of key proteins involved in regulating apoptosis in keloids. Twenty archival paraffin-embedded keloid samples were randomly selected for an immunoperoxidase assay with antibodies against fas, p53, bcl-2, and bcl-x proteins using the target antigen-retrieval technique. Apoptosis was assessed in keloids and normal skin and in keloid and normal fibroblasts by the TdT-mediated dUTP nick-end labeling (tunel) assay on tissue sections, fibroblast cultures, and by flow cytometry for cell suspensions. We found that 18 of 20 keloids expressed p53 protein; bcl-2 was expressed by keloid fibroblasts in 19 of 20 keloids, and all specimens had prominent fas expression throughout the tissue. The distribution of these three antigens was regional within each lesion and followed a consistent pattern of p53 and bcl-2 expression colocalized to the hypercellular, peripheral areas of each keloid in a perinuclear pattern (p < .001). In contrast, an inverse distribution of fas expression was shown, with staining being more diffuse across the cell surfaces and limited to the central, more hypocellular regions in 16 of 17 keloids (p < .001). There was no specific staining pattern in these keloids with antihuman bcl-x. In vitro studies on cultured keloid fibroblasts (derived from six patients) revealed maintenance of the p53+, bcl-2+ phenotype up to passage 10. Neither neonatal nor normal adult skin fibroblasts expressed either antigen but could be induced to express p53 by exposure to adriamycin. Keloid lesions and keloid fibroblasts were found to have lower rates of apoptosis than normal controls. Keloid fibroblasts displayed enhanced apoptosis rates in response to hydrocortisone, γ interferon and hypoxia treatment as compared with normal adult fibroblasts. Focal dysregulation of p53 combined with upregulation of bcl-2 may help produce a combination of increased cell proliferation and decreased cell death in the younger hypercellular areas of the keloid. This phenotype is reversed in the older areas of the keloid and may prevent malignan degeneration, thus favoring normal apoptosis as evidenced by prominent fas expression.     

丹参对瘢痕疙瘩成纤维细胞基质金属蛋白酶-1的作用

目的探讨丹参对培养瘢痕疙瘩成纤维细胞基质金属蛋白酶-1(matrix metalloprotein-ase-1,MMP-1)蛋白生成和mRNA表达的影响。方法采用酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)和Northern杂交技术检测培养瘢痕疙瘩成纤维细胞MMP-1蛋白的合成分泌和mRNA的表达。结果培养瘢痕疙瘩成纤维细胞的MMP-1自发分泌量为(95±38)pg/ml;在培养基中丹参浓度分别为5mg/ml和10mg/ml条件下,MMP-1的浓度分别为(128±29)pg/ml(t=3.425,P<0.05)和(151±43)pg/ml(t=5.075,P<0.01)。在培养基中丹参浓度为10mg/ml时,培养瘢痕疙瘩成纤维细胞的MMP-1mRNA的表达被提高到(125±11)%(t=4.628,P<0.01)。结论丹参可提高培养瘢痕疙瘩成纤维细胞MMP-1蛋白的合成分泌和mR-NA的表达,这为丹参用于瘢痕疙瘩的临床治疗提供了理论依据。     

瘢痕疙瘩分泌性卷曲相关蛋白2与骨母细胞特异性因子2的相互作用

目的 验证分泌性卷曲相关蛋白2(SFRP2)与骨母细胞特异性因子2(OSF-2)的相互作用.方法 构建能在哺乳动物细胞中表达带人膜联蛋白(HA)标签的OSF-2融合蛋白重组载体pCMV-HA-OSF-2,经酶切鉴定确认后,与Myc-SFRP2重组真核表达载体pCMV-HA-SFRP2单独或共转染人HK293细胞,利用免疫共沉淀技术及蛋白质印迹法验证OSF-2与SFRP2的相互作用.结果 重组载体经酶切电泳后,显示近800 bp处的条带为酶切后的目的 片段SFRP2编码基因,近2500 bp处的条带为酶切后目的 基因OSF-2.表明pCMV-Myc-SFRP2和pCMV-HA-OSF-2均构建成功.HK293细胞单独转染pCMV-Myc-SFRP2或pCMV-HA-OSF-2后,未检测到HA-OSF-2的表达;当pCMV-Myc-SFRP2和pCMV-HA-OSF-2共转染后,经Myc抗体进行免疫沉淀可见HA-OSF-2表达.结论 HA-OSF-2的重组载体能在哺乳动物细胞中表达,HA-OSF-2与SFRP2间存在相互作用.     

P53蛋白对人瘢痕疙瘩成纤维细胞基质金属蛋白酶1、3表达的影响

目的 探讨P53蛋白对人瘢痕疙瘩成纤维细胞（keloid fibroblast，KFB）基质金属蛋白酶1（MMP1）、基质金属蛋白酶3（MMP3）表达的影响；明确在人瘢痕疙瘩成纤维细胞中P53蛋白与MMP1、MMP3之间的相互关系。方法 采用腺病毒介导法将p53基因转染至人瘢痕疙瘩成纤维细胞中。将来源于瘢痕疙瘩的成纤维细胞随机分成二组，实验组转染p53基因转染至成纤维细胞中，对照组为空载转染组。分别用间接免疫荧光法和Western印迹法检测P53蛋白的表达；ELISA法检测MMP1、MMP3的表达。结果 p53基因转染组细胞中P53蛋白表达明显高于空载转染组；而MMP1、MMP3蛋白表达明显低于空载转染组（P〈0．01）。结论 P53蛋白能够抑制人瘢痕疙瘩成纤维细胞MMP1、MMP3蛋白的表达。     

CCN2 enhances RANKL-induced osteoclast differentiation via direct binding to RANK and OPG

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a multi-potent factor for mesenchymal cells such as chondrocytes, osteoblasts, osteoclasts, and endothelial cells. CCN2 is also known as a modulator of other cytokines and receptors via direct molecular interactions with them. We screened additional factors binding to CCN2 and found receptor activator of NF-kappa B (RANK) as one of them. RANK is also known as TNF-related activation-induced cytokine (TRANCE) receptor, and its signaling plays a critical role in osteoclastogenesis. Notable affinity between CCN2 and RANK was confirmed by using surface plasmon resonance (SPR) analysis. In fact, CCN2 enhanced the RANK-mediated signaling, such as occurs in NF-kappa B, p38 and JNK pathways, in pre-osteoclastic RAW264.7 cells; whereas CCN2 had no influence on RANK–RANK ligand (RANKL) binding. Moreover, CCN2 also significantly bound to osteoprotegerin (OPG), which is a decoy receptor of RANKL. Of note, OPG markedly inhibited the binding between CCN2 and RANK; and CCN2 canceled the inhibitory effect of OPG on osteoclast differentiation. These findings suggest CCN2 as a candidate of the fourth factor in the RANK/RANKL/OPG system for osteoclastogenesis, which regulates OPG and RANK via direct interaction.     

独角莲根茎水提取物对瘢痕疙瘩成纤维细胞的抑制作用

目的 探讨独角莲根茎水提取物(AEoTGE)对瘢痕疙瘩成纤维细胞的抑制作用,测定其半数抑制浓度及凋亡率.方法 采用组织块法体外培养瘢痕疙瘩成纤维细胞,以透射电镜鉴定细胞超微结构.四甲基偶氮唑盐比色法(MTT法)检测不同浓度AEoTGE(3.125、6.250、12.500、25.000、50.000、100.000g/L)作用瘢痕疙瘩成纤维细胞24 h后的抑制率,计算其半数抑制浓度(IC50).采用5-乙炔基-2-脱氧尿苷(EdU)染色法测定瘢痕疙瘩成纤维细胞的增殖情况,流式细胞技术检测其凋亡率.结果 ①应用不同浓度(3.125～100.000 g/L) AEoTGE作用瘢痕疙瘩成纤维细胞24 h后,随AEoTGE浓度增加对细胞抑制作用增强,与对照组比较,细胞抑制率差异有统计学意义(P＜0.05);②瘢痕疙瘩成纤维细胞经AEoTGE作用后的半数抑制浓度(IC50)为(35±0.27) g/L;③EdU染色示35 g/L AEoTGE组与对照组比较差异有统计学意义(P＜0.05);④FITC-Annexin V/PI示AEoTGE组凋亡率为(72.07±0.70)％,对照组为(23.48±1.63)％(P＜0.05).结论 体外培养人瘢痕疙瘩成纤维细胞经AEoTGE作用24 h后,半数抑制浓度(IC50)为(35±0.27) g/L,并可明显诱导细胞凋亡,凋亡率为(72.07±0.70)％.     

干扰素α-2b对瘢痕疙瘩成纤维细胞凋亡及端粒酶活性的影响

目的 观察干扰素α-2b（IFNα-2b）对瘢痕疙瘩成纤维细胞生长增殖、端粒酶活性及凋亡的影响，探讨其在瘢痕疙瘩治疗中的作用机制。方法 进行成纤维细胞原代培养，细胞分别来自8例瘢痕疙瘩标本和8例正常皮肤标本。第3到4代的细胞用于实验。以干扰素α-2b作用于体外培养的瘢痕疙瘩和正常皮肤成纤维细胞，噻唑蓝（MTF）法检测成纤维细胞生长增殖情况，聚合酶链反应-酶联免疫吸附法（PCR-ELISA法）检测不同时间成纤维细胞端粒酶活性，应用流式细胞仪观察处理后成纤维细胞凋亡。结果 IFNα-2b对瘢痕疙瘩和正常皮肤成纤维细胞有生长抑制作用，可明显下调成纤维细胞端粒酶活性，和对照组比较差异有统计学意义（P〈0．05），并呈现出明显的时间-效应关系。体外培养的瘢痕疙瘩和正常皮肤成纤维细胞经10000U／ml IFNα-2b处理后，能诱导成纤维细胞凋亡发生，与对照组比较差异有统计学意义（P〈0．01），且具有明显的时间依赖性。结论 作为一个负性调节因子，IFNα-2b能抑制瘢痕疙瘩成纤维细胞的生长增殖并诱导成纤维细胞发生凋亡，降低成纤维细胞端粒酶活性是其作用机制之一。     

Blocking transforming growth factor- receptor signaling down-regulates transforming growth factor-β1 autoproduction in keloid fibroblasts

Objective: To study transforming growth factor-β1(TGF-β1) autoproduction in keloid fibroblasts and theregulation effect of blocking TGF-β intracellular signalingon rhTGF-β1 autoproduction.Methods: Keloid fibroblasts cultured in vitro weretreated with either rhTGF-β1 (5 ng/ml ) or recombinantadenovirus containing a truncated type II TGF-β receptorgene (50 pfu/cell ). Their effects of regulating geneexpression of TGF-β1 and its receptor I and II wereobserved with Northern blot.Results: rhTGF-β1 up-regulated the gene expressionof TGF-β1 and receptor I, but not receptor II. Over-expression of the truncated receptor II down-regulated thegene expression of TGF-β1 and its receptor I, but notreceptor II.Conclusions: TGF-β1 autoproduction was observed inkeloid fibroblasts. Over-expression of the truncated TGF-βreceptor H decreased TGF-β1 autoproduction via blockingTGF-β receptor signaling.