肝再生刺激因子、表皮生长因子对肝细胞的作用

目的：观察肝再生刺激因子(HSS)及表皮生长因子（EGF）对肝细胞增殖再生作用合适剂量及其联合效应。并推测HSS作用时相。方法：以^3HTdR掺入法检测细胞DNA增殖；体外原代大鼠肝细胞培养不同时间加入HSS，MTT法检测细胞生长状况。结果与结论：1．HSS(≤100μg／ml)和EGF(≤100ng／ml)对肝衍生细胞均有显著刺激作用(P<0．01)，并存在剂量关系，但剂量过大，刺激作用反而下降。2．HSS、EGF存在协同作用，原代肝细胞、肝癌细胞株(SMMC．7721、BEL．7402)体外培养48h后，经(^3H)TdR掺入法检测cpm值分别为各自对照组的5．94，298和3．36倍。3．体外培养原代大鼠肝细胞，于贴壁后0h，20h加入HSS其刺激作用较40h组显著，提示HSS可能主要作用于肝细胞再生G1／S期。     

苯丙酸诺龙对大鼠成纤维细胞增殖作用的研究

目的　利用培养大鼠成纤维细胞 (fibroblast,FB) ,研究蛋白同化激素苯丙酸诺龙 (nandrolone phenyl-propionate,NP)对创伤后 FB的增殖作用。　方法　 Wistar大鼠致伤后取材行 FB原代培养 ,实验组培养液按 NP用药剂量 0 .5～ 15 .0μg/ ml分为 NP1 ～ NP5共 5组 ,对照组为含 5 %胎牛血清的培养液 ,7d后 MTT法测定不同剂量 NP对FB存活力的影响 ,选作用最大的一组继续实验 ,流式细胞术测定 FB增殖指数。　结果　各 NP组 FB存活值均高于同时间点对照组 ,NP4 组第 1～ 7天差异均有统计学意义 (P<0 .0 5 ) ,NP5组从第 1～ 6天与对照组相比差异有统计学意义 (P<0 .0 5 ) ,而第 7天则无差异 ;流式细胞术检测 FB增殖指数显示 NP组均高于同期对照组 ,差异有统计学意义 (P<0 .0 1)。　结论　蛋白同化激素 NP能够促进 FB的复制增殖 ,且这种增殖作用有剂量依赖性。     

肝再生刺激因子、表皮生长因子对肝细胞的作用

目的：观察肝再生刺激因子(HSS）及表皮生长因子（EGF)对肝细胞增殖再生作用合适剂量及其联合效应，并推测HSS作用时相。方法：以~3HTdR掺入法检测细胞DNA增殖；体外原代大鼠肝细胞培养不同时间加入HSS,MTT法检测细胞生长状况。结果与结论：1.HSS（≤100μg／ml)和EGF（≤100ng／ml）对肝衍生细胞均有显著刺激作用（P＜0.01）,并存在剂量关系，但剂量过大，刺激作用反而下降。2.HSS、EGF存在协同作用，原代肝细胞、肝癌细胞株（SMMC-7721、BEL-7402）体外培养48h后，经(~3H）TdR掺入法检测cpm值分别为各自对照组的5.94、2.98和3.36倍。3.体外培养原代大鼠肝细胞，于贴壁后Oh.20h加入HSS其刺激作用较40h组显著.提示HSS可能主要作用于肝细胞再生G1/S期。     

毒蕈碱受体亚型介导逼尿肌细胞收缩与IP3关系的实验研究

目的　探讨信使分子IP3 在毒蕈碱受体亚型M3 R介导逼尿肌细胞收缩中的作用。　方法　MR非选择性激动剂 (carbachol)、拮抗剂 (atropine)及M2 R拮抗剂 (methoctramine)、M3 R拮抗剂 (4 DAMP)刺激原代培养人逼尿肌细胞 ,通过 [3 H]掺入法 ,检测磷脂酰肌醇 (PI)代谢产物 [3 H] IP含量。　结果　 [3 H] IP含量随carbachol刺激浓度增加而增加 ;10 -9、10 -8、10 -7、10 -6、10 -5、10 -4mmol/L的 4 DAMP抑制carbachol后 ,[3 H] IP含量分别为 392 6 .5 7± 2 73.2 9、2 780 .5 2± 2 11.0 9、2 4 36 .84± 15 3.6 2、1973.2 2± 16 4 .71、1372 .38± 14 1.35及 110 7.98± 92 0 .4 5cpm ,相同浓度的at ropine作用后 ,[3 H] IP含量分别为 36 0 2 .6 9± 2 80 .17、2 891.31± 2 0 7.4 5、1983.97± 14 5 .74、12 6 9.5 7± 10 5 .31、110 6 .37± 75 .2 3、92 7.5 0± 77.36cpm ;而相同浓度的methoctramine作用后 ,[3 H] IP含量分别为 4 4 6 2 .74± 36 0 .6 9、3938.6 1± 32 7.13、3315 .4 5± 2 70 .36、30 6 3.19± 2 4 6 .79、2 92 7.37± 2 2 6 .4 5及 2 836 .5 5± 2 4 1.6 3cpm ,两者之间差异有非常显著性意义 (P <0 .0 1) ,表明 4 DAMP和atropine能显著抑制carbachol诱导的代谢反应 ,而methoctram     

趋化因子RANTES对人外周血单个核细胞的免疫活化作用

目的　探讨趋化因子RANTES刺激对人外周血单个核细胞 (PMNC)的免疫活化作用及其内在机制。方法　分离人外周血 ,应用不同终浓度的人重组RANTES(rhRANTES)以及抗CD3单克隆抗体 (抗CD3mAb)进行体外刺激 ,并对增殖反应显著者给予吡咯啉烷二甲基硫脲 (PDTC)或CTLA4Ig进行干预。采用3 H 胸腺嘧啶核苷掺入法检测PMNC增殖程度 ,流式细胞仪检测淋巴细胞表型的变化。结果　rhRANTES终浓度为 10 0ng/ml和 5 0 0 0ng/ml时 ,诱导PMNC增殖反应出现两次峰值 ;rhRANTES(10 0ng/ml)刺激产生的增殖反应显著高于抗CD3mAb(5 0ng/ml)的刺激 (P <0 .0 5 ) ,但两者无拮抗或协同作用 ;PDTC和CTLA4Ig对rhRANTES(10 0ng/ml)诱导的增殖反应均能产生抑制作用 ,并呈现剂量依赖性 ;rhRANTES刺激后 ,淋巴细胞CD2 5表达率显著增加 (P <0 .0 5 ) ,而人RANTES的受体CCR5表达率显著降低 (P <0 .0 5 ) ,CD2 8表达率及CD4 /CD8比值无明显改变(P >0 .0 5 )。结论　RANTES趋化信号具有不依赖于CD3信号的独特的诱导PMNC免疫活化的功能。     

粉防己碱对瘢痕成纤维细胞DNA和胶原合成的影响

目的探讨粉防己碱对瘢痕成纤维细胞DNA和胶原合成的影响.方法以体外培养的人瘢痕成纤维细胞为实验模型,将粉防己碱(5～80mg/L)加入细胞培养液中进行干预并与对照组比较,用3H-TdR掺入法和3H-脯氨酸掺入法分别测定各组瘢痕成纤维细胞3H-TdR掺入值与3H-脯氨酸掺入值,以反映其DNA和胶原合成水平的变化.结果粉防己碱预处理浓度由5mg/L升至80mg/L时(1)瘢痕成纤维细胞3H-TdR掺入值(min-1)由对照组的1740±165分别降至1162±226、412±82,抑制率达76.32%,组间差异有非常显著意义(P＜0.01);(2)瘢痕成纤维细胞3H-脯氨酸掺入值(min-1)由对照组的1126±193分别降至535±141、341±89,抑制率达69.71%,组间差异有非常显著意义(P＜0.01).结论粉防己碱对体外培养的瘢痕成纤维细胞DNA和胶原合成具有抑制作用,呈剂量依赖关系,具有防治增生性瘢痕的应用前景.     

前列腺癌患者血清胰岛素样生长因子-1检测的临床意义

目的　探讨血清胰岛素样生长因子 1(IGF Ⅰ )与前列腺癌 (PCa)发生发展的关系。　方法　采用免疫放射分析法 (IRMA)检测 3 7例PCa、3 5例良性前列腺增生 (BPH)患者和 2 0例健康人血清IGF Ⅰ ,比较各期PCa血清IGF Ⅰ水平 ,并对 8例行根治性前列腺全切术后患者手术前后IGF Ⅰ水平随访。　结果　PCa组血清IGF Ⅰ ( 3 2 5 .6± 10 0 .8)ng/ml,明显高于BPH组 ( 2 0 1.6± 5 3 .8)ng/ml和健康组 ( 179.0± 5 7.2 )ng/ml,差异有显著性意义 (P <0 .0 1) ;BPH组与健康组比较差异无显著性意义 (P >0 .0 5 ) ;8例PCa患者术前IGF I( 3 15 .8± 87.0 )ng/ml,术后 ( 2 2 4.8± 88.4)ng/ml,差异有显著性意义 (P <0 .0 5 ) ;PCa患者各期血清IGF Ⅰ比较差异无显著性意义 (P >0 .0 5 )。　结论　IGF Ⅰ有可能作为临床上一个新的PCa检测指标预测高危人群 ,进行早期诊断     

骨形成蛋白对骨骼肌卫星细胞增殖与胶原蛋白合成的影响

目的　探讨骨形成蛋白 (BMP)对骨骼肌卫星细胞增殖与胶原蛋白合成的影响。　方法　体外获取与培养 Wistar大鼠骨骼肌卫星细胞 ,分别用含 BMP浓度为 0、50、1 0 0、50 0和 1 0 0 0 ng/ml的诱导培养基培养 72小时。通过 MTT法测定细胞的增殖 ,光镜观察细胞融合率 ,3 H-脯氨酸掺入法测定细胞胶原蛋白合成量。　结果　 BMP可促进骨骼肌卫星细胞的增殖 ,降低其细胞融合率 ,同时增加胶原蛋白的合成量。这种作用在 BMP浓度为 50 0 ng/ml即可表现出来 ,并随着浓度的增加越明显。　结论　 BMP可促进骨骼肌卫星细胞的增殖 ,抑制成肌表型促进向成骨细胞分化     

改良法制备富血小板血浆促进体外原代培养成骨细胞增殖的实验研究

目的　探讨富血小板血浆 (platelet- rich plasma,PRP)来源的富生长因子血清 (serum rich in growthfactors,SRGF)对体外原代培养的大鼠和人成骨细胞增殖的影响。　方法　改良法分别制备大鼠和人 SRGF和贫生长因子血清 (serum poor in growth factors,SPGF) ,EL ISA法测定人 SRGF和 SPGF中转化生长因子 β1 (transforminggrowth factorβ1 ,TGF- β1 )和血小板源性生长因子 AB(platelet- derived growth factor AB,PDGF- AB)浓度 ;原代培养并传代大鼠和人成骨细胞 ,取第 3代大鼠成骨细胞和第 4代人成骨细胞各自分为 3组 ,分别加入 5 %相应种属的 SRGF、SPGF和无血清细胞培养基 ,2 4、4 8、72和 96 h用 MTT法检测成骨细胞的增殖。　结果　 EL ISA法测定人 SRGF中TGF-β1 浓度为 30 7.6 7± 35 .5 7ng/ml,PDGF- AB浓度为 5 2 .76± 7.89ng/ml;大鼠和人成骨细胞在相应种属的 SRGF作用下 ,细胞增殖迅速 ,细胞数量维持在较高水平 ,其吸光度 (A)值在各时间点与 SPGF组相应数值比较 ,发现大鼠成骨细胞在 4 8和 96 h细胞增殖差异有统计学意义 (P<0 .0 5 ) ,而人成骨细胞增殖差异在 4 8、72和 96 h均有统计学意义 (P<0 .0 5 ) ;两种成骨细胞在无血清培养基中增殖基本停滞 ,与同时间点 SRGF组比较 ,差异均有统计学     

不同途径制备的中药复方"肝纤方"含药血清对大鼠肝星状细胞的不同影响

目的观察通过两种不同途径制备的中药复方"肝纤方"含药血清对大鼠肝星状细胞(HSC)在各个水平上的作用,以明确其抗肝纤维化的分子学机制和研究有关中药抗肝纤维化的血清药理学实验方法.方法采用血清药理学方法制备两种含药血清-门静脉血清和外周静脉(体静脉)血清,作用于离体培养的大鼠传2代HSC,以氚标胸腺嘧啶([3H]TdR)和氚标脯氨酸([3H]Pro)核素掺入试验、流式细胞仪、逆转录-聚合酶链反应(RT-PCR)等方法检测HSC增殖及其胶原合成量、细胞增殖周期、Ⅰ型前胶原mRNA表达水平等指标.结果门静脉血清组[3H]TdR和[3H]Pro的掺入量分别为 (569.2± 19.8)cpm/孔和 (907.3± 10.3)cpm/孔,与对照组相比均减少 (P< 0.05),外周静脉血清组的为 (592.8± 10.9)cpm/孔和 1 025.2± 54.0cpm/孔,与对照组相比前者减少 (P< 0.05)而后者差异无显著性;门静脉含药血清提高HSC的G0/G1期的比例和降低G2/M及S期的比例 (P< 0.05),而外周静脉血清对各细胞周期无影响 (P> 0.05);I型前胶原mRNA的PC-mRNA/GAPDH-mRNA比值为门静脉血清组 0.42± 0.09和外周静脉血清组 0.72± 0.05,与对照组相比均降低,而门静脉血清组则更低 (P< 0.05).结论肝纤方抗肝纤维化的部分作用机制可能是通过阻断G0/G1期向S期的转变来抑制HSC增殖、通过下调Ⅰ型前胶原mRNA的表达水平来减少胶原的合成;由门静脉和外周静脉采得的含药血清在血清药理学实验中有不同的药理作用.     

脂多糖对皮肤成纤维细胞生物学特性的影响及与创面愈合的关系

目的探讨脂多糖（lipopolysaccharide，LPS）对皮肤成纤维细胞增殖和胶原合成的影响，以研究细菌内毒素与皮肤创面愈合的关系。方法取正常皮肤按黄勇等方法行成纤维细胞培养后，分为1个对照组及6个实验组。实验组分别与终浓度为0．005、0．010、0．050、0．100、0．500和1．000μg／ml大肠杆菌LPS（E．coli055：B5）培养，对照组为DMEM培养。分别通过MTT比色法及细胞计数法观察各组1～9d吸光度（A）值和细胞数量的变化；于LPS加入后7d细胞处于融合状态时，通过^3H-脯氨酸掺入、胃蛋白酶消化法检测细胞胶原合成量的变化，并绘制细胞生长曲线。结果与对照组比较，0．005～0．500μg／ml组A值增加，于5～9d差异有统计学意义（P〈0．05）；1．000μg／ml组A值降低，于3～9d差异有统计学意义（P〈0．05）。与对照组比较，0．005～0．500μg／ml组促进细胞胶原合成，且0．100μg／ml组作用达高峰（P〈0．05），1．000μg／ml LPS抑制细胞胶原合成，差异有统计学意义（P〈0．05）。与对照组比较，0．005～0．500μg／ml组细胞数量明显增加，分别于3～6d、1～6d、3～6d、2～6d及3～6d时比较，差异均有统计学意义（P〈0．05）；1．000μg／ml组细胞数量明显降低，于2～9d差异有统计学意义（P〈0．05）。结论在一定浓度范围内LPS促进成纤维细胞增殖和胶原蛋白合成，但过高浓度LPS则产生抑制效应。提示一定量的细菌内毒素可能有利于皮肤创面愈合，当内毒素过多时将对创面愈合产生负面作用。     

硒化壳聚糖促创面愈合作用的初步研究

目的：通过研究硒化壳聚糖对体外培养皮肤成纤维细胞增殖的影响来评判其对创面愈合的作用。方法：用不同剂量（25mg/L、50mg/L、100mg/L、200mg/L、400mg/L）硒化壳聚糖作用于成纤维细胞,光镜下观察药物对细胞形态的影响;MTT法、3H-TdR、3H-脯氨酸掺入法、细胞生长动力学研究用于检测药物对细胞增殖及胶原合成影响;LDH漏出率用于检测药物对细胞有无损伤;并以等体积细胞培养液处理为对照组。结果：各浓度药物处理细胞后,细胞形态未见明显改变,均可使细胞吸光度值增加,细胞倍增时间缩短,促进细胞对3H-TdR和3H-脯氨酸的掺入（P〈0.05,P〈0.01）,降低LDH漏出率（P〈0.05,P〈0.01）。结论：硒化壳聚糖可促进体外培养皮肤成纤维细胞增殖和胶原合成,进而促进创面愈合。     

The wound environment as a regulator of fibroblast phenotype

Fibroblasts are fundamental to successful wound healing. We hypothesized that the induction and regulation of various fibroblast functions (proliferation, collagen synthesis, and remodeling) are determined by the wound environment. We examined the effect of wound fluid (WF), as a reflection of the wound environment, on the phenotypic expression of normal dermal (NF) and wound-harvested fibroblasts (WHF). WF and WHF were obtained from implanted polyvinyl alcohol sponges in 10-day-old wounds. NF and WHF were used between one and three passages. Proliferative function was assayed in a microculture system using serum stimulation (n = 12). The proliferative response of both NF and WHF to serum was significantly reduced by the addition of 20% WF (17,261 +/- 1231 cpm vs 2704 +/- 1215 cpm for NF, P less than 0.05; and 15,391 +/- 3735 cpm vs 1701 +/- 816 cpm for WHF, P less than 0.05 in serum and WF, respectively). Total protein synthesis (measured by [3H]proline incorporation) was equal in both fibroblast types; however, the relative collagen synthesis (collagenase-digestible fraction) was markedly different (2.2 +/- 0.9% for NF vs 11.4 +/- 2% for WHF, P less than 0.05). Addition of WF markedly enhanced NF collagen synthesis to 9.4 +/- 2%, but had no effect on WHF. Mechanical and remodeling functions were assayed using fibroblast-populated collagen lattices. In serum, WHF contracted the lattices faster than NF (499 +/- 14 mm2 vs 770 +/- 30 mm2 at 24 hr, P less than 0.05, and 301 +/- 18 mm2 vs 540 +/- 21 mm2 at 72 hr, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

血管紧张素Ⅱ对转化生长因子β诱导的人皮肤成纤维细胞增殖的影响

目的 观察血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)对人皮肤成纤维细胞增殖及对转化生长因子β(transforming growth factor β,TGF-β)诱导的成纤维细胞增殖作用的影响,并初步探讨可能的信号机制. 方法 取自愿捐献的正常皮肤组织标本,采用胶原酶法行成纤维细胞培养.取第4～5代细胞,按实验设计分别加入不同浓度的Ang Ⅱ(1×10-10、1×10-9、1×10-8、1×10-7 mol/L)、 TGF-β(0.1、1.0、10.0 ng/ml)、1×10-10 mol/L Ang Ⅱ+ 0.1 ng/ml TGF-β共8组;对照组仅加入等量DMEM.以3H-TdR掺入法测定细胞增殖,Western blot法检测抗细胞外信号调节激酶(extracellular signal-regulated kinases,ERK)活性变化,观察不同浓度的Ang Ⅱ或/和TGF-β对培养的成纤维细胞3H-TdR掺入量和ERK磷酸化的影响. 结果 与对照组比较Ang Ⅱ(1×10-9、1×10-8、1×10-7 mol/L)或TGF-β(1.0、10.0 ng/ml)均能促进成纤维细胞的3H-TdR掺入量(P＜0.05);1×10-10 mol/L Ang Ⅱ或0.1 ng/ml TGF-β单独使用不影响成纤维细胞的3H-TdR掺入量,但二者联合使用提高了成纤维细胞的3H-TdR掺入量(P＜0.05).1×10-7 mol/L Ang Ⅱ、10.0 ng/ml TGF-β增加皮肤成纤维细胞的ERK磷酸化,与对照组比较差异有统计学意义(P＜0.01).1×10-10 mol/L Ang Ⅱ或0.1 ng/ml TGF-β单独刺激成纤维细胞并未影响ERK磷酸化,而二者联合使用增加ERK磷酸化,与对照组比较差异有统计学意义(P＜0.05).应用抗ERK抗体显示各组ERK含量一致. 结论 Ang Ⅱ不仅能作为促有丝分裂素直接促进成纤维细胞分裂增殖,同时也可作为调节因子促进TGF-β的促增殖作用.Ang Ⅱ和TGF-β通过各自特异性受体共同作用于ERK,使磷酸化增加是其产生协同作用可能的机制之一.     

单核巨噬细胞集落刺激因子对人皮肤成纤维细胞增殖及胶原合成的影响

目的：研究单核巨噬细胞集落刺激因子（granulocyte/macrophage colony-stimulating factor,GMCSF）对人皮肤成纤维细胞增殖及胶原合成的影响,探讨其促进创面愈合的机制并为临床准确定量应用该因子促进创面愈合提供理论依据。方法：分别应用浓度为0、25、50、75、100、150ng/mlGMCSF培养液孵育离体培养的人皮肤成纤维细胞,作用24h后四甲基偶氮唑盐比色法（MTT）检测细胞的活性。选择最适浓度GMCSF干预细胞,用流式细胞仪检测成纤维细胞的周期变化;应用ELISA检测上清液中Ⅰ、Ⅲ型胶原合成情况。结果：GMCSF在浓度为25～100ng/ml之间对人皮肤成纤维细胞有明显的促增殖作用,其中以50/ml最为显著;成纤维细胞在最适浓度GMCSF干预24h后,细胞大多处于DNA合成前期和合成期;与空白组比较,GMCSF组Ⅰ、Ⅲ型胶原分泌明显增加（P〈0.01）,且Ⅰ、Ⅲ型胶原比值下降（P〈0.01）。结论：GMCSF在浓度为25～100ng/ml对人皮肤成纤维细胞有明显的促增殖作用,且能刺激成纤维细胞合成大量Ⅰ、Ⅲ型胶原,这可能是其加速创面愈合的机制之一。     

肉芽组织中NK1受体表达与创面愈合关系的实验研究

目的：研究创面愈合过程中NK1受体（NK1R）在肉芽组织成纤维细胞上的表达及其与创面愈合之间的关系。方法：新生Sprague-Dawley仔鼠20只，随机分为两组，每组10只。其中一组注射辣椒素将皮肤内感觉神经纤维破坏，制成“失感觉神经支配模型“，另一组作对照。两组动物均在腹部制作圆形皮肤缺损，并各选5只分别在一定时间点沿创面周缘及底部取材，进行NK1受体的免疫组织荧光染色及HE染色，观察其相应的时间、空间规律；另外5只不取材，而是在一定时间点测量未愈合的残留创面面积，并进行比较。结果：创面愈合过程中正常大鼠与去感觉神经支配大鼠在肉芽组织成纤维细胞均有NK1R表达；损伤后NK1R表达阳性的成纤维细胞形态变肥厚，胞体增大，车增殖旺盛表现；NK1R在感觉神经支配的大鼠成纤维细胞上的表达较正常对照升高；去感觉神经的大鼠与正常大鼠相比，创面愈合速度较慢。结论：大鼠皮肤NK1R表达与创面愈合之间关系密切，由感觉神经末梢所释放的P物质等神经递质可能是调节促进创面愈合的重要因子之一。     

Interleukin-10 suppresses proliferation and remodeling of extracellular matrix of cultured human skin fibroblasts

When we previously examined the participation of local expression of interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNFalpha) in wound healing of an intestinal anastomosis under septic conditions in mice, we found that IL-10 and TNFalpha expressions were markedly enhanced around the anastomosis and that wound healing was impaired in this animal model. The purpose of the present study was to investigate the combined effect of IL-10 on proliferation and remodeling of the extracellular matrix (ECM) of cultured human skin fibroblasts. Human skin fibroblasts were cultured for 48 h with IL-10 and/or TNFalpha at various concentrations, then the proliferation rates were determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The concentration of transforming growth factor-beta1 (TGFbeta1) in cell culture supernatants was measured by enzyme-linked immunosorbent assay, and type I collagen protein and matrix metalloproteinase-I (MMP-I) were detected by indirect immunofluorescence in cultured cells incubated for 48 h with 10 ng/ml of IL-10 and/or 10 ng/ml of TNFalpha. IL-10 itself had no effect on fibroblast proliferation, but reduced TNFalpha-induced fibroblast proliferation. The concentration of TGFbeta1 in cell culture supernatants was significantly lower in the presence of TNFalpha and IL-10 than in the presence of TNFalpha alone. Immunolabeling of fibroblasts for type I collagen protein was decreased in cells incubated with IL-10 and/or TNFalpha compared to controls. MMP-I immunolabeling was increased in cells incubated with IL-10, IL-10 and TNFalpha compared to control and cells incubated with TNFalpha. It is suggested that IL-10 is an inhibitory factor for the remodeling of the ECM during wound healing.     

Cultured pressure ulcer fibroblasts show replicative senescence with elevated production of plasmin, plasminogen activator inhibitor-1, and transforming growth factor-β1

In a 16-patient study, cultured fibroblast populations from normal skin were able to replicate an average of 14.8 +/- 2.2 times before becoming senescent, while fibroblast populations from the ulcer bed reached the end of their replicative life span after 7.2 +/- 1.9 population doublings (p= 0.001). Fibroblast populations from 10 of 16 pressure ulcers became senescent after fewer than five population doublings, whereas when populations of fibroblasts from adjacent normal skin were studied, only 2 of 16 became senescent within this same time period. In addition, only an occasional fibroblast from normal skin stained positively for senescence-associated beta-galactosidase compared to approximately 50% of equally aged ulcer bed fibroblasts (p = 0.0060). Senescent ulcer bed fibroblasts secreted significantly more plasmin than early passage ulcer bed fibroblasts (p= 0.0237), nearly six times as much plasmin as early passage normal skin fibroblasts (p < 0.0001), three and a half times the level of normal skin fibroblasts of the same age (11.52 +/- 4.58 microg/mg protein; p= 0.0003), and more than one and a half times the level of senescent normal skin fibroblasts (p= 0.0525). Senescent pressure ulcer fibroblasts generated significantly more plasminogen activator inhibitor-1 (1179.27 +/- 25.37 ng/mg protein) than normal skin fibroblasts of the same age (132.16 +/- 16.20 ng/mg protein; p = 0.0357). Also, senescent ulcer bed fibroblasts produced higher levels of transforming growth factor-beta1, but these were not significantly different from senescent normal skin fibroblasts. Although senescent ulcer fibroblasts produce elevated levels of plasminogen activator inhibitor-1 and transforming growth factor-beta1, the ratio of these factors to plasmin levels suggests that this may have little influence on extracellular matrix synthesis or maintenance in the chronic wound. These data show that cultured fibroblasts from most patient pressure ulcers profile a wound environment that is associated with an increasing population of senescent fibroblasts; however, factors within the chronic wound environment that promote cellular senescence remain unclear. We have proposed that a prolonged inflammatory response may be a contributing factor to the chronic wound condition.     

Interleukin-8 levels and activity in delayed-healing human thermal wounds

There are numerous causes for slow or delayed wound healing. Because slowly healing wounds are often inflamed, we quantitated the inflammatory chemokine, interleukin-8, produced by slowly healing human burn wounds and compared this to interleukin-8 from healed wounds and normal intact skin. Interleukin-8 levels were increased significantly in unhealed wounds (19.7 ng/ml) compared to healed wounds (7.7 ng/ml) or normal skin (5.7 ng/ml). Interleukin-8 in these ranges was added to adult human keratinocytes and fibroblasts. Interleukin-8 significantly decreased keratinocyte replication but had no effect on fibroblast replication. The rate or final degree of fibroblast populated collagen lattice contraction was inhibited at interleukin-8 concentrations between 10 and 30 ng/ml, but not altered at concentrations below 10 ng/ml and above 100 ng/ml. The concurrent application of indomethacin at 10 microg/ml reversed this interleukin-8 induced inhibition. Interleukin-8 inhibited myosin ATPase activity, apparently by reducing the phosphorylation of nonmuscle myosin light chain. We conclude that elevated levels of interleukin-8 may be found during delayed healing, and these elevated interleukin-8 levels may directly contribute to retarded wound closure.     

Heparin-like inhibitory activity to fibroblast growth factor-2 in wound fluids of patients with chronic skin ulcers and its modulation during wound healing

Fibroblast growth factors are potent mitogens and angiogenic factors which play a critical role in wound healing. Fibroblast growth factors require heparan sulfates as cofactors in order to activate their cognate receptors and exert their cellular and biological effects. Heparan sulfates were extracted from wound fluids of 5 patients with chronic diabetic foot ulcers or chronic venous stasis ulcers and tested for their capacity to modulate fibroblast growth factor-receptor binding, during the course of the ulcers' resolution, until complete healing (3-8 months). Total heparan sulfates concentration measured as iduronic acid equivalents, decreased in wound fluids from 1.1 +/-0.3 microg/ml to 0.26 +/-0.1 microg/ml as wound healing progressed. These heparan sulfates exhibited a predominant inhibitory effect on fibroblast growth factor-2 binding to fibroblast growth factor receptor-1, when tested in cells deficient in cell surface heparan sulfates. During wound healing, there was a marked decrease in the relative inhibitory activity of the extracted heparan sulfates on fibroblast growth factor-2-receptor binding. Heparan sulfates extracted from chronic skin ulcers of different etiologies such as diabetic foot or chronic venous stasis ulcers showed the same pattern of alternating balance in heparan sulfates mediated activity. The presence of fibroblast growth factor inhibitory factors which possess heparin-like activity in fluids of chronic skin ulcers and their ability to modulate fibroblast growth factor-receptor activity throughout the process of wound healing, may significantly contribute to the mechanism of chronicity. Treatments to counter this inhibition may offer new possibilities for healing chronic wounds.