特异性整合素连接激酶抑制剂QLT0267对大鼠烧伤创面愈合影响的初步研究

目的 观察局部注射特异性整合素连接激酶（ILK）抑制剂QLT0267对SD大鼠深Ⅱ度烧伤创面愈合影响,初步探讨ILK在创面愈合过程中的作用及机制.方法 选取45只雄性SD大鼠,采用恒温恒压烫伤仪在其背部皮肤制备深Ⅱ度烧伤创面,并随机分成实验组、对照组及二甲基亚砜（DMSO）组,每组15只.实验组创面每天注射浓度100 μM QLT0267液100 μL;对照组创面每天注射0.9％氯化钠溶液100 μL;DMSO组创面每天注射0.3％ DMSO液100 μL.测量各组大鼠创面愈合时间和愈合率;伤后14 d取材,各组随机选择5只大鼠,用SP法检测ILK、AKT、PAKT、α-SMA在创面组织中的表达,Western Blot检测各组ILK、AKT、PAKT蛋白含量;用Masson改良法检测创面胶原.结果 实验组大鼠创面平均愈合时间为（21.1±0.6）d,比对照组（17.1±0.6）d和DMSO组（17.7 ±0.6）d长;实验组创面愈合率也低于对照组和DMSO组,比较差异有统计学意义（P＜0.05）.Western Blot检测结果显示：各组ILK、AKT蛋白表达差异无统计学意义（P＞0.05）,实验组PAKT蛋白显著低于对照组和DMSO组.SP法结果各组ILK、AKT表达差异无统计学意义（P＞0.05）,实验组ILK活性被抑制,PAKT（0.406±0.008）表达较对照组（0.901 ±0.013）、DMSO组（0.966±0.011）降低,比较差异有统计学意义（F=11.27,P=0.04）;实验组α-SMA表达（0.201±0.003）较对照组（0.339 ±0.006）、DMSO组（0.351 ±0.005）也降低,比较差异有统计学意义（F=52.86,P=0.001）;实验组细胞外基质胶原排列较对照组、DMSO组明显稀疏、紊乱.结论 ILK活性被抑制时延迟大鼠皮肤创面愈合.ILK特异性抑制剂QLT0267通过抑制ILK活性PAKT合成减少,可能影响其下游信号的传导,导致肌成纤维细胞生成减少、胶原合成减少,影响创面愈合.     

整合素连接激酶和β1整合素在大鼠烫伤创面愈合过程中表达的实验研究

目的探讨大鼠烫伤创面愈合过程中整合素连接激酶（ILK）和β1整合素的表达以及在创面愈合中的作用。方法健康雌性SD大鼠18只,随机选取其中15只作为实验组,建立深Ⅱ度烫伤模型,另外3只为对照组,不做处理。实验组分别于烫伤模型建立后第1、5、10、15、20天随机选取3只大鼠,切取背部两侧全层创面,运用免疫组织化学的方法检测创面中ILK、β1整合素的表达。对照组取背部正常皮肤作为对照。结果实验组烫伤后第5、10、15天大鼠烫伤创面中ILK的表达明显高于对照组和实验组烫伤后第20天（P〈0.05）;创面中β1整合素的表达在烫伤后第5天明显高于对照组（P〈0.05）,实验组烫伤后第10、15天则明显高于对照组和实验组烫伤后第20天（P〈0.05）。结论ILK、β1整合素在大鼠创面愈合过程中表达明显增加,ILK可能通过参与整合素信号通路,在调节创面细胞的增生以及细胞与ECM相互作用中扮演着重要的角色。     

Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis

Secreted protein, acidic and rich in cysteine (SPARC), which functions in tissue remodeling, has been reported to be expressed by myofibroblasts in liver cirrhosis and hepatocellular carcinoma. This study aimed to reveal its expression in chronic hepatitis. Immuno-light and electron microscopy demonstrated that SPARC was expressed by nerve fibers and hepatic stellate cells (HSCs) in the liver parenchyma and myofibroblasts in the fibrous septa. Reaction products were localized in the rough endoplasmic reticulum and nuclear envelope. Serial section analysis demonstrated that SPARC, platelet-derived growth factor receptor-beta, and alpha-smooth muscle actin were co-expressed by HSCs. Quantitative analysis demonstrated that, while SPARC-positive HSCs were sparse in control livers, they significantly increased in number in the livers with chronic hepatitis. There were, however, no significant differences in number among the grades of activity, the stages of fibrosis, or etiology (virus-infected or autoimmune, hepatitis B virus or hepatitis C virus). In liver cirrhosis, however, they significantly decreased in number. The present results indicate that SPARC is expressed by activated HSCs in chronic hepatitis, suggesting the involvement of SPARC in hepatic fibrogenesis after chronic injuries.     

The role of mast cells in wound healing

Mast cells are known to participate in three phases of wound healing: the inflammatory reaction, angiogenesis and extracellular-matrix reabsorption. The inflammatory reaction is mediated by released histamine and arachidonic acid metabolites. Compound 48/80 and disodium-cromoglycate are both able to increase skin breaking strength shortly after wounding. Under light and electron microscopy we found that small, granule-poor, irregular mast cells (MLMC) accumulate in the wound. This suggests that the small MLMC (mucosal-like mast cells) migrate into the skin during wound healing, and that both CTMC (connective-tissue mast cells) and MLMC are involved in tissue repair. Moreover, there is some evidence that mast cells participate in angiogenesis, since heparin is able to stimulate endothelial-cell migration and proliferation in vitro, and protamine to inhibit these processes and also angiogenesis in vivo. When the effect of protamine on wound breaking strength was examined, we encountered a decrease which was not prevented by heparin administration. Further studies are needed to demonstrate that protamine is specifically involved in inhibiting heparin-mediated angiogenesis in wounded tissue. Finally, mast cells may play a role in the extracellular matrix remodelling, on the basis of in-vitro experiments (but there are still no in-vivo data).     

Actin filaments in normal dermis and during wound healing

During wound healing, it has been suggested, modified fibroblasts rich in actin filaments are responsible for wound contraction. With the use of specific fluorescent probe (NBD-phallacidin), the distribution of actin filaments are compared in normal dermis and in several wound contraction models, including open and burn wounds and full and thin-thickness skin autografts. Fibroblasts of normal dermis are slightly stained with NBD-phallacidin. Fibroblasts with actin filaments are increased in autografts, particularly at Days 15 and 21 after grafting, and are prominent in open and burn wounds. The wound contraction rate is not directly related to the presence of actin-staining fibroblasts. After stabilization of the contraction of open or burn wounds, fibroblasts rich in actin filaments remain. The superficial layer of full-thickness skin graft contains a similar actin distribution without concomitant contraction. It is concluded that the distribution of actin-rich fibroblasts corresponds morphologically to previous areas of necrosis or injury.     

正常与糖尿病小鼠脂肪间充质干细胞移植促进皮肤创伤愈合的比较

目的比较正常和糖尿病小鼠脂肪间充质干细胞(Adipose mesenchymal stem cells,ADSCs)移植促进皮肤创伤愈合疗效差别。方法无菌条件下获取正常小鼠及糖尿病小鼠的ADSCs,应用流式细胞术对3代细胞表面抗原CD34、CD45、CD90、CD105进行表型鉴定。应用WST法和Transwell迁移实验检测细胞增殖和迁移情况。应用ELISA对正常小鼠及糖尿病小鼠的ADSCs条件培养液中VEGF、HGF和IGF-1蛋白含量进行检测。建立小鼠皮肤创伤模型并随机分为3组,分别以皮内注射方式将3代正常小鼠或糖尿病小鼠来源ADSCs的细胞混悬液移植到小鼠创面四周,空白对照组注射生理盐水,于伤后14d观察创面愈合情况;Western blot检测伤后14d创面组织Bcl-2蛋白表达。结果小鼠ADSCs表达CD90和CD105,不表达CD34和CD45,与正常小鼠相比,糖尿病小鼠来源的ADSCs增殖和迁移能力下降,条件培养液中VEGF、HGF和IGF-1蛋白含量明显降低。糖尿病小鼠ADSCs移植组于创伤后第14d伤口愈合率为(79.6%±6.2%),均显著低于正常小鼠移植组14d的(97.1%±4.1%),两者均高于对照组14d的(64.6%±2.9%,0.05)。与正常小鼠移植组相比,糖尿病小鼠移植组伤后14d创面组织Bcl-2表达水平降低,高于对照组创面组织Bcl-2表达水平。结论正常小鼠来源ADSCs较糖尿病小鼠ADSCs更能促进小鼠皮肤创面愈合。     

Macrophage mediation in normal and diabetic wound healing responses

Inflammation Research - The failure in timely healing of wounds is a central feature in chronic wounds that leads to physiological, psychological and economic burdens. Macrophages have been...     

定向诱导骨髓间充质干细胞参与创面修复的研究

目的探讨诱导骨髓间充质干细胞(MSCs)分化为血管内皮样细胞参与创面修复的可能机制。方法选择正常成人MSCs,经密度梯度离心分离、纯化及体外培养扩增后,定向诱导分化为血管内皮样细胞作为种子细胞,以大于(1～2)×10个/cm密度种植于支架上,扫描电镜观察后,应用溴脱氧尿嘧啶(BrdU)标记技术标记细胞。于兔子背部制作全层皮肤缺损创面,即刻以纤维蛋白胶为载体,将已标记的血管内皮样细胞回植到供体动物创面上。术后2周切取创面组织,行BrdU和凝血因子Ⅷ(FⅧ)免疫组织化学染色。结果 BrdU阳性的MSCs多聚集在创面肉芽组织中的小血管周围,且有个别血管内皮细胞也呈现BrdU阳性。部分MSCs细胞质中亦有FⅧ表达。结论创面愈合过程中,诱导的MSCs与肉芽组织中小血管的形成密切相关。诱导的MSCs可分化为血管内皮样细胞,并参与创面修复。     

正常和放射复合伤口血管再生中VEGF基因的表达及其意义

目的 研究VEGF基因在正常和放射复合伤口愈合中的表达及意义,探讨其在血管再生中的作用。方法 将48只Wistar二级大鼠背部皮肤造成圆形伤口后,以25Gy ^60Co γ射线局部照射,于伤后2、5、10、15、21和28d活杀取材,采用免疫组织化学、原位杂交和原位PCR等方法,研究VEGF基因的表达及意义。结果：单纯创伤组于伤后2d,新生血管内皮细胞浆内VEGF的表达呈弱阳性;而于10d后,VEGF的表达逐渐减少。创伤＋照射组则于伤后5d,VEGF的表达在血管内皮细胞胞浆内呈弱阳性,10d时阳性,15d后则呈弱阳性。原位杂交,VEGF mRNA于单纯创伤组伤后2－5d及创伤＋照射组伤后5－10d,血管内皮细胞浆内阳性。原位反转录PCR则显示,VEGF mRNA于单纯创伤组后2－15d及创伤＋照射组伤后5－28d,呈阳性反应。结论 内源性VEGF基因的表达参与创伤愈合中血管再生过程。辐射使血管内皮细胞中VEGF的表达减少。原位反转录PCR方法较原位杂交能客观地反映内源性VEGF基因的表达情况。     

Confocal laser scanning microscopy of porcine skin: implications for human wound healing studies

The structure of porcine skin as examined by light microscopy is reviewed and its similarities to and differences from human skin are highlighted. Special imaging techniques and staining procedures are described and their use in gathering morphological information in porcine skin is discussed. Confocal laser scanning microscopy (CLSM) was employed to examine the structure of porcine skin and the findings are presented as an adjunct to the information already available in the literature. It is concluded that CLSM provides valuable additional morphological information to material examined by conventional microscopy and is useful for wound healing studies in the porcine model.     

Structure and function of hepatic stellate cells

Hepatic stellate cells (vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, Ito cells) exist in the space between parenchymal cells and sinusoidal endothelial cells of the hepatic lobule and store 80% of retinoids in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. In physiological conditions, these cells play pivotal roles in the regulation of retinoid homeostasis; they express specific receptors for retinol-binding protein (RBP), a binding protein specific for retinol, on their cell surface, and take up the complex of retinol and RBP by receptor-mediated endocytosis. Hepatic stellate cells in arctic animals such as polar bears and arctic foxes store 20–100 times the levels of retinoids found in humans or rats. In pathological conditions such as liver fibrosis, hepatic stellate cells lose retinoids, and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan, and adhesive glycoproteins. Morphology of these cells also changes from the star-shaped stellate cells to that of fibroblasts or myofibroblasts. The three-dimensional structure of ECM components was found to regulate reversibly the morphology, proliferation, and functions of the hepatic stellate cells. Molecular mechanisms in the reversible regulation of the stellate cells by ECM imply cell-surface integrin binding to ECM components, followed by signal transduction processes and then cytoskeleton assembly. Stellate cells also exist in extrahepatic organs such as pancreas, lung, kidney, and intestine. Hepatic and extrahepatic stellate cells form the stellate cell system.     

整合素连接激酶在心脏中的功能意义

Integrin-linked kinase (ILK) is a widely expressed protein kinase that relate to cellular growth and differentiation. It is most abundant in the heart. Recently, many researches revealed that ILK is highly relevant to cardiac response to biomechanical stresses. Also, ILK plays important roles in regulation of the occurrence and development of cardiac hypertrophy, dilated cardiomyopathy, viral myocarditis and myocardial senescence via correlation to several classical signal transduction pathway. Meanwhile, ILK functions in protection after myocardial infarction. This article will try to summarize the effects and relevant mechanism of ILK in above-mentioned aspects, overall reveals the roles of ILK in heart and its potential clinical significance.     

骨髓间质干细胞培养液上清对肝星状细胞增殖及纤维化的影响

背景：利用骨髓间质干细胞治疗肝纤维化的研究取得了一定的效果,但研究其培养液上清治疗肝纤维化的并不多。 目的：观察大鼠骨髓间质干细胞培养液上清抑制肝星状细胞增殖及活化的可能性。 方法：实验组1,2将骨髓间质干细胞培养液上清加入6孔板内处理肝星状细胞,分别为2 mL培养液上清/孔,1 mL培养液上清+1 mL完全培养基/孔;对照组为肝星状细胞单独培养(2 mL完全培养基/孔)。流式细胞仪分析细胞周期并观察抑制效果,RT-PCR检测肝星状细胞内转化生长因子β、金属蛋白酶抑制因子2、胶原蛋白ⅠmRNA的表达情况。 结果与结论：加入骨髓间质干细胞培养液上清后,实验组1中的肝星状细胞增殖与对照组相比受到抑制,且抑制率随处理时间的延长而增多(处理72 h>处理48 h>处理24 h,P < 0.05); 实验组的肝星状细胞内转化生长因子β、金属蛋白酶抑制因子2、胶原蛋白Ⅰ mRNA的表达较对照组均下调,且下调程度随培养液上清增多而增大(实验组1>实验组2>对照组)。     

Vinculin: a novel marker for quiescent and activated hepatic stellate cells in human and rat livers

In liver injuries, the quiescent hepatic stellate cells (HSCs) promptly change to activated HSCs, which are easily identified by the intense immunoreactivity for alpha-smooth muscle actin. However, reproducible markers for quiescent HSCs in formalin-fixed, paraffin-embedded liver tissue sections have not yet been reported. We immunohistochemically examined the expression of vinculin, one major protein of dense plaques, on cultured LI90 cells and on HSCs in normal and diseased human and rat livers. In cultured LI90 cells, vinculin appeared as small linear patches. Although vinculin was consistently negative in the routine liver tissue sections, an antigen retrieval technique using microwave oven heating yielded excellent effects. Using this technique, the formalin-fixed, paraffin-embedded human and rat normal liver tissue sections showed the vinculin immunoreactivity along the sinusoidal wall. Immunoelectron microscopic observation of hepatic parenchyma demonstrated that the vinculin was exclusively expressed in quiescent HSCs. In fetal rat livers, vinculin-positive quiescent HSCs gradually increased in number with gestation. In diseased livers the activated HSCs showed more intense immunoreaction for vinculin. These results indicate that, using microwave pretreatment, vinculin is expressed in quiescent and activated HSCs in routinely processed liver tissue sections. It could allow us to evaluate the development and distribution of quiescent HSCs and to examine the relationship between quiescent and activated HSCs.     

损伤皮肤修复和伤口愈合过程中的干细胞：问题与前景

BACKGROUND:Present treatments for chronic skin wounds have certain limitations, and adult stem cells play a potential part in cutaneous repair and regeneration. OBJECTIVE:To review effects of stem cells in skin regeneration and wound healing. METHODS:The first author retrieved CNKI and Medline databases by computer for relevant articles published from 2000 to 2010. The keywords were “epidermal stem cells, hair follicle stem cells, stem cells, transplantation, dermal stem cells” in Chinese and in English, respectively. Then totally 489 papers were obtained after initial survey, and according to the inclusion criteria, 30 articles were selected for review. RESULTS AND CONCLUSION:Epidermal stem cells and other adult stem cells have been applied to treat wounds and other skin diseases. Epidermal stem cells are the crucial cell source of skin development, repair and remodeling. Epidermal stem cells are always in a resting state in vivo. Unless, skin injure or culture in vitro, cell division and proliferation will be significantly fastened. The stability of the epidermis mainly depends on the asymmetric division of a subpopulation, in which two daughter cells are produced, including one with characteristics of stem cells, and the other differentiated into transient amplifying cells that will be differentiated into post mitotic cells after a series of cell divisions (3-5 times). Afterwards, those post mitotic cells are developed into terminal differentiation cells on the basal layer, finally detach from the epidermis as dander. In addition, it is unclear whether epidermal factors are related to apoptosis, migration and differentiation in the process of wound repair and even under physiological conditions. Therefore, application of stem cells in wound healing requires a further discussion.     

The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis

The origin of myofibroblasts and the factors promoting their differentiation during liver fibrogenesis remain uncertain. During biliary-type fibrogenesis, the proliferation and chemoattraction of hepatic stellate cells (HSC) toward bile ducts is mediated by platelet-derived growth factor (PDGF), while myofibroblastic conversion of peribiliary cells distinct from HSC also occurs. We herein examined the phenotype of these peribiliary myofibroblasts as compared with myofibroblastic HSC and tested whether their differentiation was affected by PDGF. Biliary-type liver fibrogenesis was induced by common bile duct ligation in rats. After 48 hours, periductular fibrosis in portal tracts colocalized with smooth muscle alpha-actin-immunoreactive myofibroblasts, the majority of which were desmin negative. Simultaneously, in sinusoids, desmin immunoreactivity was induced in a large number of HSC, which were smooth muscle alpha-actin negative. Cultures of peribiliary myofibroblasts were expanded from isolated bile duct segments and compared with myofibroblastic HSC. Peribiliary myofibroblasts outgrowing from bile duct segments expressed smooth muscle alpha-actin, alpha1 (I) collagen mRNA, and PDGF receptor-beta subunit. Desmin immunoreactivity gradually decreased in cultured peribiliary myofibroblasts, contrasting with constant labeling of all myofibroblastic HSC. In addition, IL-6 expression in peribiliary myofibroblasts was up to 100-fold lower than in myofibroblastic HSC, whereas the expression of the complement-activating protease P100 in both cell types showed little difference and that of the extracellular matrix component fibulin 2 was similar. The expression of smooth muscle alpha-actin protein in cultured peribiliary myofibroblasts was stimulated by PDGF-BB and inhibited by STI571, a PDGF receptor tyrosine kinase inhibitor, whereas in bile duct-ligated rats, the administration of STI571 caused a significant decrease in peribiliary smooth muscle alpha-actin immunoreactivity, and to a lesser extent, a decrease in peribiliary fibrosis. These results indicate that peribiliary cells distinct from HSC undergo a PDGF-mediated conversion into myofibroblasts expressing IL-6 at lower levels than myofibroblastic HSC and contribute to the initial formation of biliary-type liver fibrosis.