PDGF在大鼠断层供皮区创面愈合过程中表达变化的研究

实验研究已经证明Ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ（ＰＤＧＦ）能够促进各种类型的伤口愈合,然而在伤口愈合过程中内源性ＰＤＧＦ表达变化的研究却少有报道,为探讨ＰＤＧＦ对伤口愈合的影响,我们应用原位杂交、斑点杂交技术观察了内源性ＰＤＧＦ在大鼠创面愈合过程中的表达变化,结果发现：在创面愈合过程中,肉芽组织中的成纤维细胞,毛细血管内皮细胞及创缘真皮内的毛囊上皮细胞均能表达ＰＤＧＦ－ＢＢ基因,在伤后６天,组织修复的高峰期,ＰＤＧＦ－ＢＢ基因表达达到最强,伤后１２天,伤口完全上皮化,ＰＤＧＦ的基因表达也恢复正常,说明ＰＤＧＦ的基因表达和伤口愈合时间有密切的关系。提示ＰＤＧＦ在创面愈合过程中可能起着重要的调控作用。     

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。     

HyTK基因转移联合ganciclovir对瘢痕成纤维细胞的体外杀伤作用

增生性瘢痕和瘢痕疙瘩的过度增生主要是由于高度增生活性的成纤维细胞的数量异常增多及细胞外基质合成增加所致．用逆转录病毒载体介导单纯疱疹病毒胸苷激酶基因（ＨＳＶｔｋ）的转移,随后应用药物９－（１,３－二羟基－丙氧基－甲基）鸟嘌呤（ｇａｎｃｉｃｌｏｖｉｒ,ＧＣＶ）可选择性地杀死增生细胞．采用组织块贴壁法在体外原代培养成功增生性瘢痕病人的成纤维细胞（ＦＢ）．重组逆转录病毒ＧＴＫ转染ＦＢ细胞后,用ｈｙｇｒｏｍｙｃｉｎＢ筛选出阳性细胞克隆（ＦＢ／ＧＴＫ）,经ＰＣＲ方法检测证明ＨｙＴＫ基因已成功地导入ＦＢ中,但不含可复制的辅助病毒．分别用不同浓度的ＧＣＶ作用于ＦＢ／ＧＴＫ及ＦＢ,光镜下观察不同时间后细胞形态变化及ＭＴＴ法检测细胞活性．结果表明,ＧＣＶ浓度大于０．１μｍｏｌ／Ｌ时即对ＦＢ／ＧＴＫ有显著的杀伤作用,且具有强有力的“旁观者效应”     

成纤维细胞生长因子与心血管疾病

成纤维细胞生长因子（ＦＧＦ）是广泛存在于体内多种组织中的一类多肽生长因子,主要分为ａＦＧＦ和ｂＦＧＦ两大类;由于对肝素有很强的亲和力,故又命名为肝素结合生长因子。近年研究发现,在血管内皮细胞,平滑肌细胞和心肌细胞中亦含有一定数量的ＦＧＦ。ＦＧＦ有很强的促进血管形成作用,能刺激血管内皮细胞分裂和迁移,促进平滑肌细胞增生,在创伤愈合和组织修复过程中起很重要的作用。ＦＧＦ也存在于胚胎和成年心肌细胞中,参     

鹿茸角柄断面伤口无疤痕愈合过程中骨形态蛋白的表达及功能分析

为研究骨形态蛋白（bone morphogenetic proteins ,BMP）在鹿茸再生早期角柄断面伤口无疤痕愈合过程中的功能,本研究通过免疫组化技术对比分析了BMP 2 和BMP 4在正常皮肤及茸皮中的表达及分布差异,同时利用添加外源性蛋白分析了BMP 4对鹿真皮成纤维细胞和毛乳头细胞的影响。结果显示：(1)原代培养的真皮成纤维细胞表达波形蛋白阳性率几乎为100%;(2) BMP 2和BMP 4强烈表达于茸皮中新生毛囊的毛基质细胞中;(3) BMP 4可促进鹿真皮成纤维细胞向脂肪细胞转分化;(4) BMP 4可促进鹿真皮毛乳头细胞成团。以此推测BMP在鹿毛囊形成及伤口愈合过程中发挥重要作用。     

TK/GCV系统体外诱导成纤维细胞凋亡

 在体外培养并鉴定增生性瘢痕成纤维细胞（ Ｆ Ｂ）的基础上,采用重组逆转录病毒 Ｇ Ｔ Ｋ 介导并联合应用 ｇａｎｃｉｃｌｏｖｉｒ 对 Ｆ Ｂ细胞进行体外杀伤,以探究 Ｔ Ｋ／ Ｇ Ｃ Ｖ 系统体外杀伤成纤维细胞的机制．经光镜、电镜及凝胶电泳等实验发现,在 Ｔ Ｋ／ Ｇ Ｃ Ｖ 对瘢痕成纤维细胞的杀伤过程中存在明显的细胞凋亡现象．这提示 Ｔ Ｋ／ Ｇ Ｃ Ｖ 系统对体外培养的瘢痕成纤维细胞的杀伤作用部分是通过细胞凋亡途径实现的．     

人前列腺生长因子的分离纯化及生物活性鉴定

从人良性增生前列腺组织中经硫酸铵沉淀和肝素－琼脂糖凝胶层析纯化出人前列腺生长因子（ｈＰＧＦ）,纯化倍数约１０００倍,ＳＤＳ－ＰＡＧＥ和等电聚焦电泳示分子量约为１７ｋＤ、等电点同标准ｂＦＧＦ,利用分离培养的人前列腺间质成纤维细胞进行活性鉴定,发现以１．３～１．７ｍｏｌ／ＬＮａＣｌ洗脱部分为ｈＰＧＦ,活性最高,对间质成纤维细胞有显著刺激增殖作用。     

缺氧内皮细胞条件培养液对肺动脉平滑肌细胞b-FGF表达的影响

应用细胞培养、免疫细胞化学和图像分析方法,探讨了缺氧猪肺动脉内皮细胞条件培养液对肺动脉平滑肌细胞碱性成纤维细胞生长因子（ｂＦＧＦ）表达的影响。本研究表明,肺动脉内皮细胞培养汇合前和汇合后均可表达ｂＦＧＦ,在缺氧时其表达增多。缺氧猪肺动脉内皮细胞条件培养液可刺激猪肺动脉平滑肌细胞表达ｂＦＧＦ,将条件培养液加热１００℃处理或加入ｂＦＧＦ抗体后,可使猪肺动脉平滑肌细胞表达ｂＦＧＦ明显减少。这提示缺氧可刺激猪肺动脉内皮细胞表达ｂＦＧＦ,缺氧猪肺动脉内皮细胞条件培养液通过ｂＦＧＦ使肺动脉平滑肌细胞表达ｂＦＧＦ,因此推测缺氧条件下肺动脉内皮细胞ｂＦＧＦ的旁分泌作用可能在肺动脉平滑肌细胞增生中起重要作用,这一过程在缺氧性肺动脉高压时肺动脉结构重建的发生机制中具有重要意义。     

miR-21在疤痕形成中的作用及其机制研究

目的:探究mi R-21在疤痕形成中的作用及其相关机制。方法:选取2016年3月-2017年8月在本院就诊的20例皮肤有疤痕患者,采集患者的正常皮肤组织(正常组)、疤痕组织(疤痕组),进行成纤维细胞的分离培养,比较正常组、疤痕组mi R-21与Smad-7的m RNA表达水平,并在疤痕组织加入mi R-21抑制剂(抑制组),比较三组成纤维细胞增殖情况、蛋白表达及磷酸化水平。结果:正常组中Smad-7的m RNA表达水平显著高于疤痕组,而mi R-21的m RNA水平显著低于疤痕组,差异均有统计学意义(P0.05)。细胞培养48 h和72 h时,疤痕组成纤维细胞增殖水平明显高于正常组和抑制组,差异有统计学意义(P0.05),而抑制组和正常组成纤维细胞增殖水平比较差异无统计学意义(P0.05)。疤痕组成纤维细胞Smad-7蛋白表达低于正常组和抑制组,差异有统计学意义(P0.05),疤痕组成纤维细胞Smad-2、Smad-3蛋白表达及磷酸化水平均高于正常组和抑制组,差异有统计学意义(P0.05)。结论:疤痕组织中mi R-21通过负调控Smad-7的表达,从而引起Smad-2与Smad-3蛋白表达发生变化,使得疤痕成纤维细胞的增殖水平明显增高,最终促成疤痕的形成。     

成纤维细胞生长因子与高血压

成纤维细胞生长因子与高血压成纤维细胞生长因子（ＦＧＦ）刺激细胞生长、分化、参与细胞肥大、增生过程,受到广泛重视,但迄今为止,ＦＧＦ不足所引起的病理生理过程尚不清楚。最近,Ｃｕｅｖａｓ等报道ＦＧＦ水平降低与高血压发病有关。他们发现自发性高血压大鼠（ＳＨ...     

Histological differences in healing following experimental transmural infarction in rats

Mechanisms of cardiac regeneration following transmural myocardial infarction were analysed in rat hearts using immunohistochemistry for a-SMA, caspase-3, Ki-67 and nestin markers. Seven weeks after experimental myocardial infarction, two different types of healing processes were revealed in rats with and without aneurysmatic bulging of the left ventricular wall. Besides thinning of the ventricular wall, three zones characterized both types of scars: the scar zone (divided into central and peripheral parts), the peri-infarct zone and the border zone. The main difference between the types of scars was the presence of a central necrotic zone inside the aneurysmatic wall, while connective tissue with myofibroblasts characterized the same zone in non-bulging wall. Apoptotic caspase-3 positive cells were found in the granulation tissue of the border zone in aneurysmatic scar, while in non-bulging scar they characterized all three zones. Proliferating Ki-67 positive cells displayed reverse expression pattern compared to apoptotic cells. Quantification of a-SMA positive cells revealed 60% a-SMA positive cells inside the central part of the aneurysmatic scar zone and 39% in invaginating areas, versus 19% in non-invaginating areas of the peripheral zone, but only 30% in the peripheral part of the non-bulging scar zone. Nestin positive cells were found in both types of scars, but with different distribution. These results suggest that even seven weeks after myocardial infarction, the healing processes in non-bulging scars are in chronic phase, while aneurysmatic scars are still in subacute phase. Histological differences in scar healing might be important for functional properties of the heart wall and for heart recovery prognosis.     

病理性瘢痕中主要氧化酶和抗氧化酶活性测定

采用化学比色法测定正常皮肤(8例)、增生性瘢痕(10例)及瘢痕疙瘩(10例)组织中黄嘌呤氧化酶(xanthine oxidase,XO)、铜锌超氧化物歧化酶(copper,zinc-superoxide dismutase,CuZn-SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)活性以及丙二醛(malonaldehyde,MDA)含量.结果表明:与正常皮肤比较,病理性瘢痕中XO和CuZn-SOD活性增加、CAT活性降低(P<0.05)而GPX活性不变,CAT/CuZn-SOD和GPX/CuZn-SOD活性比率下降(P<0.05),同时MDA含量升高(P<0.05).增生性瘢痕、瘢痕疙瘩之间比较均无差异.上述结果表明,在病理性瘢痕中,氧化酶XO,抗氧化酶CuZn-SOD、CAT以及GPX的活性改变可能是引起活性氧水平升高的原因之一,在抗氧化剂选择上,CAT可能较为合理.     

A mathematical model for the simulation of the formation and the subsequent regression of hypertrophic scar tissue after dermal wounding

A continuum hypothesis-based model is presented for the simulation of the formation and the subsequent regression of hypertrophic scar tissue after dermal wounding. Solely the dermal layer of the skin is modeled explicitly and it is modeled as a heterogeneous, isotropic and compressible neo-Hookean solid. With respect to the constituents of the dermal layer, the following components are selected as primary model components: fibroblasts, myofibroblasts, a generic signaling molecule and collagen molecules. A good match with respect to the evolution of the thickness of the dermal layer of scars between the outcomes of simulations and clinical measurements on hypertrophic scars at different time points after injury in human subjects is demonstrated. Interestingly, the comparison between the outcomes of the simulations and the clinical measurements demonstrates that a relatively high apoptosis rate of myofibroblasts results in scar tissue that behaves more like normal scar tissue with respect to the evolution of the thickness of the tissue over time, while a relatively low apoptosis rate results in scar tissue that behaves like hypertrophic scar tissue with respect to the evolution of the thickness of the tissue over time. Our ultimate goal is to construct models with which the properties of newly generated tissues that form during wound healing can be predicted with a high degree of certainty. The development of the presented model is considered by us as a step toward their construction.     

X-irradiation reduces lesion scarring at the contusion site of adult rat spinal cord

Spinal cord injury (SCI) results in cell death and tissue destruction, and ultimately cavitation followed by the formation of lesion scars at the injury site. The lesion scars include an astrocytic component (glial scar) and a fibroblastic component (connective tissue scar). The purpose of the present study is to determine if X-irradiation could minimize the formation of lesion scars and reduce the levels of chondroitin sulfate proteoglycans (CSPGs) in the contusion SCI model of the adult rat. Two weeks after SCI, a connective tissue scar formed at the injury site consisting primarily of fibroblasts and exhibits strong CSPG immunoreactivity. The fibroblasts might originate from the connective tissue of pia mater or arachnoid mater. At the same time, reactive astrocytes in the spared tissue accumulate surrounding the lesion cavity to form a thick glial scar with significant enhancement of glial fibrillary acidic protein (GFAP) and CSPG immunoreactivity. After X-irradiation (40 Gy) of the injury site 2 days post-injury, that results in an attenuated dose to the lesion, the connective tissue scar was not observed, and accordingly, almost no CSPG immunoreactivity was detected at this area. Meanwhile, the glial scar and its CSPG immunoreactivity were prominently reduced. X-irradiation did not show significant improvement in locomotor recovery, but resulted in a slight delay of body weight recovery following injury. This preparative treatment could be used to reduce secondary scarring in the lesion resulting in an enriched site for further treatment such as growth related transplantation.     

Presence of basic fibroblast growth factor receptors in bovine brain membranes

We described a protocol for purification of bovine brain membranes suitable to study the binding of iodinated basic fibroblast growth factor (FGF) to bovine brain membrane preparation. The binding of 125I basic FGF to brain membranes reached equilibrium within 30 min at 20 degrees C, was reversible, and displaced by an excess of unlabeled basic FGF. Scatchard analysis of the data revealed that two classes of binding sites could be detected with an apparent Kd of 30 pM and a capacity of 0.24 pmol/mg of membrane proteins for the high affinity binding site and Kd of 3 nM with a capacity of 51 pmol/mg of membrane proteins for the low affinity binding site. Cross-linking experiments of labeled basic FGF to brain membrane receptor yield the formation of a single major complex with an apparent molecular mass of 170 kDa which is similar to the value obtained for the high affinity binding site for basic FGF on target cells in tissue culture. Hence these data present the first biochemical evidence suggesting that membrane purified from bovine brain contain two classes of specific binding sites for basic FGF and confirm results described with cells grown in vitro.     

PI3-K/AkT信号转导通路与瘢痕形成研究进展

临床上组织损伤2—3天后即可出现肉芽组织,进而由于成纤维细胞和血管内皮细胞的增殖逐渐形成纤维性瘢痕。瘢痕的形成与血管再生和细胞增殖及凋亡密切相关。常见的病理性瘢痕主要是增生性瘢痕和瘢痕疙瘩,他们不仅影响患者关键伤口的活动,而且在美观上给患者带来莫大的痛苦。但是由于对瘢痕的形成原因及发病机制仍不甚清楚,至今临床上实行地以手术为主的对瘢痕的治疗方法仍未取得较满意效果。磷脂酰肌醇3激酶（P13K,phosphoinositide3．Kinase）／Akt（P13．K／Akt）通路广泛存在于人体的多个生理功能中,其在细胞因子作用下介导细胞生存已被证实,目前研究表明,P13-k／Akt信号通路在瘢痕形成中也发挥了重要作用,这可能会为瘢痕的治疗带来新的前景。本文将就近年来关于P13-k／Akt通路在中发挥的作用机制作一综述,并对未来利用此通路彻底治疗瘢痕的可能方式做一展望。     

FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo

At the end of gastrulation in avians and mammals, the endoderm germ layer is an undetermined sheet of cells. Over the next 24-48 h, endoderm forms a primitive tube and becomes regionally specified along the anterior-posterior axis. Fgf4 is expressed in gastrulation and somite stage embryos in the vicinity of posterior endoderm that gives rise to the posterior gut. Moreover, the posterior endoderm adjacent to Fgf4-expressing mesoderm expresses the FGF-target genes Sprouty1 and 2 suggesting that endoderm respond to an FGF signal in vivo. Here, we report the first evidence suggesting that FGF4-mediated signaling is required for establishing gut tube domains along the A-P axis in vivo. At the gastrula stage, exposing endoderm to recombinant FGF4 protein results in an anterior shift in the Pdx1 and CdxB expression domains. These expression domains remain sensitive to FGF4 levels throughout early somite stages. Additionally, FGF4 represses the anterior endoderm markers Hex1 and Nkx2.1 and disrupts foregut morphogenesis. FGF signaling directly patterns endoderm and not via a secondary induction from another germ layer, as shown by expression of dominant-active FGFR1 specifically in endoderm, which results in ectopic anterior expression of Pdx1. Loss-of-function studies using the FGF receptor antagonist SU5402 demonstrate that FGF signaling is necessary for establishing midgut gene expression and for maintaining gene expression boundaries between the midgut and hindgut from gastrulation through somitogenesis. Moreover, FGF signaling in the primitive streak is necessary to restrict Hex1 expression to anterior endoderm. These data show that FGF signaling is critical for patterning the gut tube by promoting posterior and inhibiting anterior endoderm cell fate.     

High and low affinity binding sites for basic fibroblast growth factor on cultured cells: absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells

Scatchard analysis of binding of 125I-basic fibroblast growth factor (FGF) to baby hamster kidney (BHK) cells revealed the presence of two binding sites: a high affinity site with KD of 20 pM and 80,000 sites per cell and a low affinity site with KD of about 2 nM and 600,000 sites per cell. The binding to the two sites could be separated by first washing the cells with 2 M NaCl at pH 7.5 which released the low affinity binding and then extracting the cells with 0.5% Triton X-100 to recover the 125I-basic FGF bound to high affinity sites. The binding to the high affinity site was acid sensitive, suggesting that it represented binding to the receptor. Binding to the low affinity site could be competed strongly by heparin and less strongly by heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. Treatment of BHK cells with heparinase abolished 62% of the low affinity binding, suggesting that the low affinity binding represented binding to cell-associated, heparin-like molecules. A variety of other cell types, including bovine capillary endothelial (BCE) cells, also demonstrated both low and high affinity binding sites. To test whether the low affinity binding might play a role in the basic FGF stimulation of plasminogen activator (PA) production by BCE cells, heparin was added to BCE cultures at concentrations which totally blocked binding of 125I-basic FGF to the low affinity sites. Addition of the heparin did not diminish the increased PA production induced by basic FGF. This suggests that the low affinity binding has no direct role in the stimulation of PA production in BCE cells.     

FGF8 signaling is chemotactic for cardiac neural crest cells

Cardiac neural crest cells migrate into the pharyngeal arches where they support development of the pharyngeal arch arteries. The pharyngeal endoderm and ectoderm both express high levels of FGF8. We hypothesized that FGF8 is chemotactic for cardiac crest cells. To begin testing this hypothesis, cardiac crest was explanted for migration assays under various conditions. Cardiac neural crest cells migrated more in response to FGF8. Single cell tracing indicated that this was not due to proliferation and subsequent transwell assays showed that the cells migrate toward an FGF8 source. The migratory response was mediated by FGF receptors (FGFR) 1 and 3 and MAPK/ERK intracellular signaling. To test whether FGF8 is chemokinetic and/or chemotactic in vivo, dominant negative FGFR1 was electroporated into the premigratory cardiac neural crest. Cells expressing the dominant negative receptor migrated slower than normal cardiac neural crest cells and were prone to remain in the vicinity of the neural tube and die. Treating with the FGFR1 inhibitor, SU5402 or an FGFR3 function-blocking antibody also slowed neural crest migration. FGF8 over-signaling enhanced neural crest migration. Neural crest cells migrated to an FGF8-soaked bead placed dorsal to the pharynx. Finally, an FGF8 producing plasmid was electroporated into an ectopic site in the ventral pharyngeal endoderm. The FGF8 producing cells attracted a thick layer of mesenchymal cells. DiI labeling of the neural crest as well as quail-to-chick neural crest chimeras showed that neural crest cells migrated to and around the ectopic site of FGF8 expression. These results showing that FGF8 is chemotactic and chemokinetic for cardiac neural crest adds another dimension to understanding the relationship of FGF8 and cardiac neural crest in cardiovascular defects.