胎儿和出生后机体皮肤内表皮生长因子和两种原癌基因表达变化及其与胎儿无瘢愈合的关系

胎儿皮肤创面愈合后一般不形成瘢痕,而成人创面愈合常有瘢修复,这一变化的机制目前尚不清楚.在研究中人们发现胎儿无瘢痕愈合不受胎儿所处的无菌、湿润和低氧环境的影响,而可能与伤口处的细胞因子浓度变化密切相关.表皮生长因子(epidermal growth factor,EGF)作为主要的修复生长因子,对创面愈合的多个环节都产生作用.EGFR是一种跨膜酪氨酸激酶受体,与EGF特异性结合后,通过MAPK信号通路,上调c-fos和c-myc等转录因子的基因表达后,诱导细胞核内特异性基因转录,刺激组织修复细胞增殖,趋化炎性细胞,表皮细胞和成纤维细胞向伤口聚集,加速创面愈合.目前,关于EGF,EGFR及其下游信号分子在人不同发育阶段的皮肤组织中的基因表达变化特征还缺乏研究,对这一规律的揭示,将有利于深入了解EGF调节创面愈合的机制和早期妊娠胎儿皮肤创面无瘢痕愈合的部分奥秘.用病理学技术检测不同发育阶段皮肤的结构特征后,提取18例不同胎龄(13～32周)的胎儿皮肤和6例出生后机体皮肤组织的总RNA,分离mRNA,用R-PCR方法检测这4种基因在不同组织中的表达变化规律.结果显示,在早期妊娠胎儿的皮肤中,EGF,EGFR,c-fos和c-myc基因表达较弱,随着胎儿的生长和发育,皮肤组织内这三种基因表达逐渐增强,在出生后机体的皮肤组织中,EGF,EGFR,c-fos和c-myc基因表达进一步升高,基因表达量分别为晚期妊娠胎儿皮肤的1.24倍,1.11倍,1.28倍和1.63倍.EGF、EGFR及其下游信号分子c-fos和c-myc基因在不同发育阶段的人皮肤组织内都有表达,显示EGF与其受体结合后引起的信号通路可能对皮肤的发生、结构功能的维持以及伤后修复十分重要.这4种基因在早期妊娠胎儿皮肤中低表达可能与胎儿皮肤创面无瘢痕修复密切相关.     

胎儿和成人皮肤组织中TGF-β1,2,3因子蛋白含量的变化及其与创面无瘢愈合的关系

目的观察转化生长因子β三种异构体(TGF-β1、2和3)和α-平滑肌肌动蛋白(α-ASMA)在胎儿和成人皮肤组织中的表达特征以及与胎儿创面无瘢愈合的关系.方法12例被测标本中包括不同胎龄的胎儿皮肤6例,其对应的成人皮肤组织6例.用免疫组化方法和常规病理技术确定这四种蛋白在胎儿和成人皮肤组织中的定位及表达量的变化规律.结果TGF-β1、2、3细胞因子的阳性信号主要见于表皮基底层细胞、汗腺和毛囊细胞的胞浆和胞外基质中,α-ASMA蛋白则定位于肌动成纤维细胞内.在胎儿生长发育过程中,TGF-β1、2、3和α-ASMA等蛋白在皮肤组织中的阳性细胞率逐渐升高,在成人皮肤组织中,四种蛋白的含量更加明显增大.在胎儿发育的不同时期,这四种蛋白在皮肤组织内的含量变化规律相近似.结论在成人皮肤组织内,TGF-β1和α-ASMA两种蛋白含量的升高可能与伤口愈合形成瘢痕相关,而胎儿皮肤中这两种蛋白含量的降低可能与胎儿创面无瘢愈合密切相关,而TGF-β2、3在其中所起的作用尚需进一步深入研究.     

增生瘢痕皮肤中转化生长因子基因表达

目的探讨转化生长因子(TGF)-β1和转录因子Smad 2,Smad 3三种基因影响增生性瘢痕形成和胎儿皮肤伤口无瘢痕愈合的调节机制.方法 32份被检测标本中包括增生性瘢痕8份,其对应的正常皮肤组织8份,胎儿和成人皮肤组织各8份.用逆转录-多聚酶链反应方法(RT-PCR)检测这3种基因在不同的组织细胞内的表达变化规律.结果 TGF-β1、Smad 2 和Smad 3三种基因在增生性瘢痕、胎儿和出生后机体皮肤组织细胞内都有表达.在所检测8对增生性瘢痕和其对应正常皮肤细胞中,这3种不同基因在增生性瘢痕细胞中的表达量高于正常皮肤细胞的组数分别为TGF-β1基因有5对,Smad 2基因有8对,而Smad 3基因有5对.在胎儿皮肤细胞内,TGF-β1的mRNA含量明显低于成人皮肤细胞(t=2.204,P<0.05),差异有显著意义;Smad 3基因表达也呈相似的变化规律,mRNA含量显著低于成人皮肤细胞(t=4.269,P<0.01),差异有非常显著意义;而Smad 2基因的mRNA含量却明显高于成人皮肤细胞(t=6.685,P<0.01),差异亦有非常显著意义.结论 TGF-β1和它的下游信号分子Smad 2,Smad 3可能与增生性瘢痕形成密切相关.在增生性瘢痕细胞内,这3种基因高表达可诱导胞外基质大量沉积,加速成纤维细胞增殖,促进组织纤维化.TGF-β1和Smad 3基因在胎儿皮肤组织细胞内低表达可能是皮肤伤口无瘢痕愈合的机制之一.     

抑制原癌基因c-fos表达对创伤修复结局的影响

目的:探讨原癌基因c-fos在伤后皮肤创面愈合过程中的调控作用及其对修复结局的影响.方法:利用大鼠深Ⅱ°烫伤模型,采用免疫组化和原位杂交方法分别检测PCNA蛋白及bFGF mRNA在创面组织中的表达,并观察外源性使用c-fos单克隆抗体及bFGF后二者表达的变化规律及修复结局的改变.结果:烧伤诱导c-fos及bFGF mRNA的表达,从而启动修复过程.外源性应用c-fos抗体后,伤后各时间点基本探测不到bFGFmRNA的表达,伤后14d创面皮肤虽然基本完成了再上皮化过程,但其基底细胞排列成薄层细胞,PCNA数密度明显低于对照组,同时皮肤全层变薄.而创面外源性应用bFGF后,bFGF mRNA表达有一定增加,但无明显差异.但伤后14d创面皮肤组织基底层变厚,其中含有大量PCNA阳性细胞,真皮层亦变厚,并含有大量的毛细血管.结论:c-fos在创面愈合过程中具有重要作用,它可以启动bFGF基因转录,增加细胞的增殖活性.用c-fos抗体中和内源性c-fos作用可以明显抑制生长相关基因的表达,最后导致创面修复细胞PCNA表达下降.     

创面愈合组织三种生长相关因子表达及信号转导机制的实验研究

目的探讨烫伤后创面组织中生长相关因子c-fos、c-myc和bFGF表达的变化规律以及c-fos在伤后皮肤创面愈合过程中的调控作用,研究蛋白激酶C通路对上述因子表达的影响.方法大鼠188只,制成30%深Ⅱ度烫伤模型,随机分为正常对照组(8只)、单纯烫伤组、c-fos抗体处理组、bFGF治疗组、蛋白激酶C通路抑制剂(H7)组以及蛋白激酶C通路激活剂二乙酰一肟组(各36只).采用原位杂交、逆转录聚合酶链反应(RT-PCR)和免疫组织化学方法检测创面组织c-fos、c-myc与bFGF基因及蛋白表达规律.结果(1)烫伤诱导c-fos表达增加在前,其次是bFGF,最后是c-myc,分别于伤后3h、1d以及3d到达面密度峰值(14.5±1.3,t=13.306;0.15±0.04,t=3.460和0.11±0.05,t=2.292;与单纯烫伤组比较均P＜0.05).(2)应用外源性c-fos抗体减少创面组织3种因子的表达,其中c-myc与bFGF基因表达量分别下降80%和85%.(3)?H7明显抑制?c-fos?与?bFGF?基因及蛋白的表达,而使?c-myc?基因及蛋白的表达高峰时间延迟至伤后?6h;?二乙酰一肟作用与?H7?相反,?它可以使c-myc表达高峰提前至伤后6h,同时使c-fos与bFGF表达量分别增加至0.23±0.07(t=2.942)和0.69±0.07(t=5.134,与单纯烫伤组比较均P＜0.01).结论烫伤可以激活3种生长相关因子基因和蛋白的表达;c-fos在创面愈合过程中具有重要调控作用;蛋白激酶C通路在创面修复的信号传导中具有重要作用.     

增生瘢痕中转化生长因子基因的表达

目的　探讨转化生长因子 (TGF) β1和转录因子Smad 2 ,Smad 3三种基因影响增生性瘢痕形成和皮肤伤口无瘢痕愈合的调节机制。方法　 3 2份被检测标本中包括增生性瘢痕 8份 ,其自体正常皮肤组织 8份 ,胎儿和成人皮肤组织各 8份。用逆转录 多聚酶链反应方法 (RT PCR)检测上述基因在不同的组织细胞内的表达变化规律。结果　TGF β1、Smad 2和Smad 3基因在增生性瘢痕、胎儿和成人皮肤组织细胞内都有表达。在所检测 8对增生性瘢痕和其对应正常皮肤细胞中 ,这 3种不同基因在增生性瘢痕细胞中的表达量高于正常皮肤细胞的组数分别为 :TGF β1基因有 5对 ,Smad 2基因有8对 ,Smad 3基因有 5对。在胎儿皮肤细胞内 ,TGF β1的mRNA含量明显低于成人皮肤细胞 (t =2 2 0 4 ,P <0 0 5 ) ,差异有显著意义 ;Smad 3基因表达也呈相似的变化规律 ,mRNA含量显著低于成人皮肤细胞 (t=4 2 69,P <0 0 1) ,差异有非常显著意义 ;而Smad 2基因的mRNA含量却明显高于成人皮肤细胞 (t=6 685 ,P <0 0 1) ,差异亦有非常显著意义。结论　TGF β1和它的下游信号分子Smad2 ,Smad 3可能与增生性瘢痕形成密切相关。在增生性瘢痕细胞内 ,这 3种基因高表达可诱导胞外基质大量沉积 ,加速成纤维细胞增殖 ,促进组织纤维化。TGF β1和Smad 3基     

胎儿和少儿皮肤内碱性成纤维细胞生长因子及其受体的基因表达

目的　探讨不同胎龄的胎儿和少儿皮肤中碱性成纤维细胞生长因子 (bFGF)及其两种受体 (bek和flg)基因表达的变化。 方法　提取 18例不同胎龄 ( 13～ 3 2周 )的胎儿皮肤和 6例少儿皮肤的总RNA后 ,分离mRNA ,用逆转录 聚合酶链反应 (RT PCR)方法检测这 3种基因在不同组织中的表达。结果　在早期妊娠胎儿的皮肤中 ,bFGF ,flg和bek基因表达较强 ,随着胎儿的生长和发育 ,皮肤组织内这 3种基因表达逐渐降低 ,在少儿皮肤中 3种基因的表达量分别为晚期妊娠胎儿皮肤的 62 .5 % ,5 9.5 %和 5 2 .9% ,基因表达显著降低 ( P <0 .0 5 )。结论　bFGF及其受体基因可能在皮肤的发生、结构功能的维持以及伤后修复中起重要作用。这 3种基因在胎儿皮肤中表达水平较高可能与胎儿皮肤细胞增殖较快 ,皮肤创面愈合迅速有关。     

人创面愈合过程中同源异形框基因的表达及意义

目的 探讨人胎儿及成人皮肤创面愈合过程中，几种同源异形框基因的表达及在胎儿无瘢痕愈合中的作用。方法 采用原位杂交方法，对正常成人和胎儿皮肤及创面愈合过程中PRX—2、H0XBl3、H0X2．2和H0X2．3的表达进行观察。结果 (1)在正常胎儿和成人皮肤中可见PRX—2阳性表达，以前阳性程度为强。分布部位有所不同，在正常胎儿皮肤中，阳性表达主要见于真皮乳头层毛干部周围细胞，表皮中也可见阳性表达；而在正常成人皮肤中，表皮基底层细胞呈弱阳性表达，真皮组织中未见阳性表达。胎儿皮肤创伤后，接近切口的组织中阳性表达明显增强，而成人皮肤创伤后，阳性表达未见明显变化，仍局限于表皮基底层细胞；(2)在正常胎儿及成人皮肤均可见H0XBl3阳性表达，真皮部分主要集中在毛囊细胞，表皮部分主要集中在基底层细胞，创伤后其表达明显减弱，尤其是胎儿皮肤；(3)在正常胎儿皮肤中H0X2．2和H0X2．3阳性表达主要见于表皮全层，表皮基底层阳性表达比较强，真皮中可见弱阳性表达，创伤后近切口的组织中，表达增强。在正常成人皮肤及其创面，未见到阳性表达。结论 同源异形框基因作为与发育生物学密切相关的基因，在人胎儿及成人皮肤创面愈合过程中的表达有所不同，这可能是二创面愈合差异的根本原因。     

抑制原癌基因c-fos表达对创伤修复的影响

目的　探讨原癌基因c-fos在伤后皮肤创面愈合过程中的调控作用及其对修复结局的影响。方法　利用大鼠深Ⅱ度烧伤模型36只，采用免疫组织化学和原位杂交方法检测c-fos、增殖细胞核抗原（PCNA）蛋白及碱性成纤维细胞生长因子（bFGF）mRNA在创面组织中的表达，并观察使用外源性c-fos单克隆抗体及bFGF后二者表达的变化规律及其对烫伤修复的影响。结果　烫伤导致内源性bFGFmRNA表达减少，而诱导c-fos蛋白表达增加,伤后1d其mRNA面密度为11.10±1.56。给予外源性c-fos抗体能抑制bFGFmRNA的表达，伤后1d其mRNA表达的面密度降为4.60±0.86，伤后14d创面皮肤虽然基本完成了再上皮化过程，但其基底细胞排列成薄层细胞，PCNA数密度明显低于对照组，同时皮肤全层变薄。而创面应用外源性bFGF后，bFGFmRNA表达有一定增加但差异无显著性。伤后14d创面皮肤组织基底层变厚，其中含有大量PCNA阳性细胞，真皮层亦变厚，并含有大量的毛细血管。结论　bFGF表达受原癌基因c-fos的调节，它们的相互调控关系在创面愈合中具有重要意义。     

病理性瘢痕中原癌基因c-myc和c-fos的表达

目的探讨原癌基因的表达与病理性瘢痕形成的相关性.方法应用免疫组化SP法,检测c-myc和c-fos蛋白在增生性瘢痕、瘢痕疙瘩和正常皮肤组织中的表达和分布,并用图像定量分析比较其差异.结果在增生性瘢痕和瘢痕疙瘩的成纤维细胞中c-myc、c-fos呈强阳性表达,两组间无明显差异,而与正常皮肤对照组均有显著性差异.结论增生性瘢痕与瘢痕疙瘩中c-myc、c-fos蛋白表达升高,存在c-myc和c-fos原癌基因的激活,可能参与了成纤维细胞的分化增殖或表型转化、胶原合成与降解以及对细胞因子的调控,并导致瘢痕增生.     

Maternal and fetal anaesthesia for fetal surgery

Over the last three decades, advances in early diagnosis of fetal anomalies, imaging and surgical techniques have led to a huge expansion in fetal surgery. A small number of specialist centres perform fetal surgery, which involves high-risk anaesthesia for the mother and fetus. The anaesthetist plays an integral role within the large multispecialty and multidisciplinary team, involved in planning and delivering care for complex surgical procedures. This article reviews three fetal surgical procedures, congenital diaphragmatic hernia, myelomeningocele repair and ex-utero intrapartum treatment for airway obstruction. The underlying fetal pathology, surgical management, anaesthetic considerations and risks for both the mother and fetus are described for each. Fundamental to this is the understanding that clear communication and collaboration between all team members is vital to ensure successful outcomes of patients, the mother and the fetus.     

C-reactive protein as a predictor of fetal and maternal infective morbidity and fetal mortality

The value of maternal C-reactive protein (CRP) levels as predictors of fetal and maternal infective morbidity and fetal mortality was assessed prospectively over a 6-month period in all cases of premature rupture of the fetal membranes or suspected premature labour. Statistical analysis of results showed that CRP at a level of 1.32 mg/dl is a sensitive marker of infective morbidity in mother and neonate. Furthermore, there was a significant association between raised CRP levels and low-birth-weight babies, suggesting that intra-uterine infection is a major cause of prematurity in the study population.     

Maternal anesthesia and fetal neurodevelopment

It is clear from animal studies that commonly used anesthetic agents affect early brain development both histologically and functionally. With human epidemiologic evidence suggesting an association between anesthesia and surgery early in life and late-onset learning disabilities, investigators have focused their attention on the subtle long-term effects of anesthesia exposure. Most obstetric anesthesia studies, however, have focused on either the teratogenic effects of anesthetic agents in the first trimester or on the neonatal status immediately after delivery. Not much attention has been paid to the human second trimester, a period of active fetal brain development typified by neurogenesis and neuronal migration. Of concern though, is that these events are easily perturbed by environmental and pharmacological influences. New research studies have raised significant questions about the fetal impact of maternal anesthesia for non-obstetric and fetal surgery. This review summarizes the major findings in the field of developmental neurotoxicity of anesthetic agents, discusses the susceptibility of the fetal brain to anesthetic effects in a trimester-specific style, and outlines the pitfalls in extrapolating animal research to humans.     

Antenatal screening and fetal intervention

Congenital malformations are a common cause of reduced life expectancy and disability and can affect all body systems. Therefore, they are relevant to all surgeons. One in fifty children will have a major or significant congenital anomaly at birth. Since the advent of antenatal ultrasound (US) there has been the potential to diagnose many congenital anomalies before birth. This can forewarn parents and clinicians, so that treatment can be started promptly following birth rather than waiting for the baby to become symptomatic. It has also raised the possibility of treatment in-utero with the goal of stopping or reversing the pathological effects of the anomaly on the developing tissues, which will, hopefully, reduce morbidity and mortality once the baby is born and thus improve outcomes. This article focuses on congenital anomalies relevant to surgeons, initially looking at screening and diagnosis and how this may affect treatment and surgery following delivery. The second part will look at some of the interventions that have been attempted to treat ‘surgical’ congenital anomalies in-utero.     

Antenatal screening and fetal intervention

Congenital malformations are a common cause of reduced life expectancy and disability and can affect all body systems, thus are relevant to all surgeons. One in 50 children will have a major or significant congenital anomaly at birth. Since the advent of antenatal ultrasound (US) there has been the potential to diagnose many congenital anomalies before birth. This can forewarn parents and clinicians, so that treatment can be started promptly following birth rather than waiting for the baby to become symptomatic. It has also raised the possibility of treatment in utero with the goal of stopping or reversing the pathological effects of the anomaly on the developing tissues, which hopefully will reduce morbidity and mortality once the baby is born and thus improve outcomes. This article focuses on congenital anomalies relevant to surgeons, initially looking at screening and diagnosis and how this may affect treatment and surgery following delivery. The second part will look at some of the interventions that have been tried to treat “surgical” congenital anomalies in utero.     

Labour analgesia and fetal bradycardia

Canadian Journal of Anesthesia/Journal canadien d'anesthésie - Labour analgesia induction can result in fetal heart rate changes and even severe fetal bradycardia. This is most likely due...     

Echography and fetal urinary malformations

A great number of malformations of the foetal urinary tract can now be discovered during the antenatal period, due to the technological development of ultrasound in the light of foetal physiology: firstly the case of a severe oligohydramnios indicating a bilateral renal malformation and underlining its seriousness which is lethal in most cases: early severe oligohydramnios leads to very serious pulmonary hypoplasia of which the child will die at birth even if he only presents a surgically curable malformation such as lower urinary tract valves. This severe oligohydramnios is seen in bilateral renal agenesis, infantile polycystic kidney disease, multicystic bilateral dysplasia, lower urinary tract obstructions. Absence of oligohydramnios: unilateral malformation (it must be taken into consideration that normality of the contralateral kidney is not always easy to confirm by ultrasound). This is the case of unilateral hydronephrosis, unilateral renal agenesis, unilateral multicystic dysplasia, solid renal dysplasia, unilateral primary megaureter. Finally, complex malformations, where the diagnosis and management are problematic because the intensity of the effects is difficult to evaluate, the complexity of the malformation makes the diagnosis uncertain and lastly there exists an oligohydramnios which is more or less severe which makes for a difficult examination. This is the case of bilateral hydronephrosis, lower urinary tract obstructions, Prune Belly syndrome, bilateral primary megaureters, and megabladders.