Keratin 5 knockout mice reveal plasticity of keratin expression in the corneal epithelium

We have recently demonstrated that the keratin K3 gene, which is active in the suprabasal human corneal epithelium, is missing in the genome of the mouse. We show that a normal K3 gene exists in a wide variety of mammals while in rodents the gene is converted to a pseudogene with a very strong sequence drift. The availability of K5-/- mice provides a unique opportunity to investigate type-specific keratin function during corneal differentiation in the absence of both K5 and K3. Here, we report that the deletion of K5, which in wild-type mice forms a cytoskeleton with K12, does neither cause keratin aggregation nor cytolysis in the cornea. This is due to the induction of K4 in corneal epithelial cells, normally restricted to corneal stem stem cells residing in the limbus. Using a combination of antibodies and RT-PCR, we identified additional keratins expressed in the mouse cornea including K23 which was previously thought to be specific for pancreatic carcinomas. This reflects an unexpected complexity of keratin expression in the cornea. Our data suggest that in the absence of mechanical stress, corneal differentiation does not depend on distinct keratin pairs, supporting a concept of functional redundancy, at least for certain keratins.     

PCT及CRP在急性肠梗阻大鼠血清中的表达水平及其临床意义

目的:探讨降钙素原(Procalcitonin,PCT)及C反应蛋白(C-reactive protein,CRP)在急性肠梗阻大鼠血清中的水平及其临床意义。方法:将83只Wistar大鼠分为对照组(n=13)、假手术组(n=35)和急性肠梗阻组(n=35)。对照组大鼠采集标本后处死,肠梗阻组行开腹手术结扎回肠末端,假手术组仅行开腹手术。检测8 h、24 h、48 h、72 h及96 h血清PCT及CRP水平,观察急性肠梗阻大鼠回肠组织的病理学改变情况。结果:假手术组PCT与CRP水平在术后24 h内显著升高,48 h至96 h逐渐下降;各时间点PCT水平明显高于对照组,而CRP水平在实验结束时已恢复至正常水平。肠梗阻组PCT和CRP水平在各时间点均明显高于对照组,并逐渐增加,到实验结束时达到高峰;肠梗阻组PCT和CRP水平在48 h-96 h均显著高于假手术组,差异具有统计学意义(P0.05)。组织病理学检查显示,对照组大鼠肠壁粘膜结构正常,假手术组可见轻度病理改变,肠梗阻组大鼠回肠组织可见粘膜结构明显破坏,绒毛坏死,严重水肿和炎症细胞浸润。结论:血浆PCT和CRP水平能够反映肠梗阻的状态和肠粘膜受损程度。     

奥曲肽治疗急性粘连性肠梗阻疗效观察

目的:探讨生长抑素对腹部手术后急性粘连性肠梗阻的治疗作用。方法:70例急性粘连性肠梗阻患者随机分为观察组与对照组各35例。对照组予胃肠减压、灌肠、补液及抗感染治疗等常规治疗;观察组在此基础上加用奥曲肽(生长抑素类似物)0.1 mg皮下注射,每8h一次,治疗72 h。观察两组患者腹痛评分、腹痛缓解时间、胃肠减压量、肛门恢复排气时间、立卧位腹部平片、临床缓解情况。结果:观察组34例(97.1%)临床缓解,明显高于对照组的28例(80.0%)(P<0.05)。与对照组相比,观察组在治疗第1、2天后腹痛评分明显下降(均为P<0.01),腹痛缓解时间显著缩短(P<0.01),治疗第1、2、3天的胃肠减压量均显著减少(均为P<0.01),恢复排气时间也明显缩短(P<0.01)。结论:急性粘连性肠梗阻在常规治疗基础上加用生长抑素,可明显改善临床症状,提高疗效。     

双歧杆菌三联活菌胶囊在术后早期炎性肠梗阻治疗中的应用

目的 探究双歧杆菌三联活菌胶囊辅助治疗术后早期炎性肠梗阻的临床效果。方法 收集兰州大学第一医院2014年9月至2016年9月收治的术后早期炎性肠梗阻患者56例为研究对象,随机分为对照组和干预组,每组28例,对照组予以常规治疗,干预组在对照组治疗基础上加用双歧杆菌三联活菌胶囊。收集整理两组患者临床数据并分析比较临床治疗效果。结果 两组患者治疗总有效率差异无统计学意义（P>0.05）。干预组患者腹胀消失时间、肛门排气时间、肠鸣音恢复时间均较对照组短（P＜0.05）;两组患者治疗前后降钙素原下降及淋巴细胞绝对值增加差异均有统计学意义（P＜0.05）,且干预组治疗前后降钙素原降低及淋巴细胞绝对值增加幅度与对照组相比差异均有统计学意义（P＜0.05）。结论 双歧杆菌三联活菌胶囊用于术后早期炎性肠梗阻的治疗,可缩短治疗时间,促进肠道免疫功能恢复,且使用安全,值得临床推广。     

High-affinity binding of the NC1 domain of collagen VII to laminin 5 and collagen IV

Anchoring functions of collagen VII depend on its ability to form homotypic fibrils and to bind to other macromolecules to form heterotypic complexes. Biosensor-based binding assays were employed to analyze the kinetics of the NC1 domain-mediated binding of collagen VII to laminin 5, collagen IV, and collagen I. We showed that collagen VII interacts with laminin 5 and collagen IV with a Kd value of 10(-9) M. In contrast, the NC1-mediated binding to collagen I was weak with a Kd value of 10(-6) M. Binding assays also showed that the NC1 domain utilizes the same region to bind to both laminin 5 and collagen IV. We postulate that the ability of the NC1 domains to bind with high affinities to laminin 5 and collagen IV facilitates stabilization of the structure of the basement membrane itself and that the NC1-collagen I interaction may be less important for stabilization of the dermal-epidermal junction.     

The probiotic bacterium Lactobacillus plantarum species 299 reduces intestinal permeability in experimental biliary obstruction

AIMS: Extrahepatic biliary obstruction is associated with the failure of intestinal barrier function, allowing bacteria and other substances from the intestine to enter the circulation and initiate a systemic inflammatory response, causing impairment of organ function. Probiotic bacteria have been shown to have beneficial effects on intestinal barrier function in other conditions, but their effects have never been studied in biliary obstruction. Methods and Results: This study examined the effects of enteral administration of Lactobacillus plantarum species 299 (LP299) in oatmeal fibre compared with sterile oatmeal fibre in water or water alone in an animal model of biliary obstruction. Administration of LP299 was associated with reduced intestinal permeability compared with sterile oatmeal alone (0.262 +/- 0.105%vs 0.537 +/- 0.037%, P=0.019, percentage excretion of (14)Carbon), but there was no evidence of reduced endotoxin exposure or blunting of the systemic inflammatory response. Animals receiving sterile oatmeal fibre alone also failed to develop the hyperpermeability after biliary obstruction seen in animals receiving water only (0.512+/- 0.05%vs 0.788 +/- 0.18%), suggesting that oatmeal itself may have some beneficial effects on intestinal barrier function. Conclusion: Enteral administration of the probiotic bacterium LP299 reduces intestinal hyperpermeability associated with experimental biliary obstruction. Significance and Impact of the Study: This study provides insight to direct further work into the modulation of intestinal barrier function by probiotic bacteria.     

腹腔镜手术治疗急性粘连性肠梗阻的临床效果分析

目的:探讨腹腔镜手术处理急性粘连性肠梗阻的临床效果,为普外科手术提供参考。方法:选取2012年2月-2013年3月我院收治的74例急性粘连性肠梗阻患者的临床资料进行回顾分析。根据手术方式不同,将病例分为对照组和腹腔镜组,每组37例,对照组实施开腹手术,腹腔镜组行微创治疗。观察并比较两组患者的术中出血量、手术时间、下床活动时间、肠蠕动恢复时间、住院时间、复发率及术后并发症等。结果:腹腔镜组手术时间为(67.82±9.57)min,术中出血量为(296.48±33.24)mL,肠蠕动恢复时间为(11.12±1.33)d,下床活动时间为(6.05±1.85)d,住院时间为(8.44±1.63)d,复发率为10.81%,并发症发生率为13.51%;对照组手术时间为(88.16±8.94)min,术中出血量为(482.32±24.21)mL,肠蠕动恢复时间为(18.18±1.09)d,下床活动时间为(8.47±1.23)d,住院时间为(11.28±1.91)d,复发率为19.44%,并发症发生率为30.55%;腹腔镜组各项指标均优于对照组,差异显著具有统计学意义(P0.05)。结论:腹腔镜手术用于治疗急性粘连性肠梗阻具有手术时间短、出血少及并发症发生率低等优势,效果显著值得临床推广。     

腹腔镜手术治疗粘连性肠梗阻的疗效及安全性研究

目的：探讨腹腔镜粘连松解术治疗粘连性肠梗阻（AIO）的疗效,减少再梗阻率。方法：将120 例AIO 患者随机分为两组,每组60 例,开腹组实施开腹手术,腹腔镜组实施腹腔镜粘连松解术,观察两组术后恢复及并发症发生情况,对再梗阻危险因素进行Logistic 回归分析。结果：腹腔镜组术中失血量（73.48± 9.32）mL,少于开腹组的（207.45± 33.21）mL（P<0.05）;腹腔镜组手术、术后镇痛、下床活动、肠恢复蠕动、肛门恢复排气、拔除尿管及住院时间分别为（69.15± 10.13）min、（14.67± 7.23）h、（27.14± 7.04）h、（3.11± 0.96）d、（3.24± 1.02）d、（3.37± 1.23）d、（7.95± 3.05）d,均短于开腹组的（83.84± 9.24）min、（27.38± 8.02）h、（36.23± 5.87）h、（4.05± 1.35）d、（4.35± 1.74）d、（5.02± 2.13）d、（10.35± 3.71）d（P<0.05);腹腔镜组并发症发生率、再梗阻率分别为10.00%、10.00%,均低于开腹组的33.33%、40.00%（P<0.05);多因素Logistic 回归分析显示开腹手术、手术时间≥ 60 min 是再梗阻发生的独立危险因素。结论：腹腔镜手术治疗AIO 疗效优于开腹手术,而且并发症与再梗阻率低。     

Diseases of epidermal keratins and their linker proteins

Epidermal keratins, a diverse group of structural proteins, form intermediate filament networks responsible for the structural integrity of keratinocytes. The networks extend from the nucleus of the epidermal cells to the plasma membrane where the keratins attach to linker proteins which are part of desmosomal and hemidesmosomal attachment complexes. The expression of specific keratin genes is regulated by differentiation of the epidermal cells within the stratifying squamous epithelium. Progress in molecular characterization of the epidermal keratins and their linker proteins has formed the basis to identify mutations which are associated with distinct cutaneous manifestations in patients with genodermatoses. The precise phenotype of each disease apparently reflects the spatial level of expression of the mutated genes, as well as the types and positions of the mutations and their consequences at mRNA and protein levels. Identification of specific mutations in keratinization disorders has provided the basis for improved diagnosis and subclassification with prognostic implications and has formed the platform for prenatal testing and preimplantation genetic diagnosis. Finally, precise knowledge of the mutations is a prerequisite for development of gene therapy approaches to counteract, and potentially cure, these often devastating and currently intractable diseases.     

Non-protein-bound iron detection in small samples of biological fluids and tissues

Interest in the pro-oxidative nature of non-protein-bound-iron (NPBI) led to the development of an assay for its detection. The aim was to set up a reliable method of detecting NPBI in small samples of biological fluids and tissue. The method was based on preferential chelation of NPBI by a large excess of the low-affinity ligand nitrilotriacetic acid. To separate NPBI, a two-step filtration procedure was used. All glassware and plasticware were treated to minimize iron contamination. Measurements were performed in plasma, amniotic fluid, bronchoalveolar lavage, and brain tissues. The analytic system detected iron as ferric nitrate standard down to a concentration of 0.01 μM. The 1,2-dimethyl-3-hydroxy-4(1H)-pyridone-Fe(DHP-Fe) complex eluted with a retention time of about 2.6 min. The standard curve for the DHP-Fe complex was linear between 0.01 and 400 μM in water as well as in plasma, bronchoalveolar lavage, brain tissue, and amniotic fluid. The detection limit was 0.01 μM for all biological fluids and brain tissue. The data show that reliable measurements of NPBI are possible in studies on oxidative stress under experimental and clinical conditions. The possibility of investigating NPBI involvement in free-radical injury might be useful in all human diseases in which oxidative stress occur.     

Sustained phenotypic reversion of junctional epidermolysis bullosa dog keratinocytes: Establishment of an immunocompetent animal model for cutaneous gene therapy

Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 (alpha3beta3gamma2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the alpha3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretion of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials.     

不同来源乳铁蛋白对幼鼠肠道发育的影响

目的 探究哺乳期乳铁蛋白（lactoferrin,LF）的缺失及不同来源LF补充后对幼鼠肠道发育的影响。方法 以LF基因敲除型雌鼠作为哺乳母鼠造成幼鼠哺乳期无LF的摄入,且从幼鼠出生第3~21天每日人工饲喂100 mg/kg 牛血清白蛋白（BSA）、牛源乳铁蛋白（bovine Lactoferrin,bLF）及重组人源乳铁蛋白（recombinant human Lactoferrin,rhLF）,于幼鼠21日龄取样,测定各组小鼠小肠发育指标。结果 在本实验周期下,哺乳期rhLF的补充显著性增加小鼠回肠绒毛长度/隐窝深度值（P<0.05）,且上调回肠Occludin和ZO-1基因的表达（P<0.05）,增加小鼠十二指肠、空肠和回肠麦芽糖酶酶活/乳糖酶酶活比值（P<0.05）,表明哺乳期rhLF的补充能够增强小鼠肠道消化吸收能力和肠屏障功能;哺乳期bLF的补充显著增加小鼠十二指肠及回肠麦芽糖酶活性/乳糖酶活性比值（P<0.05）。结论 对于哺乳期无LF摄入的乳鼠来说,哺乳期间LF的补充能够增强乳鼠肠道对营养物质的消化吸收能力、促进肠道的发育成熟、增强肠道屏障功能,并且,本实验中rhLF表现出比bLF更加有效的作用。     

粘连性肠梗阻再手术治疗35例临床分析

目的观察粘连性肠梗阻再手术治疗的效果。方法对我院收治的35例粘连性肠梗阻再手术治疗患者的临床资料进行回顾性分析。结果粘连性肠梗阻患者再手术治疗的效果满意,住院时间为（9．8±5．7）天,随访6个月～4年,除1例患者出现肠梗阻复发,其余患者无复发。结论手术治疗术后粘连性肠梗阻应及时进行,可降低复发率,达到较好的疗效和预后。     

Growth and development of the term and premature pig-tailed macaque (Macaca nemestrina): Cardiovascular and respiratory changes during the first 3 weeks of life

Growth and development of the infant pigtailed macaque (Macaca nemestrina) over the first 3 weeks of life have been characterized by the variables of body weight, blood pressure, heart rate, ventilation, blood gas tensions, and lung mechanics. The effects of prematurity on postnatal developmental trends were assessed at gestational ages of 135–145 days (0.80 of term) and 150–155 days (0.91 of term). There was no indication that cesarean section, restraint, or instrumentation had any significant influence on the measurements. Gestational age at delivery had no effect on the minute ventilation per kilogram body weight, the hematocrit, or the heart rate; however, body weight, tidal volume, respiratory frequency, arterial gas tensions, blood pressure, and lung compliance did vary with gestational age at delivery. Postnatal maturational changes in these variables were similar between term and premature animals. The data for infant macaques and newborn humans were compared. The newborn macaque appears to be an excellent model of human developmental trends (and/or disease state) over the first 3 weeks of life, though some potentially important differences have been found.