两种脱细胞方法制备组织工程气管基质的比较

BACKGROUND: Acellular tracheal matrix is similar to the native trachea with structure and biological performance preserved after decellularization, and it is an important aim in tissue engineering to find an effective method of decellularization. OBJECTIVE: To select the optimal decellularization method through comparing chemical extraction method with detergent-enzymatic method for preparing rabbit tissue engineering trachea matrix. METHODS: Thirty tracheas from New Zealand white rabbits were randomly divide into three groups. Twenty of rabbit tracheas were subjected to decellularization using 2% TritonX-100 combined with deoxyribonuclease I and ribonuclease (chemical extraction method group), and sodium deoxycholate combined with deoxyribonuclease I (detergent-enzymatic group), respectively. The other ten were given no intervention as controls. Samples were collected and observed by hematoxylin-eosin staining, Masson-trichrome staining, safranin O staining, DAPI staining and scanning electronic microscope. RESULTS AND CONCLUSION: Hematoxylin-eosin staining demonstrated that compared with the control group, almost all cellular components of the mucosal epithelium were removed in the detergent-enzymatic and chemical extraction groups, and there were few remnant chondrocytes. Masson-trichrome staining indicated that compared with the control group, components of the mucosal layer chondrocytes in the chemical extraction and detergent-enzymatic groups were completely removed, with only part of remained chondrocytes in the cartilage zone. Glycosaminoglycan was slightly decreased both in the chemical extraction and detergent-enzymatic groups shown by Safranin O staining, but more reduction was found in the chemical extraction group. DAPI staining reveled that only a small amount of cartilage cells remained in the dense layer of cartilage and lacuna both in this two methods. Scanning electronic microscope showed that using the detergent-enzymatic method there were the hierarchical structures of native trachea, but slight disruption using the chemical extraction method. In conclusion, decellularized rabbit trachea matrix obtained by detergent-enzymatic method is better, with little disruption to the matrix. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

人胚胎气管壁组织发生的研究

目的：了解气管壁的组织发生及探讨婴幼儿呼吸道疾病的相关因素。方法：应用ＨＥ及ＰＡＳ反应,光镜观察。结果：８周的胚胎,气管壁由２－３层复层柱状上皮细胞及外周的间充质构成,上皮细胞的ＰＡＳ反应为阳性。     

Differential effects of phosphoramidon and captopril on NK1 receptor-mediated plasma extravasation in the rat trachea

We sought to confirm the identity of the tachykinin receptor subtype that mediates plasma extravasation in the rat trachea, and assess the respective contributions of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) in regulating this tachykinin-induced response. To achieve these aims, we determined the relative potencies of several natural tachykinins and receptor-selective synthetic agonists, both before and after inhibiting NEP with phosphoramidon and ACE with captopril. We also determined the effects of these peptidase inhibitors, and the NK-1 receptor antagonist L-703,606, on the plasma extravasation produced by capsaicin, which releases tachykinins endogenously from sensory nerve endings. We found that the rank order of potency for producing plasma extravasation in the rat trachea was NK-1 receptor agonist ([Sar⁹, Met(O₂)¹¹] SP)>substance P>neurokinin A> neurokinin B. The NK-2 ([Nle¹⁰]NKA (4–10)) and NK-3 ([MePhe⁷]NKB) receptor agonists were without effect. We observed no change in the relative potencies of these peptides after giving rats phosphoramidon or captopril, which suggests that the different peptide potencies are not simply the consequence of different rates of enzymatic degradation. Nevertheless, the responses to substance P and neurokinin A were clearly potentiated in rats given phosphoramidon, indicating that NEP effectively degrades tachykininsin vivo. No significant potentiation was evident for any peptide in rats given captopril. Similarly, the plasma extravasation produced by capsaicin was potentiated in rats given phosphoramidon, but not in those given captopril. Pretreating rats with L-703,606 abolished the response to capsaicin. We conclude from these observations that NK-1 receptors mediate tachykinin-induced plasma extravasation in the rat trachea, and that NEP regulates this response with little or no contribution from ACE.     

福尔马林对小鼠气管、肺、肝组织损伤的病理形态学观察

本实验观察了吸入福尔马林后小鼠气管、肺、肝组织的病理形态学改变。经福尔马林作用后,气管、肺、肝组织细胞均受到不同程度的损伤。其中对呼吸系统的影响是显著的。主要表现为气管纤毛的粘连、倒伏及脱失;Ⅱ型肺泡上皮细胞内成熟及接近成熟的板层体明显减少,部分出现空泡化现象。肝细胞内线粒体数目明显减少,肿胀及崤消失。损伤的可能机理是福尔马林对细胞生物膜的直接损伤作用。     

Biomechanical and angiogenic properties of tissue-engineered rat trachea using genipin cross-linked decellularized tissue

In this study, the obtainment and characterization of decellularized rat tracheal grafts are described. The detergent-enzymatic method, already used to develop bioengineered pig and human trachea scaffolds, has been applied to rat tracheae in order to obtain airway grafts suitable to be used to improve our knowledge on the process of tissue-engineered airway transplantation and regeneration. The results demonstrated that, after 9 detergent-enzymatic cycles, almost complete decellularized tracheae, retaining the hierarchical and mechanical properties of the native tissues with strong in vivo angiogenic characteristics, could be obtained. Moreover, to improve the mechanical properties of decellularized tracheae, genipin is here considered as a naturally derived cross-linking agent. The results demonstrated that the treatment increased mechanical properties, in term of secant modulus, without neither altering the pro-angiogenic properties of decellularized airway matrices or eliciting an in vivo inflammatory response.     

气管支气管损伤的外科治疗

目的探讨严重胸部损伤中气管、支气管损伤的及时诊断和治疗方法。方法回顾分析23例气管支气管损伤的临床特点、损伤部位、及时诊断和治疗的方法。结果4例叶支气管断裂行肺叶切除术，1例左主支气管完全断裂行切除左全肺，另1例左主支气管完全断裂行左全肺切除。剩余17例，3例气管膜部裂伤经保守治疗痊愈，14例气管支气管损伤者手术治疗修补断裂的气管支气管，术后复查胸片肺膨胀均良好。全组23例无死亡，术后无支气管胸膜瘘发生，均痊愈出院。结论严重胸部创伤的患者通常伴有气管或支气管损伤。如不能及时明确诊断，极易导致管腔狭窄或闭锁，影响患者肺功能。纤维支气管镜检查是尽早明确气管支气管损伤诊断和损伤部位的有效方法。     

Assessment of upper airway mechanics during sleep

Obstructive sleep apnea, which is the most prevalent sleep breathing disorder, is characterized by recurrent episodes of upper airway collapse and reopening. However, the mechanical properties of the upper airway are not directly measured in routine polysomnography because only qualitative sensors (thermistors for flow and thoraco-abdominal bands for pressure) are used. This review focuses on two techniques that quantify upper airway obstruction during sleep. A Starling model of collapsible conduit allows us to interpret the mechanics of the upper airway by means of two parameters: the critical pressure (P_crit) and the upstream resistance (R_up). A simple technique to measure P_crit and R_up involves the application of different levels of continuous positive airway pressure (CPAP) during sleep. The forced oscillation technique is another non-invasive procedure for quantifying upper airway impedance during the breathing cycle in sleep studies. The latest developments in these two methods allow them to be easily applied on a routine basis in order to more fully characterize upper airway mechanics in patients with sleep breathing disorders.     

Structure of the guinea-pig trachea at rest and in contraction

Summary The trachea of the guinea-pig measures about 47.5 mm in situ, and it shrinks to 38 mm when excised. It can be stretched to the in situ length with a load of 2–4 grams. The transverse area of its lumen measures about 4.5 mm² in the cervical portion, whereas in the lowermost thoracic portion it measures 2.8 mm², a difference of 37%. The lumen has an oval shape with the transverse diameter always exceeding the sagittal diameter. The separation between the ends of a cartilage in the dorsal region of the trachea is greater in the cervical than in the thoracic region. Elastic fibres are abundant in the tunica propria and run longitudinally; the collagen is in some areas arranged with a criss-cross pattern. The muscle is inserted on the concave aspect of each cartilage at some distance from the ends of the cartilage and it is therefore much longer than the gap between cartilage ends. Upon contraction in vitro (induced by carbachol) the muscle shortens by about 50%; there is a marked decrease of the transverse diameter of the trachea, and a certain decrease also of the sagittal diameter due to a straightening of the muscle and a change in shape and a movement of the mucosa. The cartilage ends are brought together and in the thoracic region they are bent and overlap extensively. The lumen of the trachea becomes circular and its area is reduced to 2.2 mm² in the cervical portion and 1.7 mm² in the thoracic portion.     

An image-processing technique for measuring the dynamic movement of cell membranes

An automated method has been developed to measure and compare the dynamic movement of cell membranes. Using the red blood cell as a common example the method locates the edge of the cell, with sub-pixel precision at multiple points on the periphery. This method is a different implementation to a technique used for giant unilamellar vesicles and addresses issues relating specifically to biological cells, in particular relating to finding a local minima, calculating equi-spaced measuring points for arbitrary shapes and using the perpendicular direction to the edge for position measurement. Parameters have been defined to characterise the cell's membrane behaviour and the analysis program allows the automatic compilation of multiple tests under varying conditions, and statistical comparison of identical populations of cells.     

间充质干细胞构建组织工程化气管的研究与应用

背景：间充质干细胞诸多特有的良好生物学特性使得其在组织工程化气管研究中成为理想的种子细胞来源。 目的：总结间充质干细胞在构建组织工程化气管领域的研究现状、进展、存在的问题及展望。 方法：由作者检索PubMed数据库及CNKI数据库1979至2012年,在英文标题和摘要中以“mesenchymal stem cells, tissue-engineered trachea”和“tracheal chondrocytes, tracheal epithelial cells, tracheal vascular endothelial cells” 检索,中文文献检索中以“间充质干细胞、组织工程化气管、气管软骨细胞、气管上皮细胞和气管血管内皮细胞”为关键词,选择与组织工程化气管相关的文章,同一领域文献则选择近期发表或发表在权威杂志的文章,共纳入51篇。 结果与结论：组织工程化气管种子细胞研究主要包括软骨细胞、上皮细胞和血管内皮细胞。实验已证实, 间充质干细胞可定向分化为软骨细胞,细胞因子在诱导间充质干细胞分化为软骨过程中起着关键作用。目前尚未发现合适的诱导因子能特异性诱导间充质干细胞定向分化气管上皮细胞。有研究发现间充质干细胞具有向上皮细胞分化的潜能。间充质干细胞在体内或体外特殊条件下可诱导分化为血管内皮细胞,为间充质干细胞作为种子细胞分化血管内皮细胞应用于组织工程化气管起到借鉴作用。     

Effects of lipopolysaccharide on the histomorphology and expression of toll-like receptor 4 in the chicken trachea and lung

Endotoxin or lipopolysaccharide (LPS) exposure can cause injury to the respiratory airways and in response, the respiratory epithelia express toll-like receptors (TLRs) in many species. However, its role in the innate immunity in the avian respiratory system is poorly understood. The aim of the present study was to evaluate the effects of LPS on the chicken trachea and lung. After intraperitoneal LPS or saline injection, the trachea and lungs were harvested at 0, 12, 36 and 72?h (n?=?6 at each time point) and histopathologically analysed using haematoxylin and eosin and periodic acid-Schiff staining, while TLR4 expression was determined by immunohistochemistry and secretory Immunoglobulin A (SIgA) levels by enzyme-linked immunosorbent assay. After LPS stimulation, we observed a remarkable decrease in the number of goblet cells along with obvious disruption and desquamation of the ciliated epithelium in the trachea, blurring of the boundary between pulmonary lobules, narrowed or indistinguishable lumen of the pulmonary atria and leukostasis in the lungs. Following LPS stimulation, TLR4 protein expression was up-regulated in both the trachea and the lungs and was found on the ciliated columnar cells as well as in the submucosa of the trachea, and in the lungs on parenchymal and immune cells. However, SIgA levels were only up-regulated in the trachea at 12?h following LPS stimulation. Hence, this report provides novel information about the effects of LPS on the microstructure of the lower respiratory tract and it is concluded that its intra-peritoneal administration leads to TLR4-mediated destruction of the tracheal epithelium and pulmonary inflammation along with increased SIgA expression in the tracheal mucosa.     

Assessment of a procedure for detecting minute levels of tooth erosion

Over a period of some years, the components of a system for discerning erosion on children's teeth have been progressively developed, for use in an extensive project seeking correlations between erosion and various perceived risk factors. The aim was the detection of minute levels of erosion, based on mappings of the palatal surfaces of the maxillary central incisors in children. Significant challenges were encountered, the primary problem being the impracticality of placing control marks that would aid the realignment of successive measurements. The paper describes the erosion detection system and initial experiences based on the results of the first 100 subjects measured after 9 months. The procedures detected the occurrence of erosion of 50 μm magnitude on about one-quarter of the teeth over the 9 month period, at a precision estimated to be ±15 μm. The occurrence of some anomalous incidents prevented the procedure from being fully automatic, and it necessitated human examination of a graphical diagram derived from the surface matching program, but it was nevertheless superior to current practices of examining impressions or replicas entirely by eye.     

Experience in the use of an image-processing workstation for a photostimulable phosphor radiographic system

Photostimulable phosphor radiographic (computed radiographic) systems are being installed and evaluated by radiology departments. The use of interactive image-processing workstations are a major advantage for any computed radiography system. An interactive image-processing workstation enables rapid image retrieval, reduces the examination repeat rate, provides for image enhancement, and rapidly sets the desired display parameters for laser-printed images. The authors have conducted over 2,500 radiographic examinations using a computed radiographic system equipped with an interactive image-processing workstation. Their experience supports the necessity of such a workstation.     

Evaluation of respiratory mechanics and lung histology in a model of atelectasis

To develop a reproducible model of atelectasis, 15 mechanically ventilated Wistar rats were wrapped around the thorax/abdomen with a sphygmomanometer. The cuff was inflated to transpulmonary pressures (PL) of -4 cmH2O (group A) and -8 cmH2O (group B) for 5 sec. Group C was not compressed. Airflow, volume, tracheal and oesophageal pressures were registered. Respiratory system (rs), lung (L), and chest wall resistive (DeltaP1), viscoelastic/inhomogeneous pressures (DeltaP2), DeltaPtot (=DeltaP1 + DeltaP2), static (Est) and dynamic (Edyn) elastances, and DeltaE (=Edyn - Est) were determined before and after compression. In A, respiratory mechanics remained unaltered. In B, Est,rs (+99%), Est,L (+111%), DeltaE,rs (+41%), DeltaE,L (+73%), DeltaP1,rs (+45%), DeltaP1,L (+44%), DeltaP2,rs (+41%), DeltaP2,L (+69%), DeltaPtot,rs (+40%), and DeltaPtot,L (+58%) increased after compression. Mean alveolar diameter and bronchiolar lumen decreased in A, and were even smaller in B. In conclusion, chest wall compression with PL of -8 cmH2O yielded a reproducible alveolar collapse, which resulted in increased elastic, resistive and viscoelastic/inhomogeneous pressures.     

Globule leukocytes and mast cells in the rat trachea: their number, distribution, and response to compound 48/80 and dexamethasone

Summary Globule leukocytes in the epithelium of the rat trachea may be counterparts of mucosal mast cells that are located in the gastrointestinal tract. If they are indeed similar to mucosal mast cells, globule leukocytes would be expected to decrease in number in rats treated with dexamethasone but not in rats treated with compound 48/80, an agent which causes non-antigenic degranulation of connective tissue mast cells. In this study, we determined the number and compared the distribution of globule leukocytes and connective tissue mast cells in the tracheas of pathogen-free rats. We then determined whether the number of these two types of cells changes in rats treated for 5 days with compound 48/80, dexamethasone, a combination of compound 48/80 and dexamethasone, or saline. We identified globule leukocytes and mast cells in whole mounts and histological sections of rat tracheas by using a histochemical reaction that demonstrates the chymotrypsin-like protease (chloroacetate esterase) present in mast cell granules. Using this method, we found that aproximately 225000 globule leukocytes were present in the epithelium of the trachea. These cells were most abundant in the rostral trachea. Rats treated with dexamethasone had a 91% reduction in the number of globule leukocytes with protease-containing granules, but rats treated with compound 48/80 had a normal number of these cells. We found some 55000 connective tissue mast cells in the same tracheas. Mast cells were most abundant in the posterior membrane of the caudal trachea and in the lamina propria between cartilaginous rings. Rats treated with compound 48/80 had a 96% reduction in mast cells with protease-containing granules, but rats treated with dexamethasone had a normal complement of mast cells. We conclude that globule leukocytes are abundant in the tracheas of healthy rats, are similar in morphology and pharmacological responses to mucosal mast cells located in other organs of rats, and are more numerous than and have a different distribution than connective tissue mast cells. Globule leukocytes in the tracheal epithelium may have a role in respiratory defenses similar to that of mucosal mast cells in other organs. Funded in part by National Institutes of Health Pulmonary Program Project Grant HL-24136. Dr. Tam is a fellow of the Parker B. Francis Foundation     

Changes in epithelial secretory cells and potentiation of neurogenic inflammation in the trachea of rats with respiratory tract infections

Summary In rats respiratory tract infections due to Sendai virus and coronavirus usually are transient, but they can have long-lasting consequences when accompanied by Mycoplasma pulmonis infections. Morphological alterations in the tracheal epithelium and a potentiation of the inflammatory response evoked by sensory nerve stimulation (neurogenic inflammation) are evident nine weeks after the infections begin, but the extent to which these changes are present at earlier times is not known. In the present study we characterized these abnormalities in the epithelium and determined the extent to which they are present 3 and 6 weeks after the infections begin. We also determined the magnitude of the potentiation of neurogenic inflammation at these times, whether the potentiation can be reversed by glucocorticoids, and whether a proliferation of blood vessels contributes to the abnormally large amount of plasma extravasation associated with this potentiation. To this end, we studied Long-Evans rats that acquired these viral and mycoplasmal infections from other rats. We found that the tracheal epithelium of the infected rats had ten times as many Alcian blue-PAS positive mucous cells as did that of pathogen-free rats; but it contained none of the serous cells typical of pathogen-free rats, so the total number of secretory cells was not increased. In addition, the epithelium of the infected rats had three times the number of ciliated cells and had only a third of the number of globule leukocytes. In response to an injection of capsaicin (150 g/kg i.v.), the tracheas of the infected rats developed an abnormally large amount of extravasation of two tracers Evans blue dye and Monastral blue pigment, and had an abnormally large number of Monastral blue-labeled venules, particularly in regions of mucosa overlying the cartilaginous rings. This abnormally large amount of extravasation was blocked by dexamethasone (1 mg/day i.p. for 5 days). We conclude that M. pulmonis infections, exacerbated at the outset by viral infections, result within three weeks in the transformation of epithelial serous cells into mucous cells, the proliferation of ciliated cells, and the depletion of globule leukocytes. They also cause a proliferation of mediator-sensitive blood vessels in the airway mucosa, which is likely to contribute to the potentiation of neurogenic inflammation that accompanies these infections.Funded in part by National Institutes of Health Pulmonary Program Project Grant HL-24136 from the US Public Health Service. Dr. Huang is the recepient of an award from the National Science Council of the Republic of China     

Age-related characteristics of epithelial-muscular interactions in the rat trachea

The interactions between epithelial and muscular cells of tracheal wall were studied by a mechanographic technique in rats of different ages. The state of the epithelium was assessed using hematoxylin-eosin stained sections. The epithelium-dependent relaxation was found to be most promounced in 10–12-week-old rats. It was not observed in younger rats probably due to the low sensitivity of guanylate cyclase to activating stimuli. Destructive changes in the respiratory epithelium after 24 weeks of life reduced the amplitude of epithelium-dependent relaxation in old rats. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 12, pp. 625–627, December, 1998     

Re-acceleration of the down-regulated contraction kinetics in the rat tracheal smooth muscle

The contraction kinetics of smooth muscle show a down-regulation after the transient rise found during sustained contraction. We tried to find out therefore if the contraction kinetics of rat tracheal smooth muscle can be re-accelerated during sustained activation. A 2 s length vibration (100 Hz sinusoidal; amplitude=6% of the muscle length) produces an immediate fall in the force developed by the activated muscle. A biexponential function was fitted to the force recovery. The reciprocal of the time constant,t ₂, describing the slow component of force recovery, reflects the kinetics of contraction. The contraction kinetics reach their highest levels (t ₂=4.9±0.1 s,n=166) about 30 s after the onset of electrical field stimulation. Three experimental groups were activated by either 10 M serotonin (5-HT), 100 M acetylcholine (ACh), or by 2 M ACh for 50 min. Approximately 10 vibrations were applied to each preparation after an 8 min activation in order to observe stabilized down-regulated contraction kinetics.t ₂ values were calculated from the force recovery after vibration and averaged 11.2±0.2 s (n=141), 11.5±0.2 s (n=137), and 11.1±0.3 s (n=84), respectively. After 50 min of continuous chemical activation, the preparation was stimulated additionally by the neurogenic release of acetylcholine. Thet ₂ of post-vibration force recovery, as measured after 30 s of neural activation, showed no change in the specimens basically activated by 100 M ACh (11.0±0.4 s,n=51). A decline int ₂, indicating accelerated kinetics, was observed in the groups which had been stimulated by 10 M 5-HT (5.9±0.2 s,n=51) and 2 M ACh (5.6±0.2 s,n=47). The re-accelerating effect of the second stimulus could be reproduced recurrently. The down-regulated contraction kinetics can be re-accelerated either by activating another receptor type in addition to the one already maximally stimulated or by increasing the stimulus mediated by one of the receptor types from half maximal to maximal strength. However, this is only possible if the additional activation is strong enough, as indicated by an increase in active force. It could be demonstrated that the slowing of the cross-bridge cycling rate is the result of a regulatory process and not the result of substrate deficiencies or refractoriness in the regulatory of contractile proteins.