大鼠糖尿病溃疡动物模型的初步研究

目的构建大鼠糖尿病溃疡动物模型,观察评价该模型的临床及病理特点。方法利用磁片循环压迫的方法,构建大鼠糖尿病溃疡动物模型,并从整体,组织和生化三个层次对糖尿病溃疡进行了研究。结果构建出了一个可以复制的糖尿病溃疡动物模型,该模型具有组织坏死、白细胞聚集以及高浓度晚期糖化终末产物等特征。结论利用缺血再灌注法构建了大鼠糖尿病溃疡动物模型。其病理改变与人极为相似,是一种很好的用于糖尿病溃疡发病机制和治疗研究的动物模型。     

Alterations in cytokeratin expression precede histological changes in epithelia of vitamin A-deficient rats

Summary Normal epithelial cell differentiation is charactezied by the production of distinct cytokeratin proteins. It is well known that epithelia of several organs show squamous metaplasia in a vitamin A-deficient status. It is not yet known whether these histological changes are concomitant with a change in cytokeratin expression. Therefore, 3-week-old female rats (BN/BiRij) were fed a vitamin A-deficient diet for 8 weeks. The cytokcratin expression in epithelia of various organs was monitored immunohistochemically during the induction of vitamin A deficiency. Therefore, monoclonal antibodies specific for human cytokeratin 4, 5, 5+8, 7, 10, 14, 18 and 19 were used. In a normal vitamin A status, the distributional pattern for the different cytokeratins in rats was similar to that reported for human tissue. No change in cytokeratin expression was seen in trachea, skin, liver and colon at any time point studied. Squamous metaplasia in urinary bladder and salivary glands was observed after six weeks on the vitamin A-deficient diet. This was concomitant with a substitution of cytokeratins 4, 5+8, 7, 18 and 19 by cytokeratin 10. The latter cytokeratin is specific for keratinzed squamous epithelium. A change in cytokeratin expression was observed in bladder, ureter, kidney, salivary glands, uterus and conjunctiva before histological alterations appeared. In conclusion, the changes in cytokeratin expression observed under vitamin A deficiency in epithelia in vivo are in agreement with those described in other studies for epithelial cells in vitro. The changes in cytokeratin expression and the subsequent differentiation into squamous cells occurs in basal cells of the bladder but not in transitional cells. Furthermore, histological alterations are preceded by changes in cytokeratin expression indicating that vitamin A status controls cytokeratin expression in vivo.     

Role of p38 mitogen-activated protein kinase in the healing of gastric ulcers in rats.

p38 belongs to the mitogen-activated protein kinase family and plays a crucial role in cellular responses to both cytokines and various stresses. We investigated the role of p38 in the healing of experimental gastric ulcers. Gastric ulcers were induced by submucosal injection of acetic acid solution into male rats. Western blotting and a kinase assay examined the p38 activity and phosphorylation state in ulcerated tisue. After orally administering FR167653 (p38 kinase inhibitor) for 3 to 14 days, the production level of cytokines and the protein-level expression of cyclooxygenase and inducible nitric oxide synthase were examined by enzyme-linked immunosorbent assay and Western blotting. Only in fibroblasts and macrophages/monocytes in ulcerated tissue, p38 was found to be phosphorylated with an elevated kinase activity level. FR 167653 inhibited the activity of p38, yet had no effect on its phosphorylation state. The drug significantly impaired ulcer healing (without affecting acid secretion) and angiogenesis in the ulcer base. The production of interleukin-1beta and tumor necrosis factor-alpha were significantly reduced after FR167653 treatment. In addition the expression of cyclooxygenase-2 and inducible nitric oxide synthase proteins increased PGE2 generation and NOx secretion in the ulcerated stomach were suppressed by FR167653. From these findings, we conclude that p38, activated by gastric ulceration, might play some role in the healing of gastic ulcers in rats.     

Immunohistochemical distribution of leptin in kidney tissues of melatonin treated diabetic rats

Abstract

We examined using immunohistochemistry the distribution of leptin in kidney tissues of melatonin treated, streptozotocin (STZ) diabetic rats. The animals were divided into five groups: control, sham, melatonin-treated, diabetic and melatonin-treated diabetic. Kidney sections were prepared and stained with hematoxylin and eosin, and Crossman's triple staining for histological examination. The immunohistochemical localization of leptin in the kidney tissue was determined using the streptavidin-biotin-peroxidase method. We determined that on days 7 and 14, the leptin immunoreactivity of the diabetic and melatonin-treated diabetic groups was weaker than for the other groups. Weak immunoreactivity was found in the proximal and distal tubules of the kidney in the diabetic and melatonin-treated diabetic groups on days 7 and 14, and strong immunoreactivity was found in the control, sham and melatonin groups. Melatonin application had no significant effect on leptin production in the kidney tissues of diabetic rats.     

Arterial disease in chronic leg ulceration: an underestimated hazard? Lothian and Forth Valley leg ulcer study.

Six hundred patients with chronic leg ulcers were interviewed and examined for evidence of arterial impairment. There were 827 ulcerated legs. Pedal pulses could not be felt in 94 (11%). A Doppler resting pressure index of 0.9 or less was found in 176 legs (21%). Risk factors for arterial impairment included age, ulceration affecting the foot, and a history of claudication, ischaemic heart disease, or cerebrovascular disease. Roughly half the patients with arterial impairment also showed the clinical features of chronic venous insufficiency. Careful assessment for arterial disease is mandatory before patients with chronic leg ulcers are treated with elastic compression.     

Expression of natural antimicrobial peptide beta-defensin-2 and Langerhans cell accumulation in epidermis from human non-healing leg ulcers

Chronic wounds like venous calf and diabetic foot ulcers are frequently contaminated and colonized by bacteria and it remains unclear whether there is sufficient expression of defensins and recruitment of epidermal Langerhans cells in the margin of ulcer compared to normal skin. The aim of this study was to examine immunohistochemically the expression of beta-defensin-2 (hBD2), GM-CSF, VEGF growth factors and accumulation of CD1a+ Langerhans cells (LC) in epidermis from chronic skin ulcers and to compare it to normal skin from the corresponding areas. Studies were carried out in 10 patients with diabetic foot, 10 patients with varicous ulcers of the calf and 10 patients undergoing orthopedic surgery (normal skin for control). Biopsy specimens were immunostained using specific primary antibodies, LSAB+ kit based on biotin-avidin-peroxidase complex technique and DAB chromogen. Results were expressed as a mean staining intensity. Statistical analysis of staining showed significantly higher staining of hBD2 in both normal and ulcerated epidermis from foot sole skin compared to calf skin (normal and ulcerated, p < 0.05). Chronic ulcers showed the same expression of hBD2 as normal skin. There was significantly lower accumulation of CD1a+ LC in normal epidermis from foot sole skin compared to normal calf skin (p<0.05). Accumulation of CD1a+ LC and GM-CSF upregulation at the border area of diabetic foot ulcer and reduction of LC concentration at the margin of venous calf ulcer compared to normal skin were observed. It seems that normal calf and sole epidermis is, unlike in the mechanisms of innate immunity, influenced by the different keratinocyte turnover and bacterial flora colonizing these regions. Insufficient upregulation of hBD2 in both diabetic foot and venous calf ulcers may suggest the pathological role of this protein in the chronicity of ulcers.     

Involvement of heat shock proteins in the healing of acetic acid-induced gastric ulcers in rats.

The present study examined the expression of 73-kDa of heat shock cognate protein (HSC70), 72-kDa of heat shock protein (HSP70) and 47-kDa of HSP (HSP47) observed in the ulcer healing process in rats. Gastric ulcers were induced by a luminal application of acetic acid in male Donryu rats. During the ulcer healing process, the expression of HSPs in the ulcerated tissue was determined. A high level of HSC70 expression was observed both in the normal mucosa and ulcerated tissue, but the level did not change upon ulceration and ulcer healing. While HSP70 and HSP47 were markedly expressed in the ulcer base during ulceration, and decreased with ulcer healing. HSP70 expression in the ulcer margin was gradually increased with ulcer healing. Omeprazole accelerated the healing of gastric ulcers with strong inhibition of gastric acid secretion, while indomethactin delayed in ulcer healing despite slight inhibition of gastric acid secretion. Omperazole enhanced the expression of HSP70 both in the ulcer margin and base, but it reduced HSP47 expression in the ulcer base Indomethacin markedly enhanced HSP47 expression only in the ulcer base. In conclusion, the expression of HSP70 and HSP47 is changed during ulcer healing. Furthermore, it was suggested that the enhanced expression of HSP70 is involved in acceleration of ulcer healing, but overexpression of HSP47 is involved in delayed ulcer healing.     

Expression of 'visceral' cytokeratin and ultrastructural findings in solid cell nests of the thyroid

Solid cell nests of the thyroid display histological features that resemble squamous epithelium. In the present study we investigated whether these ultimobranchial nests express a renal tubular cell cytokeratin which is found to be widely distributed in epithelia but does not appear in squamous tissue. All 17 solid cell nests studied, in contrast to squamous epithelium lining an intrathyroid thyroglossal cyst, stained positively for this intermediate filament. These findings, together with the gland-like features shown ultrastructurally by these ultimobranchial remnants, further support the view that solid cell nests of the thyroid should not be regarded as a kind of intrathyroid squamous inclusion. In addition, they were electron-microscopically comparable to mammalian ultimobranchial tissue.     

Method of healing diabetic forefoot ulcers.

Six diabetic patients with neuropathic ulceration of the sole of the foot (seven feet, eight ulcers) were treated by the application of a below knee walking plaster with a rubber rocker. All the ulcers healed with this treatment, greatly reducing the usual period of hospital inpatient stay. After healing, study of the forces acting on the sole of the foot showed that these ulcers occur at the site of maximal horizontal shear force and confirmed that they occur at the site of maximal vertical force. This treatment is highly effective for neuropathic ulcers of the sole not affecting bone or complicated by deep sepsis. There may be a high rate of recurrence, however, reflecting inadequacy of methods of protecting damaged neuropathic feet.     

The diabetic rat as an impaired wound healing model: stimulatory effects of transforming growth factor-beta and basic fibroblast growth factor

Two models of wound repair compared the effect of defined, recombinant growth factors on the rate of wound repair in both normal and streptozotocin-induced diabetic rats: subcutaneous implantation of polyvinyl alcohol sponges and incisional wounding. Transverse incisional wounds were made on the dorsal surface of rats and closed with steel sutures. Three days postwounding the rats received a single injection of either transforming growth factor-beta or vehicle alone directly into the wound site. Animals were sacrificed 7, 14, and 21 days postwounding, and fresh and formalin-fixed wound tensile strength were measured. Diabetic rats had expected defects in wound repair, including decreased granulation tissue and reduced amounts of collagen, protein, and DNA. Fresh tensile strength of the diabetic incisions was 53% of normal on Day 7 (p < or = .01) and 29% of normal on Day 21. Fixed tensile strength was 41% of normal on Day 7 (p < or = .01) and fell to 78% of normal by Day 21 (p < or = .01), suggesting that collagen concentrations of diabetic wounds increased towards normal but did not undergo maturation. TGF beta produced a moderate increase in tensile strength of fresh and fixed wounds of diabetic rats, but not to the levels of wounds in untreated normal rats. Sponges fill with granulation tissue, their reproducible rate of organization being measured by histological and biochemical methods. A single injection into sponges 3 days postimplantation of basic fibroblast growth factor, transforming growth factor-beta, or vehicle only, was evaluated at 7 and 9 days postimplantation. In the sponge model, bFGF and TGF beta were each able to induce significant increases in the accumulation of granulation tissue in both diabetic and normal rats. TGF beta increased the collagen content of sponges by 136% in sponges from diabetic animals (p < or = .001), thereby raising the collagen content to that of normal control wounds, while stimulating a 49% (p < or = .02) increase in sponges from normal animals on Day 9. By contrast, the response to bFGF was predominantly an increase in the protein and DNA content of the sponges. These results emphasize the differential effects of the two cytokines in accelerating healing under conditions of defective wound repair.     

Differential diagnosis of Charcot arthropathy and osteomyelitis

Foot problems are common causes of morbidity in patients with diabetes mellitus. Foot ulcers are the leading cause of hospitalization in diabetic patients. Bones may be involved in two different clinical conditions: osteomyelitis and Charcot osteoarthropathy. Osteomyelitis usually develops by spreading from contiguous soft tissue to underlying bone. Charcot foot is deformation of foot as a result of muscle athrophy, bone and joint structure changes in a joint as a secondary complication of neuropathy. To distinguish bone infection from non-infectious bone disorders as in Charcot joint may be difficult, especially if there is no skin ulceration. So, the mere absence of skin ulcers does not exclude the diagnosis of osteomyelitis.     

Distribution of cytokeratin polypeptides in epithelia of the adult human urinary tract

Cytokeratin expression was studied in the epithelia lining the normal human urine conducting system using immunohistochemistry on frozen sections employing a panel of 14 monoclonal antibodies. Eleven of these anticytokeratin antibodies reacted specifically with one of the 19 human cytokeratin polypeptides. Profound differences were found in the cytokeratin expression patterns between the different types of epithelium in the male and female urinary tract. In the areas showing morphological transitions of transitional epithelium to columnar epithelium and of nonkeratinizing squamous epithelium to keratinizing squamous epithelium gradual shifts of cytokeratin expression patterns were observed, often anticipating the morphological changes. However, also within one type of epithelium, i.e. the transitional epithelium, two different patterns of cytokeratin expression were found. Expression of cytokeratin 7 was homogeneous in the transitional epithelium of renal pelvis and ureter but heterogeneous in the transitional epithelium of the bladder. Furthermore, intraepithelial differences in cytokeratin expression could be shown to be differentiation related. Using a panel of chain-specific monoclonal antibodies to cytokeratins 8 and 18 conformational and/or biochemical changes in the organization of these intermediate filaments were demonstrated upon differentiation in columnar and transitional epithelium.     

Study of Disease Progression and Relevant Risk Factors in Diabetic Foot Patients Using a Multistate Continuous-Time Markov Chain Model

The diabetic foot is a lifelong disease. The longer patients with diabetes and foot ulcers are observed, the higher the likelihood that they will develop comorbidities that adversely influence ulcer recurrence, amputation and survival (for example peripheral arterial disease, renal failure or ischaemic heart disease). The purpose of our study was to quantify person and limb-related disease progression and the time-dependent influence of any associated factors (present at baseline or appearing during observation) based on which effective prevention and/or treatment strategies could be developed. Using a nine-state continuous-time Markov chain model with time-dependent risk factors, all living patients were divided into eight groups based on their ulceration (previous or current) and previous amputation (none, minor or major) status. State nine is an absorbing state (death). If all transitions are fully observable, this model can be decomposed into eight submodels, which can be analyzed using the methods of survival analysis for competing risks. The dependencies of the risk factors (covariates) were included in the submodels using Cox-like regression. The transition intensities and relative risks for covariates were calculated from long-term data of patients with diabetic foot ulcers collected in a single specialized center in North-Rhine Westphalia (Germany). The detected estimates were in line with previously published, but scarce, data. Together with the interesting new results obtained, this indicates that the proposed model may be useful for studying disease progression in larger samples of patients with diabetic foot ulcers.     

Dynamics of changes in vascular endothelial growth factor (VEGF) expression and angiogenesis in stress-induced gastric ulceration in rats.

Angiogenesis and VEGF play a major role in many repair processes such as healing of gastric ulceration.The present study was undertaken to assess the dynamics of changes in VEGF expression and angiogenesis in stress-induced gastric ulcers in rats. Acute gastric ulceration was induced using a water-immersion and restraint stress method. The VEGF expression, angiogenesis, size of area and depth of ulcers in gastric specimens were evaluated. The study shows that as early as one day after the development of ulcers there is a significant increase in both the expression of the VEGF protein and the number of newly formed microvessels, while an abrupt decrease in VEGF expression, observed on the fifth day, results in a decreased intensity of angiogenesis. Moreover, it has been demonstrated that the increase in VEGF expression and angiogenesis is accompanied by a reduction in the size of area and depth of stress-induced ulcers in rats. Six days after ulcer development both VEGF expression and angiogenesis return to normal levels.     

Kinetics and collagenolytic role of eosinophils in chronic gastric ulcer in the rat

 An association between eosinophils and tissue damage has been observed in numerous disorders. However, few reports have addressed the role of infiltrating eosinophils in gastric ulcer healing. The aim of this study was to investigate the kinetics and role of eosinophils infiltrating experimental chronic gastric ulcers in the rat. We developed a monoclonal antibody against human matrix metalloproteinase 1 (MMP1) purified from conditioned culture medium of human skin fibroblasts. Acetic acid-induced gastric ulcers were resected from rats on days 1, 3, 5, 10, 20, 40, and 180 after the days of induction (day 0). Tissue specimens were immunostained with this antibody and examined with an electron microscope. Few eosinophils were observed in the granulation tissue until day 20. By days 40 and 180, MMP1-positive eosinophils had increased in the granulation tissue of open ulcers. Azan staining revealed dispersed collagen fibers around infiltrating eosinophils. In contrast, scars demonstrated few eosinophils in fibrous tissue on days 40 and 180. Eosinophils which express MMP1 infiltrate granulation tissue at the chronic stage of gastric ulceration. The results suggest that eosinophils may play a role in tissue remodeling and deterioration of ulceration. Accepted: 18 March 1997     

自体富血小板凝胶联合冷沉淀制剂对糖尿病足溃疡患者创面愈合、血管生成因子及生活质量的影响

目的:探讨自体富血小板凝胶联合冷沉淀制剂对糖尿病足溃疡患者创面愈合、血管生成因子及生活质量的影响。方法:选取西宁市第一人民医院于2016年8月～2018年8月间收治的糖尿病足溃疡患者54例,依据数表法将患者随机分为对照组(n=27)和观察组(n=27)。对照组采用常规基础治疗,观察组在对照组基础上序贯使用自体富血小板凝胶联合冷沉淀制剂治疗。比较两组疗效、溃疡愈合时间、住院时间,比较两组患者治疗前及治疗20d后的肉芽组织中碱性成纤维细胞生长因子(bFGF)和血管内皮细胞生长因子(VEGF)表达水平以及糖尿病患者生存质量特异性量表评分。结果:观察组总有效率高于对照组(P0.05)。观察组溃疡愈合时间和住院时间均明显短于对照组,差异有统计学意义(P0.05)。治疗20d后,两组患者肉芽组织bFGF、VEGF表达水平均明显升高(P0.05),且观察组肉芽组织bFGF、VEGF表达水平均明显高于对照组(P0.05)。治疗20d后,两组患者生理功能、社会关系、心理或精神以及治疗影响四个维度评分均明显下降(P0.05),且观察组低于对照组(P0.05)。结论:自体富血小板凝胶联合冷沉淀制剂治疗糖尿病足溃疡疗效确切,可提高患者生活质量,可促进肉芽组织中血管生成和创面愈合。     

Cyclooxygenase-2/prostaglandin E2 accelerates the healing of gastric ulcers via EP4 receptors

We examined the involvement of cyclooxygenase (COX)-1 as well as COX-2 in the healing of gastric ulcers and investigated which prostaglandin (PG) EP receptor subtype is responsible for the healing-promoting action of PGE2. Male SD rats and C57BL/6 mice, including wild-type, COX-1(-/-), and COX-2(-/-), were used. Gastric ulcers were produced by thermocauterization under ether anesthesia. Gastric ulcer healing was significantly delayed in both rats and mice by indomethacin and rofecoxib but not SC-560 given for 14 days after ulceration. The impaired healing was also observed in COX-2(-/-) but not COX-1(-/-) mice. Mucosal PGE2 content increased after ulceration, and this response was significantly suppressed by indomethacin and rofecoxib but not SC-560. The delayed healing in mice caused by indomethacin was significantly reversed by the coadministration of 11-deoxy-PGE1 (EP3/EP4 agonist) but not other prostanoids, including the EP1, EP2, and EP3 agonists. By contrast, CJ-42794 (selective EP(4) antagonist) significantly delayed the ulcer healing in rats and mice. VEGF expression and angiogenesis were both upregulated in the ulcerated mucosa, and these responses were suppressed by indomethacin, rofocoxib, and CJ-42794. The expression of VEGF in primary rat gastric fibroblasts was increased by PGE2 or AE1-329 (EP4 agonist), and these responses were both attenuated by coadministration of CJ-42794. These results confirmed the importance of COX-2/PGE2 in the healing mechanism of gastric ulcers and further suggested that the healing-promoting action of PGE2 is mediated by the activation of EP4 receptors and is associated with VEGF expression.     

Cytokeratin expression in human thymus: immunohistochemical mapping

Cytokeratin expression in normal postnatal human thymus was studied immunohistochemically by using monoclonal antibodies against various cytokeratin polypeptides. An attempt was made to characterize cell populations giving rise to the cornified structures of Hassal's corpuscles. Monoclonal antibody KB-37, a marker of squamous epithelium basal cells, was applied to distinguish the earliest cells capable of undergoing squamous differentiation. Parts of the subcapsular epithelium were extensively stained with this reagent. This epithelium, like the basal layer of certain squamous epithelia, exibited a high incidence of cytokeratins 13 and 14, and pronounced expression of cytokeratin 19. Simple epithelium cytokeratins 8, 18, and 19 were present in the cortex. Scattered cells reacted with KB-37 antibody. All stellate epithelial cells in the medulla were positive for cytokeratin 19. Most of the medullar epithelial cells were positive for cytokeratins 13, 14 and 17 of complex epithelium, in contrast to the cortex, where only a few cells were positive for these cytokeratins. A significant proportion of the medullar cells was positive for KB-37 antigen. Cytokeratins 8 and 18 were expressed in single cells and in groups of cells surrounding Hassal's corpuscles. The outermost cells of these corpuscles were positive for cytokeratin 19 and KB-37. In the peripheral parts of Hassal's corpuscles, simple epithelium cytokeratins 7, 8, 18, and cytokeratins 4, 13, 14, and 17, characteristic of stratified nonkeratinizing epithelia, were coexpressed with keratinization-specific cytokeratins 10/11. The inner parts of the swirls were uniformly positive for cytokeratins was reduced.