神经肽P物质对糖尿病动物模型伤口愈合影响的研究

目的 研究外用神经肽P物质（SP）对糖尿病大鼠动物模型伤口愈合的影响及意义。方法 2005年3月至2005年6月对重庆市第三军医大学西南医院中心实验室的外用SP及其拮抗剂，观测不同时相点糖尿病动物模型伤口愈合速度的影响。结果 加入SP实验组糖尿病大鼠伤口愈合最快，拮抗剂组伤口愈合更加延迟，与SP结合起作用的主要是NK1受体，加入NK1受体拮抗剂可阻断SP主要作用。结论 外用SP可促糖尿病伤口愈合。     

P物质NK1受体对大鼠结肠动力的调节作用

[目的]探讨P物质(substance P,SP)不同受体对大鼠结肠动力的调节作用。[方法]取大鼠近端结肠制备纵行肌(LMS)及环形肌(CMS)肌条,用张力换能器及多通道生理信号采集处理系统观察SP及其受体拮抗剂对SP诱导的LMS和CMS收缩活动的影响。[结果]SP显著促进大鼠结肠LMS和CMS自发性收缩活动[LMS:正常(0.85±0.06)g∶SP(1.02±0.05)g;CMS:正常(0.22±0.03)g∶SP(0.81±0.07)g,P0.01];CMS孵育河豚毒素TTX后加入SP收缩幅度显著增加[正常(1.15±0.11)g∶SP(2.25±0.18)g,P0.05];NK1受体特异性阻断剂L-703孵育肌条后加入SP,平滑肌收缩无显著增强,NK2受体特异性阻断剂GR159孵育后,SP能显著诱导平滑肌收缩增强(P0.05)。[结论]NK1和NK2受体均参与SP对结肠动力的调节,且对CMS的调节过程中,NK1受体发挥更重要的作用,SP对LMS的调节可能主要通过肠神经系统。     

芦荟凝胶促进糖尿病创面溃疡愈合的机制

目的在分子水平探讨芦荟凝胶促进糖尿病创面溃疡愈合的机制。方法实验分为3组,A组为正常组,B组和C组应用链脲佐菌素诱导的糖尿病大鼠模型并建立全皮伤口模型,B组给予生理盐水处理创面,C组给予芦荟凝胶干预,4次/d,通过胶原纤维-Masson三色法和免疫组织化学法评估伤口形成后3、7、14 d皮肤组织的组织病理学改变,采用逆转录-聚合酶链反应(RT-PCR)检测大鼠皮肤基质金属蛋白酶(MMPs)-9和金属蛋白酶抑制剂(TIMPs)-1 mRNA的表达情况。结果 B组大鼠伤口愈合明显迟缓。芦荟凝胶干预可加速糖尿病大鼠大鼠伤口愈合。伤口愈合过程中,B组MMP-9 mRNA水平持续高于A组(P0.01),而TIMP-1 mRNA水平持续低高于A组(P0.01);给予芦荟凝胶干预的C组MMP-9 mRNA和TIMP-1 mRNA水平仅在伤口形成第3天与A组相比具有统计学差异,其余时间点均与A组相近。芦荟凝胶干预可降低大鼠伤口组织MMP-9 mRNA/TIMP-1 mRNA比值。结论糖尿病大鼠皮肤伤口愈合速度明显减慢,芦荟凝胶干预可加速糖尿病大鼠伤口愈合,芦荟凝胶的这种作用可能是通过调控MMP-9/和TIMP-l水平实现的。     

A型肉毒毒素对P物质引发的离体食管下括约肌收缩的抑制作用

目的观察A型肉毒毒素(BTX-A)对电场刺激(EFS)、内源性及外源性P物质(SP)引发的SD大鼠离体食管下括约肌(LES)收缩的影响。方法 SD大鼠叩击头部致昏后剪取食管下段与贲门交界处组织制备肌条。使用EFS诱发肌条内源性递质释放,阿托品阻断胆碱受体作用后,观察NK1受体拮抗剂(APTL-SP)、BTX-A对LES收缩的影响;观察不同浓度APTL-SP、BTX-A,对外源性SP引发的LES收缩变化影响。采用Biolap BL-420E生物机能实验系统同步记录肌条的收缩变化曲线。结果 EFS可增强LES的收缩张力及振幅(P<0.01),BTX-A、阿托品、APTL-SP均可单独抑制其效应(P<0.05或P<0.01),而阿托品作用后BTX-A可进一步降低LES的收缩张力及振幅(P<0.05);EFS的增强LES收缩效应,在被阿托品抑制后,可被后加入的APTL-SP进一步抑制(P<0.05);阿托品与APTL-SP对EFS引发的LES收缩的协同抑制效应和BTX-A的抑制效应对比,二者无统计学差异(P>0.05)。外源性加入的SP可显著增强LES收缩张力(P<0.01)及振幅(P<0.05)。结论 BTX-A可抑制EFS诱发的内源性SP引发的LES缩增强,其对外源性SP引发的LES收缩也具有抑制效应。     

氯沙坦及苯那普利对糖尿病大鼠肾脏细胞凋亡的影响

糖尿病肾脏组织中细胞凋亡增多 ,并伴随着凋亡相关基因的改变[1] 。本研究观察了血管紧张素转换酶抑制剂(ＡＣＥＩ)苯那普利和血管紧张素Ⅱ受体 1拮抗剂 (ＡＴ1Ｒａ )氯沙坦对糖尿病大鼠肾功能及肾脏细胞凋亡的影响 ,以观察这两种药物的作用是否为部分通过影响肾脏细胞凋亡而实现的。一、材料与方法1.动物模型及分组 :选取体重 12 0～ 140ｇＳＤ大鼠 96只(河北省实验动物中心提供 ) ,所有动物均用戊巴比妥钠 (4 0ｍｇ/ｋｇ)腹腔注射麻醉 ,行右肾切除术 ,2周后切口完全愈合 ,将大鼠随机分为Ａ组 :右肾切除对照组 ;Ｂ组 :糖尿病 (ＤＭ)组 ;Ｃ…     

脊髓损伤大鼠体内P物质变化对成骨细胞分化的影响

目的探讨P物质（SP）对脊髓损伤（SCI）大鼠骨髓间充质干细胞（MSCs）源性成骨细胞分化的影响及机制。方法取SCI大鼠双侧胫骨、股骨冲洗髓腔后收集细胞悬液,进行骨髓MSCs源性成骨细胞诱导培养。观察SP不同浓度（10^-12、10^-10、10^-8、10^-7、10^-6M）、不同作用时间（1、2、3周）对成骨细胞碱性磷酸酶（ALP）活性的影响,观察SP与Spantide（SP抑制剂）、L703606（NK1受体抑制剂）配伍作用后细胞ALP活性。结果与对照组比较,不同浓度的SP作用后细胞ALP活性均明显降低,10^-8M浓度时最低,P均〈0.05;随着作用时间的延长ALP活性降低明显,P〈0.05。与SP单独作用比较,SP与Spantide和L703606配伍后细胞ALP活性均明显升高,P〈0.05。结论SP通过NK1受体对成骨细胞的分化产生影响;SP抑制成骨细胞的分化,使成熟的成骨细胞减少,可导致骨质疏松的发生。     

牛奶和1型糖尿病——关于粘膜免疫功能的争论[Leonard CH. Diabetes,1999,48:1501]

通过对生物喂养模型(BB)大鼠和非肥胖糖尿病(NOD)小鼠饮食与糖尿病发病率的研究,以及在人类流行病学的调查结果表明,在婴幼儿期喂牛奶和／或非母乳喂养是1型糖尿病的危险因素之一。美国儿科学会1994年和1997年推荐在有1型糖尿病家族史的婴儿中进行母乳喂养并推迟接触牛奶的时间。毋须置疑母乳喂养的好处,但对于牛奶与1型糖尿病的关系,目前尚无确凿证据,然而牛乳的争论引发了基因型、粘膜免疫功能和1型糖尿病之间的可能联系。 一、饮食和啮齿类1型糖尿病动物模型 在BB大鼠和NOD小鼠,用合成氨基酸和酪蛋白降解产物喂养组与未经处理的酪蛋白喂养组相比,糖尿病发生率明显降低,但在BB大鼠,如果加入25％的酪蛋白作为唯一蛋白质来源或小牛血清白蛋白或牛奶蛋白,并不能逆转这种保护作用。Scott等人的研究表明,植物尤其是小麦和大豆中的某些成分,是导致BB大鼠发生糖尿病的主要饮食因素。在NOD小鼠则尚无此类依据。Elliott等人的进一步研究表明,A1酪蛋白的降解产物β-casomorphin(鸦片受体激动剂)有致糖尿病作用,并能被鸦片受体拮抗剂纳洛酮完全阻断。     

丝胶对2型糖尿病大鼠肝脏胰岛素受体底物-2表达的影响

目的研究丝胶对2型糖尿病大鼠肝脏胰岛素受体底物(IRS)-2表达的影响。方法 36只雄性SD大鼠随机分为正常对照组、糖尿病模型组、丝胶治疗组,每组12只。采用链脲佐菌素连续腹腔注射法制作2型糖尿病大鼠模型。待模型成功建立后,丝胶治疗组大鼠给予丝胶(2.4 g·kg~(-1)·d~(-1))灌胃。SP免疫组织化学染色、免疫印迹法和RT-PCR检测大鼠肝脏组织IRS-2的表达。结果丝胶可明显提高糖尿病模型大鼠肝脏组织IRS-2阳性细胞数、IRS-2蛋白和mRNA的表达(P0.01)。结论丝胶可通过上调糖尿病大鼠肝脏IRS-2的表达,影响胰岛素的信号转导和改善胰岛素抵抗,起到降低血糖的作用。     

缬沙坦对2型糖尿病大鼠肾脏保护作用的研究

目的　了解血管紧张素受体拮抗剂缬沙坦对 2型糖尿病大鼠肾脏病变的保护作用及其机制。　方法　高糖、高脂饮食加小剂量链脲佐菌素 (STZ) 30mg/kg制作 2型糖尿病动物模型 ,将实验动物分为正常对照组、糖尿病组及缬沙坦治疗组 ,检测各组大鼠的血糖、血胰岛素、血肌酐、尿素氮、尿白蛋白、肾脏肥大指数的变化 ,免疫组化检测PAI 1的蛋白表达水平。　结果　缬沙坦治疗组较非治疗组尿白蛋白明显减少 (P <0 .0 1) ,尿素氮水平下降 (P <0 .0 5 ) ,肾脏肥大指数改善 (P <0 .0 5 ) ,免疫组化显示 ,糖尿病组大鼠肾小球PAI 1蛋白表达增多 ,治疗组PAI 1蛋白表达明显降低。　结论　缬沙坦对 2型糖尿病肾脏病变有一定的保护作用 ,其机制可能是通过下调糖尿病大鼠PAI 1的表达     

吡格列酮对糖尿病大鼠肾脏的保护作用

目的　探讨吡格列酮对糖尿病大鼠肾脏的保护作用及其机制。方法　采用链脲佐菌素诱导制备糖尿病大鼠动物模型 ,给予吡格列酮3mg· kg- 1· d- 1和 9mg· kg- 1· d- 1治疗 ,共 1 2 w。结果　给予治疗 1 2 w后 ,糖尿病治疗组大鼠尿蛋白定量 ,肾小球肥大均较糖尿病组显著降低 ,并可降低肾小球内细胞总数和减少肾小球内增殖细胞的数量 ,抑制系膜基质的增生和减轻尿蛋白的排泄。结论　吡格列酮对糖尿病大鼠肾脏具有保护作用。其防治糖尿病肾病发生、发展的作用机制可能部分是通过抑制糖尿病大鼠肾脏 PCNA过度表达。     

Promotion of corneal epithelial wound healing in diabetic rats by the combination of a substance P-derived peptide (FGLM-NH<Subscript>2</Subscript>) and insulin-like growth factor-1

AIMS/HYPOTHESIS: The healing of corneal epithelial wounds is often delayed in individuals with diabetes. The effect of the combination of a substance P-derived tetrapeptide (phenylalanine-glycine-leucine-methionine amide, or FGLM-NH(2)) and insulin-like growth factor-1 (IGF-1) on corneal epithelial wound healing was investigated in rats with streptozotocin-induced diabetes. METHODS: The corneal epithelium of diabetic and non-diabetic rats was removed, and the animals were treated by the application of eye drops containing FGLM-NH(2) and IGF-1, or vehicle alone as a control, six times a day for 3 days. The area of the corneal epithelial wound was measured at various times up to 72 h after removal of the corneal epithelium. RESULTS: The rate of corneal epithelial wound healing was slower in diabetic rats treated with vehicle than in non-diabetic rats. However, the rate of wound closure in diabetic rats treated with FGLM-NH(2) and IGF-1 was markedly increased compared with that in diabetic rats treated with vehicle. The wound healing process seemed similar in normal rats and in diabetic rats treated with FGLM-NH(2) and IGF-1. CONCLUSION/INTERPRETATION: The combination of FGLM-NH(2) and IGF-1 promotes corneal epithelial wound healing in diabetic rats, suggesting that such a treatment might prove effective in humans with diabetic keratopathy.     

Wnt/β-catenin信号通路在糖尿病创面难愈中的作用

目的 探讨Wnt/β-catenin信号途径在糖尿病创面难愈中的作用.方法 在1型糖尿病大鼠制作背部皮肤缺损,分为对照组、糖尿病组、氯化锂组和表皮生长因子(EGF)组,观察背部创面愈合情况及β-catenin表达变化.结果 伤后各组大鼠创面未见感染征象.对照组、氯化锂组及EGF组大鼠与糖尿病组大鼠相比,创面愈合时间短,创面愈合率高,伤腔容积缩小显著,肉芽组织成熟,且β-catenin阳性细胞率高,差异有统计学意义(P＜0.05或P＜0.01).结论 Wnt/β-catenin信号通路参与了糖尿病大鼠创面愈合过程.

Abstract：

Objective To investigate the role of Wnt/β-catenin signaling pathway in impaired wound healing of diabetes mellitus.Methods The back skin defect was produced in rats with type1diabetes.All of these rats were divided into normal group, diabetes group, lithium chloride group, and epidermal growth factor (EGF) group.The back wound healing and β-catenin expression were observed.Results There were no signs of infection in the wound of rats after injury.Compared with diabetic group, the wound healing time was shorter,wound healing rate was higher, wound cavity volume was smaller, granulation tissue was more mature, and β-catenin positive cell rate was higher in normal group, lithium chloride group, and EGF group(P＜0.05 or P＜0.01).Conclusions Wnt/beta-catenin signaling pathway is involved in the process of wound healing in diabetic rats.     

糖尿病大鼠全层皮肤缺损创面愈合过程中皮肤神经末梢再生的研究

目的观察糖尿病大鼠与正常大鼠全层皮肤缺损刨面愈合过程中神经末梢的再支配情况与创面愈合的关系。方法非糖尿病大鼠（n=12）与糖尿病大鼠（n=12）制成相同的全层皮肤缺损创面愈合模型。于伤后3d、7d和14d采取创面皮肤标本，用组织病理HE和SP抗体免疫组织化学染色技术检测创面肉芽再生与再上皮化情况，并对肉芽创面内周围神经末梢再生情况进行观察。结果糖尿病大鼠全层皮肤缺损创面愈合的时间延长，其周围神经末梢的数量较正常组明显减少，再生速度缓慢，肉芽组织中微血管和胶原形成的能力明显降低。结论糖尿病大鼠创面的愈合障碍与神经组织再生延迟相关。     

Collagen ultrastructure and TGF-beta1 expression preserved with aminoguanidine during wound healing in diabetic rats

Advanced glycoxidation end products have been implicated in delayed diabetic wound healing. In this study, we evaluated the effects of aminoguanidine, which is an advanced glycation and nitric oxide (NO) synthase inhibitor, on extracellular matrix protein expression, collagen configuration, and nitrite/nitrate levels in wounds of diabetic rats. Sixteen Wistar male rats were made diabetic by streptozotocin. Of these, eight rats were given AG (aminoguanidine bicarbonate (AG) (group DAG) in their drinking water, and eight rats were followed as diabetic paired controls (group D). Eight healthy rats were followed as the healthy control group (group H). At the eighth week, a 2 x 2 cm area full-thickness skin defect was created. The degree of contraction of the open wounds was evaluated for 2 weeks duration. On the 15th postoperative day, wound surface areas were measured, and wound specimens and blood samples were collected. The shrinking percentage of the wounds was small in both groups H and DAG compared with group D (p < 0.05). Similar to healthy rats, the aminoguanidine-treated diabetic rats had very strong transforming growth factor (TGF)-beta1 expression in granulation tissue and intact skin in comparison with diabetic controls. In the diabetic group, the intact skin demonstrated sparsely distributed regular collagen fibers in the granulation zone, and the regular pattern of collagen fibers was lost. In conclusion, aminoguanidine improves wound healing, restores growth factor TGF-beta1 expression, and preserves collagen ultra structure, whereas it has no prominent effect on NO levels within wound tissue in diabetic rats.     

表皮生长因子纳米微粒对糖尿病大鼠皮肤创面愈合的影响

目的 观察表皮生长因子（EGF）纳米微粒对糖尿病大鼠皮肤创面的愈合作用,探讨EGF纳米微粒促进愈合的机制.方法 制备EGF纳米微粒,测定EGF纳米微粒载药率、包封率和释药行为.用打孔器制备糖尿病大鼠皮肤创面模型.分为4组：EGF纳米组（给予含1μg/d EGF的纳米微粒）,EGF组（给予1μg/d EGF）,空微粒组（给予不含EGF纳米微粒）,磷酸盐缓冲液（PBS）对照组,于3、7、14、21 d照相并取材,计算创面愈合率并比较创面病理学变化及EGF受体阳性细胞面积.结果 EGF纳米微粒平均粒径为193.5 nm,载药率为0.02％,包封率为85％.药物释放达24h.EGF纳米组创面愈合比EGF组快[14 d,（93.8±1.8）％比（89.3±2.3）％,P＜0.05;21 d（96.6±1.6）％比（92.1±4.3）％,P＜0.05].EGF纳米组创面内EGF受体阳性细胞面积高于EGF组[7d,（49.4±6.5）％比（35.1±2.8）％,P ＜0.05;14 d,（80.2±3.8）％比（73.4±1.7）％,P＜0.05].结论 EGF纳米微粒上调创面内细胞EGF受体表达,加速创面的愈合,效果优于EGF.     

Injection of stem cells derived from allogeneic adipose tissue,a new strategy for the treatment of diabetic wounds

A diabetic wound is one of the major complications of Diabetes mellitus. Considering the impact of these wounds on the health and quality of life of diabetic patients, the need for a suitable treatment is essential. Adipose-derived stem cells (ASCs) play a role in healing diabetic wounds. The purpose of this study is to examine the effect of ASCs on skin wound healing in diabetic rats. Rats were divided into three groups, diabetics treated with ASCs, non-diabetic, and diabetic (treated with phosphate-buffered saline). Skin wounds and its margin were examined to measure the level of vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β) and histopathological examinations on three, six, and nine days after wound formation and treatment. As a result, the administration of ASCs can reduce the healing time of skin wounds in diabetic rats by controlling inflammation and increasing angiogenesis.     

L-Arginine supplementation enhances diabetic wound healing: involvement of the nitric oxide synthase and arginase pathways

Diabetes impairs wound healing and there are few therapeutic options to reverse it. Previous work has demonstrated the importance of nitric oxide for successful wound healing. In diabetes, NO synthesis is reduced in the wound milieu. The amino acid L-arginine is the only substrate for NO synthesis. We hypothesized that L-arginine supplementation would enhance wound healing by restoring NO synthesis. Thirty-six male Sprague-Dawley rats (body weight, 225 to 250 g) were separated in 4 groups: 20 rats were rendered diabetic 7 days prior to wounding by intraperitoneal streptozotocin (STZ) injection (70 mg/kg). Sixteen rats served as controls. Half of the animals of each group received 1 g/kg supplemental L-arginine administered by gavage twice daily. Control rats were gavaged with water. Treatment was started 3 days before wounding. All rats underwent a dorsal skin incision and subcutaneous implantation of polyvinyl alcohol (PVA) sponges. The rats were killed 10 days post wounding and wound breaking strength, hydroxyproline content of the sponges, nitrite/nitrate (NO(x)) concentration, arginase activity, and amino acid composition of the wound fluid and plasma were analyzed. Wound fluid NO(x) concentrations and wound breaking strength were significantly reduced in the diabetic group compared to the controls. L-Arginine treatment restored diabetic NO(x) levels toward normal values and significantly enhanced wound breaking strength. Wound fluid arginase activity and ornithine concentrations were significantly lower in the diabetic animals but unaffected by treatment. The data demonstrate that the impaired NO synthesis in the diabetic wound milieu can at least partially be reversed by arginine supplementation. In view of previous results on the importance of NO for wound healing, the data suggest that arginine supplementation restores impaired healing in this acute wound model by normalizing the NO pathway but without affecting arginase activity.     

Effect of activated autologous monocytes/macrophages on wound healing in a rodent model of experimental diabetes

Aim

We aimed to evaluate the effectiveness of the application of activated autologous monocytes/macrophages (Mo/Mp) on wound healing in diabetic rats.

Methods

Sixty male SD rats were equally divided into the following: control group (normal, nondiabetic), PBS-treated diabetic group, and tumor necrotic factor alpha (TNF-α) plus interferon-γ (IFN-γ)-stimulated or unstimulated Mo/Mp-treated diabetic group. Full-thickness round wounds (1 cm × 1 cm) were created in the right hind foot of rats and the wounds were treated with PBS or Mo/Mp on day 1 after injury. In the following 14 days, the percentage of wound contraction was measured, histologic examination was performed with hematoxylin and eosin staining, and vascular endothelial growth factor (VEGF) in the wound was evaluated by Western blot analysis.

Results

Diabetic rats exhibited impaired wound healing with delayed angiogenesis and VEGF expression. The early application of TNF-α plus IFN-γ-stimulated autologous Mo/Mp to diabetic wounds significantly improved the delayed wound healing through the stimulation of angiogenesis and re-epithelization, as well as restoring the defect in VEGF expression.

Conclusions

Mo/Mp activated by TNF-α and IFN-γ promotes diabetic wound healing and normalizes the defect in VEGF regulation associated with diabetes-induced skin-repair disorders.     

应用负压封闭引流技术治疗糖尿病足溃疡的临床疗效观察

目的 探讨应用负压封闭引流（VSD）技术治疗糖尿病足溃疡的临床疗效.方法 将58例2型糖尿病足溃疡患者分为非缺血性组和缺血性组,均进行有效清创,应用负压封闭引流治疗,同时进行全身有针对性的综合治疗.结果 非缺血性组患者30例,其中显效26例,有效4例,总有效率100.00％,其中显效率86.70％.创面平均愈合时间为（65.63±14.53）天.缺血性组患者28例,其中显效17例,有效9例,无效2例,总有效率92.86％,其中显效率60.71％.创面平均愈合时间为（93.65±18.67）天.两组之间疗效比较,差异有统计学意义（P＜0.05）,创面愈合时间比较差异有统计学意义（P＜0.01）.结论 负压封闭引流能有效控制感染,促进创面肉芽生长,改善微循环,促进创面愈合,对非缺血性和缺血性足溃疡治疗均有效,非缺血性组优于缺血性组;非缺血性组创面愈合时间比缺血性组明显缩短.     

Adipose tissue derived mesenchymal stem cell (AD-MSC) promotes skin wound healing in diabetic rats

Aims

Stem cells are a new hope to ameliorate impaired diabetic wound healing. The purpose of this study was to evaluate the effect of adipose tissue derived mesenchymal stem cells (AD-MSCs) on wound healing in a diabetic rat model.

Methods

Twenty-six rats became diabetic by a single intraperitoneal injection of streptozotocin. Six rats served as non-diabetic (non-DM). Diabetic rats were divided into two equal groups randomly; control and treatment. Six weeks later, a full-thickness circular excisional wound was created on the dorsum of each rat. AD-MSCs were injected intra-dermally around the wounds of treatment group. PBS was applied to control and non-DM groups. The wound area was measured every other day. After wound healing completion, full thickness skin samples were taken from the wound sites for evaluation of volume density of collagen fibers, length and volume density of vessels, and numerical density of fibroblasts by stereological methods.

Results

AD-MSCs accelerated wound healing rate in diabetic rats, but did not increase length and volume density of the vessels and volume density of the collagen fibers. AD-MSCs decreased the numerical density of fibroblasts.

Conclusions

We concluded that AD-MSCs enhances diabetic wound healing rate probably by other mechanisms rather than enhancing angiogenesis or accumulating collagen fibers.