糖尿病难愈创面与晚期糖基化终末产物的关系

创面愈合延迟或不愈在糖尿病患者中很常见,但其发病机制目前尚未明了,其中非酶促糖基化反应可能是影响创面愈合的一个重要因素.非酶促糖基化反应系还原性糖、醛基与各种蛋白质N端游离氨基酸或赖氨酸残基的ε-氨基团间在非酶催化下形成可逆的Schiff碱基,再经进一步反应,和化学结构重排转变为性质更为稳定的Amadori产物(Amadori products,AP),这些早期糖基化产物可继续缩合,经十分复杂的重组、脱氢、交联,最终形成不可逆的晚期糖基化终末产物(Advanced glycation end-products,AGEs)[1].     

晚期非酶糖基化终末产物与糖尿病肾病

非酶糖基化终末产物（ＡＧＥｓ）可以造成组织细胞结构和功能的损害。ＡＧＥｓ可以引起肾脏细胞外基质的堆积，影响细胞代谢，诱发蛋白尿和肾小球肥大，在糖尿病肾病的发生和发展中起着重要作用。     

晚期糖基化终末产物与其受体对糖尿病创面氧化应激反应的影响

目的 观察糖尿病患者皮肤和创面中晚期糖基化终末产物(AGE)的蓄积和炎症反应,并结合体外干预实验推测两者之间可能存在的关系. 方法 采集10例2型糖尿病患者(糖尿病组)和12例非糖尿病患者(非糖对照组)的皮肤和创面组织标本,部分于光学显微镜下观察胶原排列、细胞分布(HE染色),AGE及其受体(RAGE)表达(免疫组织化学染色);部分匀浆后采用ELISA法检测丙二醛含量.体外采集、分离健康人外周血中性粒细胞,并分为正常对照组(添加RPMI-1640培养液培养)、低浓度干预组、中浓度干预组和高浓度干预组,3个干预组分别在RPMI-1640培养液中添加AGE修饰的人血清白蛋白,其中AGE的浓度依次为0.315、0.625、1.250 mg/mL;噻唑蓝法检测细胞存活率,荧光探针二氢二氯荧光黄双乙酸钠测定细胞内活性氧水平.对数据进行t检验. 结果 与非糖对照组比较,糖尿病组皮肤组织中胶原萎缩,排列紊乱;创面组织中炎性细胞弥散分布,胶原排列稀疏紊乱.糖尿病组皮肤组织中AGE和RAGE表达均多于非糖对照组;2组创面组织中RAGE呈阳性表达的细胞数量均多于所在组皮肤组织,以糖尿病组更显著,其创面内可见大量RAGE呈阳性表达的炎性细胞.糖尿病组患者皮肤和创面组织中每毫克蛋白丙二醛含量分别为(6 3±1.0)、(7.1±2.4)nmol,均明显高于非糖对照组相应组织[(2.9±1.0)、(3.6±1.4)nmol,t值分别为8.017、4.349,P ＜0.05或P＜0.01].与正常对照组[(34±5)％]比较,低浓度干预组、中浓度干预组、高浓度干预组中性粒细胞存活率均明显上升[(59±8)％、(77±5)％、( 67±6)％,t值分别为7.195、14.890、11.130,P＜0.05或P＜0.01].与正常对照组(0.58±0.06)比较,低浓度干预组、中浓度干预组、高浓度干预组中性粒细胞内活性氧水平均升高(1.67±0.14、2.13±0.17、3 48±0.48,t值分别为20.195、24.905、16.864,P＜0.05或P＜0.01). 结论 异常的氧化应激水平导致糖尿病皮肤具有异常的创伤修复起点,创面愈合过程中AGE-RAGE效应是影响糖尿病创面氧化应激水平的重要因素.     

晚期非酶糖基化终末产物与糖尿病肾病

非酶糖基化终末产物可以造成组织细胞结构和功能的损害。ＡＧＥＳ可以引起肾脏细胞外基质的堆积，影响细胞代谢，诱发蛋白尿和肾小球肥大，在糖尿病肾病的发生和发展中起着重要作用。     

烧伤创面愈合

本文重点阐述有关原理。并讨论组织修复和创面愈合。有关热力损伤内容原则上各种原因烧伤。     

晚期糖基化终末产物及受体在实验性2型糖尿病大鼠种植体周围的变化及表达

目的 建立2型糖尿病大鼠动物模型,探讨晚期糖基化终末产物及其受体在实验性2型糖尿病大鼠种植体骨整合过程中的变化及表达.方法 45只3个月龄SD健康雄性大鼠,将大鼠随机分为糖尿病模型组25只和正常对照组20只.首先建立2型糖尿病大鼠模型,建模成功后将模型大鼠随机分为DM组和DM种植组,每组10只.将20只正常组大鼠随机分为正常对照组和正常种植组,每组10只.分别于正常种植组和DM种植组的胫骨近骺端植入纯钛种植体,植入10周后于下腔静脉采血,保存所采集标本,用RF-5301PC型荧光分光光度计测定血清中AGEs含量的变化.硬组织标本采用不带种植体脱钙切片,以正常组为对照,HE染色后用免疫组织化学方法检测种植体周围RAGE的表达.结果 10周后,DM种植组和DM组与正常对照组和正常种植组相比,血清中AGEs的变化差异有统计学意义（P ＜0.05）,正常种植组和DM种植组与正常对照组的种植体周围骨组织RAGE表达比较,差异均有统计学意义（P ＜0.05）;DM种植组与DM组比较,差异亦有统计学意义（P ＜0.05）.结论 种植体骨组织愈合过程中AGEs和RAGE相互作用是影响2型糖尿病种植体骨结合的机制之一.     

晚期糖基化终末产物与其受体相互作用对足细胞凋亡的影响

目的 探讨可溶性与复合型晚期糖基化终末产物(AGE)与晚期糖基化终末产物受体（RAGE）的相互作用对足细胞凋亡的影响。 方法 以可溶性（CML-BSA、AGE-BSA）和复合型（AGE修正胶原Ⅳ）AGE刺激小鼠足细胞，并用浓度分别为10、50、100 mg/L的AGE刺激细胞，应用TUNEL染色和荧光激活细胞分类（FACS）法来计数凋亡和坏死的足细胞。用RAGE iRNA转染足细胞后，以同样剂量的可溶性和复合型AGE刺激足细胞，观察凋亡情况的改变。 结果 可溶性和复合型AGE均可诱导小鼠足细胞凋亡，复合型AGE引起的足细胞凋亡是可溶性AGE的2~3倍（均P < 0.01）。AGE呈剂量依赖性引起足细胞凋亡。用RAGE iRNA转染足细胞，降低60%~70%RAGE基因活性后，可溶性AGE引起的凋亡率明显下降，复合型AGE诱导的凋亡有下降趋势，但不明显。只有在AGE 100 mg/L刺激后才发生细胞坏死。结论 可溶性AGE主要通过与RAGE相互作用引起足细胞凋亡，复合型AGE部分通过与RAGE相互作用诱导足细胞凋亡。减少AGE生成和RAGE表达可能是预防肾脏病进展的重要途径。     

糖基化终末产物与椎间盘退变

椎间盘组织中糖基化终末产物的含量与椎间盘的退变程度有相关性。它可能通过生成非酶糖化交联而直接影响椎间盘基质的理化特性,还可能间接通过氧化应激和一些细胞因子的作用,减少蛋白多糖等基质的含量,影响椎间盘细胞的功能及细胞外基质平衡,并诱导其发生凋亡,在椎间盘退变过程中起作用。     

晚期非酶糖基化终末产物（AGEs）及其临床意义

晚期非酶糖基化终末产物在糖尿病及肾功能不全时水平升高，现有大量研究表明它与肾功能衰竭的多种并发症有关，本文就ＡＧＥｓ的生成，生化特性，致病机理，清除方式以及其与透析的关系作一介绍。     

晚期糖基化终末产物与慢性肾功能衰竭

晚期糖基化终末产物 (AGE)是蛋白质与还原糖发生非酶促糖基化反应产物 ,AGE作为一种新的尿毒症毒素 ,与尿毒症及透析多种并发症或合并症有关。本文综述AGE在慢性肾功能衰竭中的致病作用及可能采取的防治措施。     

L-精氨酸对糖尿病烧伤创面促愈作用的研究

目的探讨精氨酸对糖尿病烧伤创面修复的影响及其可能的机制. 方法 SD大鼠随机分为正常对照组(A组)、糖尿病对照组(B组)、糖尿病精氨酸干预组(C组)和糖尿病甘氨酸对照组(D组).糖尿病模型通过腹腔注射STZ建立,然后喂养8周后给予深Ⅱ度烫伤,于伤后0、1、3、7、14、21 d时相点对创面愈合面积、组织形态学改变、创面组织糖含量、羟脯氨酸(OHP)含量及局部组织释放转化生长因子-β1(TGF-β1)含量进行检测. 结果应用精氨酸后,皮肤组织糖含量降低,创面局部炎症反应出现较早,坏死组织脱落及上皮匍行提前,OHP含量、组织释放TGF-β1能力均增加,创面愈合明显加快(P﹤0.05～0.01). 结论 L-精氨酸可通过降低皮肤组织糖含量及增加TGF-β1的合成和释放,促进糖尿病烧伤创面愈合.     

人类关节滑膜细胞表达晚期糖基化终产物受体

目的 进一步探讨晚期糖基化终产物修饰的β２－微球蛋白（ＡＧＥ－β２ｍ）对关节固有细胞的生物学作用,确定人类关节滑膜细胞是否表达对ＡＧＥ特异的受体。方法 分离、培养人关节Ａ型和Ｂ型滑膜细胞,用免疫组织化学法及流式细胞仪法分别观察滑膜细胞表面ＡＧＥ受体１（ＡＧＥ－Ｒ１）,ＡＧＥ受体２（ＡＧＥ－Ｒ２）、ＡＧＥ受体３（ＡＧＥ－Ｒ３）及ＡＧＥ受体（ＲＡＧＥ）的表达,用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术     

烧伤创面愈合的信号转导机制

After 50 years of development in science of bums care in China, we have basically solved coverage of deep wounds of burn trauma, as well as role of multiple growth factors and stem cell in wound healing, making great contribution to improving the treatment of patients with large area of deep bums. Surgeons are paying close attention to problems of wound healing, especially in the fields of starless healing and rehabilitation. To solve these problems, we need to do further investigation on multiple growth factors as well as proliferation/differentiation of stem cells in regulation of cell growth and differentiation in wound healing. Therefore ,we are facing a even more serious challenge.     

Implications of chlorhexidine use in burn units for wound healing

Chlorhexidine is known to be a potent antiseptic with evidence of a beneficial role in burn care. Nevertheless, several in vitro studies have reported cytotoxicity on cultured cells, while in vivo and clinical data seem to show more controversial results. In the frame of this work, we aimed to evaluate the use of chlorhexidine in burn units worldwide be sending a survey to professionals of the field. We associated survey results to those perspectives reported in the literature to update recommendations for the use of chlorhexidine in specific protocols for burn management. The survey results showed that there is no clear consensus on the use of chlorhexidine regarding the concentrations, the type of excipient and the cleansing after application. Literature searches showed evidence that the skin of premature infants appears to be more sensitive to chlorhexidine that adult skin, with more reported cases of adverse effects. It was also determined that aqueous formulations of chlorhexidine do not appear to be necessarily less efficient than with alcohol as an excipient, and that lower concentrations are as efficient as higher concentrations. In view of this study, we have adjusted our protocols for the use of aqueous formulations at low concentrations and investigated further the role of washing after application in order to standardize the indication of chlorhexidine and minimize the probability of adverse effects.     

Advanced glycation end products in degenerative nucleus pulposus with diabetes

Diabetes mellitus (DM) has been clinically proved as a risk factor of disc degeneration, and the accumulation of advanced glycation end products (AGEs) is known to be potentially involved in diabetes. The purpose of this study is to investigate the effect of AGEs in the degeneration process of diabetic nucleus pulposus (NP) in rats and humans. Diabetic NP cells from rat coccygeal discs were treated with different concentrations of AGEs (0, 50, and 100 µg/ml) for 3 days, and mRNA expressions of MMP‐2 and RAGE were measured by real‐time RT‐PCR. In addition, conditioned medium from NP cells was used to analyze protein expression of MMP‐2 activity and ERK by gelatin zymography and Western blot. These experiments were repeated using human intervertebral disc samples. The immunohistochemical expression of AGEs was significantly increased in diabetic discs. In response to AGEs, an increase of MMP‐2, RAGE, and ERK at both mRNA and protein expression levels was observed in diabetic NP cells. The findings suggest that AGEs and DM are associated with disc degeneration in both species. Hyperglycemia in diabetes enhances the accumulation of AGEs in the NP and triggers disc degeneration. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:238–244, 2014.     

晚期糖化终末产物在老年大鼠骨质疏松发病中的作用

目的探讨晚期糖化终末产物（ＡＧＥ）在老年大鼠骨质疏松发病中的作用。方法选用２０月龄老年大鼠和３月龄大鼠,测定其骨密度及骨胶原中晚期糖化终末产物的含量及血、尿生化指标。结果老年大鼠骨密度明显低于３月龄大鼠（Ｐ〈０．０５）,而骨胶原中晚期糖化终末产物的含量明显升高（Ｐ〈０．００１）。同时发现老龄组大鼠血清甲状旁腺激素（ＰＴＨ）明显升高（Ｐ〈０．０５）,尿钙与肌酐比值升高（Ｐ〈０．０２）,而尿磷与肌酐比     

P物质与表皮干细胞联用对糖尿病大鼠创面愈合与神经再生的影响

目的 观察感觉神经肽P物质与表皮干细胞(ESC)联合应用对糖尿病大鼠创面愈合与神经再生的作用. 方法 分离培养SD大鼠ESC(经鉴定),接种于羊膜滋养层上构建羊膜-ESC备用.选择48只糖尿病模型大鼠,每只背部制作4个全层皮肤缺损创面.按随机抽签法将此192个创面分为ESC+P物质组、ESC组、P物质组、对照组,每组48个创面.ESC+P物质组和ESC组创面均移植羊膜-ESC,P物质组、对照组创面移植羊膜.移植后,ESC+P物质组、P物质组在创周及创面中央注射1×10-7 mol/L的P物质250μL,ESC组、对照组在创周及创面中央注射PBS 250μL作对照,各组每日注射2次,连用4d.于大鼠伤后4、7、10、14、17、23 d,观察并计算创面愈合率(每时相点8个创面),HE染色观察创面组织结构改变.伤后4、7、10 d,行Masson染色观察创面组织总胶原分布,免疫组织化学染色观察Ⅰ、Ⅲ型胶原沉积量.伤后14、23 d,用免疫组织化学染色法观察创面组织中蛋白基因产物9.5(PGP 9.5)及P物质阳性神经纤维分布情况.对数据行单因素方差分析和t检验. 结果(1)ESC+P物质组伤后14 d创面愈合率达100.0％,明显早于ESC组、P物质组、对照组完全愈合时间(伤后17、17、23 d).HE染色显示ESC+P物质组创面愈合质量明显优于其余3组.(2)伤后10 d,ESC+P物质组与P物质组创面组织中胶原着色深、面积广;其余2组胶原染色较浅、面积较小.随着伤后时间推移,各组创面Ⅰ型胶原沉积量逐渐升高,Ⅲ型胶原沉积量逐渐下降.伤后4、7、10 d,ESC+P物质组Ⅰ型胶原沉积量明显高于ESC组(t值分别为32.72、118.21、26.71,P值均小于0.01)和对照组(t值分别为44.37、22 76、30.32,P值均小于0.01);ESC+P物质组与P物质组水平相对接近.伤后4、7、10d,ESC+P物质组创面Ⅲ型胶原沉积量明显高于ESC组(t值分别为32.27、28 68、14.51,P值均小于0.01)和对照组(t值分别为35 68、22.52、22 24,P值均小于0.01).(3)ESC +P物质组与P物质组创面组织中有大量PGP 9.5和P物质阳性神经纤维再生,创面深层部分神经纤维末梢向表皮延伸.ESC组、对照组仅见创面深层有少量PGP 9.5和P物质阳性神经纤维,且未向表皮延伸.伤后14、23 d,ESC+P物质组创面PGP 9.5阳性神经纤维面积占(3.86±0.25)％、(7 03±0.28)％,明显高于ESC组[(1.48±0.30)％、(3.01±0 43)％,t值分别为23 95、30 27,P值均小于0.01]和对照组[(1 46±0 23)％、(2.84±0.29)％,t值分别为27.35、40.32,P值均小于0.01].伤后14、23 d,ESC+P物质组创面P物质阳性神经纤维面积占(2.01±0 14)％、(1.19±0 11)％,明显高于ESC组[(0.85±0 17)％、(1.34±0 21)％,t值分别为20.50、2.60,P＜0.05或P＜0.01]和对照组[(0.74 ±0.15)％、( 1.30 ±0.17)％,t值分别为23 98、2.41,P＜0.05或P＜0.01]. 结论 感觉神经肽P物质和ESC联合应用,可以有效促进糖尿病大鼠创面愈合与神经再生.     

烧伤创面与糖尿病溃疡创面的比较

目的比较烧伤创面与糖尿病溃疡创面的差异，初步分析糖尿病患者溃疡创面难愈的机制。方法分别切取非糖尿病烧伤患者的足部创面（对照组）和糖尿病患者的足部溃疡创面（试验组）组织，行组织块培养。用酶联免疫吸附测定（ELISA）法、反转录-PCR法分别检测创面组织释放的成纤维细胞生长因子2（FGF2）、血管内皮生长因子（VEGF）蛋白质及其mRNA水平；免疫组织化学法检测创面微血管密度（MVD）的变化。人脐静脉内皮细胞分别在含5mmol／L葡萄糖的培养液（正常培养液组）、含30mmol／L葡萄糖的培养液（高糖组）、含30mmol／L甘露醇的培养液（甘露醇组）中培养7d，以ELISA法测定VEGF蛋白质水平。结果对照组患者FGF2、VEGF蛋白质水平分别为（59±3）ng／ml、（56±7）pg／ml，试验组2种蛋白质水平分别为（89±6）ng／ml、（108±5）pg／ml，组间比较差异均有统计学意义（P〈0．05或P〈0．01），mRNA比较结果与蛋白质相似；2组的MVD水平，差异亦有统计学意义（P〈0．05）。体外细胞培养时当培养液含FGF2，高糖组与正常培养液组的VEGF蛋白质水平相近（P〉0．05）；移去FGF2后2、5d，正常培养液组该指标明显高于高糖组（P〈0．05或P〈0．01）。结论糖尿病患者溃疡创面难愈与血管化受到抑制以及调控血管生长的因子低表达密切相关。     

Advanced glycation end-product expression is upregulated in the gastrointestinal tract of type 2 diabetic rats

AIM:To investigate changes in advanced glycation end products(AGEs) and their receptor(RAGE) expression in the gastrointestinal(GI) tract in type 2 diabetic rats.METHODS:Eight inherited type 2 diabetic rats GotoKakizak(GK) and ten age-matched normal rats were used in the study.From 18 wk of age,the body weight and blood glucose were measured every week and 2 wk respectively.When the rats reached 32 wk,twocentimeter segments of esophagus,duodenum,jejunum,ileum,and colon were excised and the wet weight was measured.The segments were fixed in 10% formalin,embedded in paraffin and five micron sections were cut.The layer thickness was measured in Hematoxylin and Eosin-stained slides.AGE [N epsilon-(carboxymethyl) lysine and N epsilon-(carboxyethyl)lysine] and RAGE were detected by immunohistochemistry staining and image analysis was done using Sigmascan Pro 4.0 image analysis software.RESULTS:The blood glucose concentration(mmol/L) at 18 wk age was highest in the GK group(8.88 ± 1.87 vs 6.90 ± 0.43,P 0.001),a difference that continued to exist until the end of the experiment.The wet weight per unit length(mg/cm) increased in esophagus,jejunum and colon from the normal to the GK group(60.64 ± 9.96 vs 68.56 ± 11.69,P 0.05 for esophagus; 87.01 ± 9.35 vs 105.29 ± 15.45,P 0.01 for jejunum; 91.37 ± 7.25 vs 97.28 ± 10.90,P 0.05 for colon).Histologically,the layer thickness of the GItract was higher for esophagus,jejunum and colon in the GK group [full thickness(μm):575.37 ± 69.22 vs 753.20 ± 150.41,P 0.01 for esophagus; 813.51 ± 44.44 vs 884.81 ± 45.31,P 0.05 for jejunum; 467.12 ± 65.92 vs 572.26 ± 93.60,P 0.05 for colon].In esophagus,the AGE and RAGE mainly distributed in striated muscle cells and squamous epithelial cells.The AGE distribution was much stronger in the GK group compared to the normal group both in the striated muscle layer and mucosa layer(immuno-positive area/ total measuring area %:4.52 ± 0.89 vs 10.96 ± 1.34,P 0.01 for muscle; 8.90 ± 2.62 vs 22.45 ± 1.26,P 0.01 for mucosa).No visible difference was found for RAGE distribution between the two groups.In the intestine AGE and RAGE distributed in epithelial cells of villi and crypt.RAGE was also found in neurons in the myenteric and submucosal plexus.The intensity of AGE staining in mucosa of all segments and RAGE staining in neurons in all segments were strongest in the diabetes group.Significant difference for AGE was found in the epithelial cells of villi and crypt in duodenum(immunopositive area/total measuring area %:13.37 ± 3.51 vs 37.48 ± 8.43,P 0.05 for villi; 0.38 ± 0.12 vs 1.87 ± 0.53,P 0.05 for crypt) and for RAGE in neurons of all segments(e.g.,for jejunum:no staining neurons% 0 vs 0,mild 36.0 ± 5.2 vs 28.7 ± 3.5,moderate 53.2 ± 4.8 vs 55.8 ± 5.4,strong 10.7 ± 1.1 vs 15.4 ± 2.0,P 0.05).In the colon,RAGE was primarily found in neurons in the myenteric and submucosal plexus.It was stronger in the diabetes group than in the normal group(no staining neurons% 6.2 ± 0.2 vs 0.3 ± 0.04,mild 14.9 ± 2.1 vs 17.6 ± 1.5,moderate 53.1 ± 4.6 vs 44.7 ± 4.4,strong 25.6 ± 18 vs 43.6 ± 4.0,P 0.05).In the rectum,RAGE was primarily found in the mucosa epithelial cells.CONCLUSION:The AGE and RAGE expression was upregulated in the GI tract of GK diabetic rats and may contribute to GI dysfunction in type 2 diabetic patients.