外敷氨基胍霜剂对糖尿病大鼠皮肤组织的影响

目的 观察外敷氨基胍霜剂对糖尿病大鼠皮肤组织晚期糖基化终末产物(AGE)形成、KC细胞增殖及氧化应激的影响.方法 将硬脂酸、液状石蜡、凡士林、羊毛脂、肉豆蔻酸异丙酯、甘油、50 g/L尼泊金醇、盐酸氨基胍等试剂按一定比例混合制成氨基胍霜剂,以不含有氨基胍的霜剂为基质.取健康大鼠背部皮肤,分别用5、10 g/L氨基胍霜剂和5 g/L氨基胍+10g/L氮酮霜剂处理,于用药后2、4、7、10、12、24 h测定药物透皮效果.将30只SD大鼠按随机数字表法分为正常对照组6只、糖尿病组8只、氨基胍治疗组8只、基质治疗组8只.后3组大鼠腹腔注射链脲佐菌素65 mg/kg,诱导糖尿病模型;对照组大鼠注射0.05 mmol/L柠檬酸缓冲液.注射1周后,正常对照组与糖尿病组大鼠不行任何治疗,氨基胍治疗组与基质治疗组大鼠背部分别连续外用10 g/L氨基胍霜剂与基质治疗4周.取各组皮肤组织,胶原提取液荧光强度检测法测定AGE含量,流式细胞仪分析表皮KC周期,检测氧化应激相关指标超氧化物歧化酶(SOD)、丙二醛、总抗氧化能力、髓过氧化物酶(MPO)含量.对实验数据行t检验.结果 10 g/L氨基胍霜剂透皮效果优于5g/L氨基胍和5 g/L氨基胍+10 g/L氮酮的霜剂.1只基质治疗组大鼠未诱导成功.建模后4周,糖尿病组与氨基胍治疗组大鼠分别死亡4只和1只.糖尿病组大鼠皮肤组织AGE含量为每毫克羟脯氨酸(OHP)中(36.8±2.6)U,明显高于正常对照组的每毫克OHP中(24.6±2.7)U(t=7.2,P＜0.01);氨基胍治疗组AGE含量为每毫克OHP中(28.6±3.7)U,明显低于糖尿病组(t=-3.9,P＜0.05);基质治疗组AGE含量[每毫克OHP中(32.2±5.2)U]与糖尿病组相近(t=1.6,P＞0.05).糖尿病组大鼠S期KC比例为(5.3±0.6)%,低于正常对照组的(7.6±0.9)%(t=4.50,P＜0.01);氨基胍治疗组大鼠S期和G2/M期KC比例均明显高于糖尿病组(t值分别为6.80、3.17,P值均小于0.01);基质治疗组大鼠S期KC比例[(9.2±1.5)%]显著高于糖尿病组(t=4.90,P＜0.01).糖尿病组大鼠皮肤组织氧化应激指标含量均高于正常对照组,其中SOD和MPO差异有统计学意义(t值分别为4.4、3.7,P值均小于0.05);氨基胍治疗组各氧化应激指标含量均较糖尿病组降低,其中SOD含量显著低于糖尿病组(t=-1.4,P＜0.05);基质治疗组MDA、MPO含量显著低于糖尿病组(t值分别为2.6、2.9,P值均小于0.05).结论 外用氨基胍霜剂可以在一定程度上阻碍糖尿病大鼠皮肤组织中AGE的形成,改善表皮KC细胞增殖能力,适当降低皮肤组织氧化应激状态;单用霜剂基质也可适当降低皮肤组织氧化应激状态.

Abstract：

Objective To investigate the effects of aminoguanidine cream on the proliferation of keratinocytes (KC), content of advanced glycosylation end products (AGE) and oxidative stress in skin tissue of rats with diabetes. Methods Stearic acid, liquid paraffin, vaseline, lanolin, isopropyl myristate fat,glycerol, 50 g/L alcohol paraben, aminoguanidine hydrochloride etc. were mixed in certain proportion to make aminoguanidine cream, and cream without aminoguanidine was used as matrix. The dorsal skin of normal rats were harvested and treated by aminoguanidine cream with dose of 5, 10 g/L, or 5 g/L together with 10 g/L azone. The transdermal effect was respectively measured at post treatment hour 2, 4, 7, 10, 12,24. Thirty SD rats were divided into normal control(NC, n = 6) , diabetes(D, n = 8) , aminoguanidine cream-interfered(AI, n = 8), matrix cream-interfered groups(MI, n = 8) according to the random number table. Diabetes was reproduced by intraperitoneal injection of STZ (65 mg/kg) in rats of D, AI, and MI groups, and rats in NC group were injected with 0. 05 mmol/L citrate buffer as control. One week later, dorsal skin of rats in AI and MI groups were respectively treated with 10 g/L aminoguanidine cream and matrix cream by external use for 4 weeks. AGE content was determined with fluorescence detection from skin collagen extract. KC cell cycle was detected by flow cytometry. Skin tissue specimens were obtained for determination of levels of superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), and total antioxidant capacity. Data were processed with t test. Results Transdermal effect of aminoguanidine cream with dose of 10 g/L was better than that with 5 g/L or 5 g/L + 10 g/L azone cream. One rat was not induced successfully in MI group. Four weeks after model reproduction, 4 rats died in D group and 1 rat died in AI group. The AGE content in D group was obviously higher than that in NC group [(36.8 ± 2.6),(24. 6 ±2.7) U per milligram hydroxyproline, respectively, t = 7.2, P ＜0. 01], and that in AI group [(28.6 ±3.7) U per milligram hydroxyproline] was also lower as compared with that in D group(t = -3.9,P ＜ 0.05). There was no significant difference in AGE content between MI [( 32.2 ± 5.2) U per milligram hydroxyproline] and D groups(t = 1.6, P ＞ 0. 05). The percentage of KC in S phase was obviously lower in D group than in NC group [(5.3 ±0.6)%, (7.6±0.9)%, respectively, t =4.50, P ＜0. 01], while that in MI group [(9. 2 ± 1.5) %] was higher as compared with that in D group(t = 4.90, P ＜ 0. 01). It was more higher in AI group than in D group on KC percentage in S and G2/M phase (with t value respectively 6.80, 3.17, P values all below 0. 01). The oxidative stress indexes of skin tissue in D group were all higher than those in NC group, in which levels of MPO and SOD showed statistical difference(with t value respectively 4.4, 3.7, P values all below 0. 05). The oxidative stress indexes were all lower in AI group than in D group, especially in SOD level(t = -1.4, P ＜0. 05). Levels of MAD, MPO in MI group were significantly lower than those in D group(with t value respectively 2.6, 2.9, P values all below 0. 05).Conclusions Aminoguanidine cream can promote KC proliferation and appropriately reduce oxidative stress through inhibiting AGE formation to a certain extent in skin tissue of rats with diabetes. Signal use of matrix cream can also reduce oxidative stress in skin tissue of rats with diabetes.     

糖基化终末产物对糖尿病大鼠烧伤创面愈合的影响

目的 了解糖基化终末产物(AGE)蓄积对糖尿病大鼠烧伤创面愈合的影响. 方法 将75只SD大鼠按完全随机化方法 分为对照组、糖尿病组及氨基胍干预组,每组25只.将大鼠造成深Ⅱ度烫伤(以下称烧伤)后,后2组制作成糖尿病模型,氨基胍干预组给予管饲氨基胍100 mg·kg~(-1)·d~(-1).于伤后0(伤后当天)、3、7、14、21 d处死大鼠.描取创面形状,并取全层皮肤组织待测.同时取大鼠背部表皮行KC培养及鉴定.观察各组创面愈合率及皮肤组织糖含量,创面组织形态学变化,皮肤组织中AGE分布.观察不同浓度AGE对KC增殖,凋亡的影响,以仅加表皮细胞培养液的KC为对照组. 结果 伤后7、14、21 d糖尿病组创面愈合率显著低于对照组(P<0.01),而氨基胍干预组却明显高于前2组(P<0.01).糖尿病组皮肤组织含糖量为(2.62±0.19)mmol/g,氨基胍干预组为(2.58±0.07)mmol/g,均高于对照组(1.04±0.09)mmol/g(P<0.01).对照组大鼠创面炎性细胞浸润强烈而局限,坏死组织形成、脱落及时,创面愈合无明显延迟;糖尿病组大鼠炎性细胞浸润缓慢、弥散而持久,坏死组织形成、脱落较迟,创面愈合明显延迟;氨基胍干预组大鼠炎性细胞浸润及时、强烈,坏死组织形成、脱落以及创面愈合时间较糖尿病组早.对照组中有少量散在的AGE沉积,糖尿病组AGE蓄积显著增加,而氨基胍干预组AGE含量显著降低.AGE作用48 h后,KC的增殖显著降低,呈现浓度依赖性,各剂量AGE干预组的吸光度值均低于对照组(P<0.01).100μg/mLAGE干预组KC早期凋亡连接素V阳性细胞比例为(15.1±2.3)%,明显高于对照组[(11.2±1.2)%,P<0.05];终未期凋亡双阳性细胞比例为(14.3±3.5)%,与对照组(15.2±2.4)%比较,差异无统计学意义(P>0.05). 结论 高血糖可能是通过AGE蓄积,抑制KC等修复细胞增殖导致创面难愈;减少AGE蓄积,可改善糖尿病创面愈合延迟现象.     

左旋精氨酸对糖尿病大鼠烧伤创面血管形成的影响

目的　观察左旋 (L)精氨酸对糖尿病大鼠烧伤创面修复过程中血管形成的影响。 方法雄性SD大鼠 ,设烫伤对照 (A)组、糖尿病烫伤 (B)组、甘氨酸对照 (C)组和L 精氨酸干预 (D)组 ,每组各 2 5只。B、C、D组大鼠通过腹腔注射链脲佐菌素 (STZ)建立糖尿病模型 ,C、D组分别管饲甘氨酸和L 精氨酸 2 0 0mg·kg-1·d-1。8周后取各组大鼠 (每组 5只 )背部皮肤组织测定糖含量。之后各组大鼠给予 2 0 %TBSA深Ⅱ度烫伤 ,于伤后 3、7、14、2 1d测量创面面积 ,计算创面愈合率 ;采用CD34免疫组织化学染色计算微血管密度 (MVD);检测创面组织释放一氧化氮 (NO)、血管内皮生长因子(VEGF)和转化生长因子 (TGF)β1含量。　结果　与B组相比 ,D组大鼠创面愈合率从伤后第 7天起显著增加为 [( 4 4.10± 3.5 0 ) % ,P <0 0 5 ],创面MVD值在伤后各时相点均显著增加 ( P <0 0 5 ),创面组织中释放NO、VEGF和TGF β1量增加 ,皮肤组织糖含量降低为 ( 1.380± 0 .12 0 )mg/g。　 结论L 精氨酸可通过增加NO、VEGF和TGF β1的合成与释放 ,降低皮肤组织糖含量 ,增加糖尿病大鼠烧伤创面的血管形成 ,并促进创面愈合     

糖尿病大鼠表皮角质形成细胞生物学变化的机制研究

目的 通过体内、外2部分实验,研究糖尿病状态下表皮角质形成细胞(KCs )增殖能力的变化及其信号转导途径.方法 STZ(streptozotocin,链脲佐菌素)诱导正常SD大鼠,成模后8周(晚期糖尿病大鼠模型),荧光自显影技术检测皮肤组织中晚期糖基化终末产物(advanced glycation end-products,AGEs)的沉积;ELSA法检测表皮层中EGF(epidermal growth factor表皮生长因子)含量;提取大鼠KCs,用二甲基噻唑二苯基四唑溴盐(MTT)法测定细胞增殖速度;检测细胞周期;观察KCs核内NF-κB活性.分离培养正常SD大鼠KCs,加入不同浓度AGEs干预细胞,选择细胞反应敏感,差异显著的100mg/L AGEs干预组进行实验:测定细胞增殖速度;细胞对EGF的利用;检测KCs周期的变化,观察KCs核内NF-κB的活化状态.抑制NF-κB活性后观察细胞周期、细胞对EGF利用的变化.结果 晚期糖尿病大鼠KCs细胞周期循环阻滞,细胞的增殖能力受到了抑制;体外100mg/L AGEs可以明显抑制KCs的增殖.抑制NF-κB的活性后,部分恢复细胞的增殖能力.结论 AGEs可以通过NF-κB途径影响糖尿病大鼠表皮角质形成细胞的增殖能力.     

基质金属蛋白酶在慢性创面中的表达及其意义

皮肤创面愈合时，基质金属蛋白酶（MMPs)对细胞外基质的蛋白分解系修复和塑型的一种必需过程，MMPs及其相关的生物活性物质的动态平衡调控失衡可能是创面难愈的重要因素之一。本综述了慢性创面中部分MMPs的异常表达，探讨其异常表达对创面愈合过程的可能影响机制。     

糖尿病大鼠深Ⅱ度烫伤创面成纤维细胞生物学行为的改变

目的 研究糖尿病深Ⅱ度烫伤对真皮成纤维细胞(FB)生物学行为的影响.方法 以链脲菌素(STZ)诱导糖尿病的深Ⅱ度10%TBSA烫伤大鼠模型,观察创面愈合情况.流式细胞术检测真皮组织中FB细胞周期;TUNEL法检测FB凋亡;伤后0、3、7、21 d检测皮肤组织的羟脯氨酸含量.结果 糖尿病大鼠深二度烫伤后愈合延迟,糖尿病组烫伤后进入S期的成纤维细胞数量明显低于正常组(P<0.01,P<0.05),创面凋亡细胞数量高于正常组(P<0.01,P<0.05);烫伤前和烫伤后糖尿病组皮肤组织单位面积羟脯氨酸含量均低于正常大鼠(P<0.01,P<0.05).结论 在糖尿病病理状态下,皮肤损伤后主要的修复细胞FB增殖异常,凋亡增多,胶原含量减少,可能是创面修复延迟的原因之一.     

早期和晚期急性呼吸窘迫综合征为两种完全不同的临床综合征

本研究回顾分析了5.5年间普雷斯利地区创伤中心收治的178例创伤后并发急性呼吸窘迫综合征(ARDS)的患者。患者入院后即维持气道通畅,并维持呼吸和循环,经气管插管或气管内置管接受PB7200呼吸机正压通气。ARDS的诊断标准为急     

糖尿病合并创面难愈的机制研究

糖尿病合并难愈创面是导致创面修复"失控"难愈的因素中最值得重视的领域之一.其患病率日渐升高,治疗手段局限,使相关预后较差,从而需要耗费大量的卫生保健资源.糖尿病下肢溃疡,特别是糖尿病足溃疡在糖尿病合并难愈创面中占有重要的地位,国内外对其相关机制进行了大量的研究.