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.     

创面愈合过程中脂肪细胞内转化生长因子-β各亚型的表达特点及其生物学意义

目的　观察深Ⅱ度烫伤大鼠上皮组织修复过程中皮下脂肪组织内脂肪细胞转化生长因子 (TGF) β1、2、3亚型的表达特点及生物学意义。 方法　将 3 6只Wistar大鼠随机分为正常对照组 (A组 ,n =6)和单纯烫伤组 (B组 ,n =3 0 )。利用大鼠 5 %深Ⅱ度烫伤模型 ,于伤后 1、3、7、14和2 1d采取全层创面皮肤标本 ,采用免疫组织化学染色法检测真皮下脂肪细胞TGF β1、2、3的表达 ,并以病理学染色观察肉芽组织的形成及创面的收缩情况。结果　正常皮肤组织内脂肪细胞中均有TGF β1、2、3的表达。单纯烫伤创面愈合的过程中 ,伤后 1d ,脂肪组织内脂肪细胞膜表面TGF β1表达较弱 ,但TGF β2、3的表达较强 ,伤后 3d ,脂肪细胞TGF β1表达有所增强 ,TGF β2、3的表达进一步增强。此时创面深层脂肪组织内有新的微小血管形成。至 14d ,创面收缩 ,脂肪组织TGF β1表达维持在较高水平 ,TGF β2、3的表达也较高。真皮浅层大量的微小血管形成 ,密度明显增强。 2 1d创面完全愈合 ,在新愈合组织内有一定量的血管形成 ,TGF β各亚型的表达有所减弱 ,但TGF β2、3的表达仍强于TGF β1。 结论　组织修复过程中 ,脂肪细胞释放的TGF β可能有助于肉芽组织充填和微小血管网的形成 ,在创面愈合中扮演重要角色。     

海水浸泡对烫伤大鼠创面炎性反应与愈合的影响

目的观察海水浸泡对烫伤大鼠创面炎性反应及愈合的影响。方法将144只雄性Wistar大鼠随机分为烫伤对照组和海水浸泡组,每组72只,均造成背部10%TBSA浅Ⅱ度烫伤。海水浸泡组大鼠伤后固定四肢,立即用盛海水的方盆浸泡双前肢以下部分,持续4h；烫伤对照组大鼠则用空方盆模拟浸泡过程。于伤后0(即刻,下同)、6、12、24h采用电解质分析仪测定血清中K~+、Na~+、Cl~-的浓度。于伤前及伤后0、6、12h采用酶联免疫吸附测定法检测血清中肿瘤坏死因子(TNF)α及白细胞介索(IL)6的含量。对两组大鼠创面行大体和组织病理学观察,并记录创面愈合时间。结果海水浸泡组大鼠血清中K~+、Na~+、Cl~-的浓度大多高于烫伤对照组。伤后6h海水浸泡组大鼠血清TNF-α、IL-6含量分别为(140±22)、(160±41)ng/L,均明显高于伤前值(29±15)、(62±17)ng/L及烫伤对照组(120±12)、(124±22)ng/L(P＜0．05)。与烫伤对照组比较,海水浸泡组大鼠创面水肿及局部组织炎性反应加重,创面再上皮化和表皮各层的分化延迟；海水浸泡组创面愈合时间为(16．3±1．6)d,明显迟于烫伤对照组(14．1±1．8)d(P＜0．05)。结论大鼠烫伤后经海水浸泡,可加重创面炎性反应,使创面愈合延迟。     

芦荟提取物对烫伤大鼠创面组织一氧化氮及内皮素含量的影响

目的观察芦荟凝胶和芦荟粗多糖对烫伤大鼠创面组织含水量及一氧化氮(NO)和内皮素(ET)含量的影响。方法将42只Wistar大鼠背部造成4个直径为3 cm的深Ⅱ度烫伤创面。伤后创面分别外敷质量分数5％芦荟粗多糖膏、质量分数10％芦荟凝胶膏、质量分数1％磺胺嘧啶银(SD-Ag)霜和等渗盐水。根据创面用药的不同分为芦荟粗多糖组、芦荟凝胶组、SD-Ag组、等渗盐水组。伤后4、12、24、48 h及7、14、21 d每时相点处死6只大鼠,取创面全层皮肤测定组织含水量、No和ET含量,计算No／ET值。另取6只大鼠不烫伤,作为正常对照组,检测指标同前。结果伤后12、24、48h,芦荟粗多糖组[(73.4±3．8)％、(76．6±3．0)％、(70．6±3．8)％]和芦荟凝胶组[(74.5±2.6)％、(77．1±3．6)％、(71．2±3．1)%]创面组织含水量显著低于SD-Ag组[(80．1±4．1)％、(80．5±3．9)％、(76．1±3．8)％,P<0．05];伤后7-21 d,除SD-Ag组伤后7 d仍显著高于正常对照组(P<0．05)外,其他各组均基本恢复到正常水平。伤后12 h各烫伤组创面组织NO含量升高达峰值,随后下降,到伤后21d仍显著高于正常对照组(P<0．05);伤后12、24 h,芦荟粗多糖组和芦荟凝胶组创面组织NO含量明显低于SD-Ag组及等渗盐水组(P<0．05)。伤后7 d或14 d各烫伤组创面组织ET含量增加达高峰,随后下降;伤后7、14d均显著高于正常对照组(P<0．05)。伤后12 h各组NO／ET值达峰值,随后下降,伤后14 d基本恢复至正常水平,其中芦荟粗多糖组伤后7 d NO／ET值即恢复至正常水乎,但其他3组仍显著高于正常对照组。结论芦荟粗多糖和芦荟凝胶能有效减少烫伤后早期创面组织NO的释放、优化NO／ET值、减轻血管炎性反应、减少渗出和水肿。     

局部应用L-精氨酸对糖尿病小鼠创面愈合的影响

目的研究局部应用L-精氨酸对糖尿病小鼠创面治疗效果的影响。方法建立链脲霉素(Streptozotocin,STZ)诱导的Ⅰ型糖尿病小鼠创面模型;昆明小鼠随机分为正常对照组(A组)、糖尿病对照组(B组)、糖尿病精氨酸治疗组(C组),糖尿病小鼠创面模型建立后,观察及测量伤后0、4、8、14、16d时创面愈合面积、肉芽生长情况、组织形态学改变、羟脯氨酸(OHP)含量。结果C组创面愈合明显加快(P<0.05),创面成纤维细胞数量、胶原生成增多,OHP含量增加。结论局部应用L-精氨酸,通过加快肉芽生成,增加创面成纤维细胞数量,能促进糖尿病创面愈合。     

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.     

糖尿病大鼠深Ⅱ度烫伤后真皮成纤维细胞的生物学特征

目的观察糖尿病（DM）大鼠深Ⅱ度烫伤肝创面皮肤组织及真皮成纤维细胞（Fb）生物学特征，探讨其与DM创面愈合延迟的病理关系。方法将90只SD火鼠分为DM组（50只）和NM组（NM，40只），DM纽大鼠制成链脲菌索绣导的DM模型，之后将两纰大鼠造成10％TBSA深Ⅱ度烫伤。各组大鼠均于伤前及伤后3、7、14、21d（每组每时相点6只）留收创面皮肤组织标本，对其各项生物学特征进行检测。结果与NM组大鼠比较，DM组伤前真皮厚度明显变薄（P〈0．01）、胶原排列紊乱、真皮内出现大量的慢性炎性细胞浸润，皮肤糖含量升高（DM组2．77mg／g，NM组0．85mg／g，P〈0．01）和高级糖基化终产物（AGEs）蓄积。深Ⅱ度埂伤后，DM组大鼠Fb的S期细胞百分比和羟脯氨酸合成量明显低于NM组，而Fb的凋亡率却明显高于NM组（P〈0．05或0．01）。结论DM大鼠合并深Ⅱ度烫伤后真皮中Fb的乍物学特征异常，可能与局部皮肤组织糖含量升高和AGEs的蓄积有关，推测是DM患者容易并发慢性溃疡的病理基础之一。     

喂饲左旋精氨酸对烫伤大鼠肠道保护作用机制的研究

目的探讨喂饲左旋精氨酸(L-Arg)对烫伤大鼠肠道缺血再灌注损伤的作用机制。方法将66只SD大鼠随机分为正常对照组(6只,不作烫伤和其他处理)、精氨酸组(30只,烫伤后2h喂饲70g/LL-Arg,1ml/次,2次/d)和普通喂养组(30只,烫伤后喂饲等量凉开水)。检测正常对照组及两组烫伤大鼠伤后6、12、24、48、72h肠组织内皮素(ET)水平、一氧化氮(NO)含量、ET/NO比值以及血浆内毒素水平的变化,并取回肠组织标本作病理学观察。结果伤后6、12、24h,精氨酸组大鼠肠组织ET水平分别为(0.80±0.26)、(0.75±0.30)、(0.63±0.22)ng/g,低于普通喂养组(1.26±0.38)、(1.34±0.37)、(0.97±0.19)ng/g(P<0.05);其NO含量显著高于普通喂养组(P<0.01);ET/NO比值和血浆内毒素水平均低于普通喂养组(P<0.05或0.01)。病理学观察显示,精氨酸组大鼠肠黏膜损伤情况明显轻于普通喂养组。结论喂饲L-Arg可减轻烫伤大鼠肠组织缺血再灌注损伤,有利于保护肠黏膜屏障功能。其机制为喂饲L-Arg后增加了肠黏膜局部NO的含量,有助于维持ET/NO比值的稳定。     

局部联合应用NGF及胰岛素对糖尿病大鼠烫伤创面血管形成及Bcl-2、Bax表达的影响

目的探讨局部应用NGF联合胰岛素对糖尿病大鼠烫伤创面血管中凋亡相关因子Bcl-2、Bax表达的影响及创面愈合的机制。方法取75只清洁级雄性Wistar大鼠,体重200～220 g,随机分为正常对照组(A组)、糖尿病对照组(B组)、胰岛素治疗组(C组)、NGF治疗组(D组)、NGF联合胰岛素治疗组(E组),每组15只。B、C、D、E组大鼠采用两步给药法腹腔注射链脲佐菌素(streptozotocin,STZ)建立糖尿病模型,STZ剂量分别为第1天10 mg/kg,第3天50 mg/kg;A组给予相同剂量柠檬酸缓冲液。模型制备后1个月,采用水蒸气烫伤法于各组大鼠背部制备2个深Ⅱ度烫伤创面。烫伤模型制备后,A、B组创面外敷3层生理盐水纱布;C组创面外敷3层浸润5 U胰岛素诺和灵30R的纱布,并每日腹部皮下注射诺和灵30R 4～6 U/kg;D组创面外敷3层浸润5 mL NGF溶液(25 U/mL)的纱布;E组联合C、D组方法处理。观察大鼠一般情况,伤后7、11、15、21 d大体观察各组创面愈合情况并计算创面愈合率,伤后3、7、11、15、21 d取创面组织行组织学及免疫组织化学染色观察,检测创面Bcl-2、Bax、CD34的表达并计算微血管密度。结果各组大鼠均存活至实验完成。随时间延长,各组创面逐渐缩小,其中E组创面愈合速度、皮肤角化、毛发生长及肉芽组织和胶原纤维生长均优于其余各组。伤后各时间点E组创面愈合率均高于其余各组,差异有统计学意义(P<0.05)。伤后随时间延长,各组CD34、Bcl-2表达逐渐增强,至15 d达高峰,21 d表达减弱;E组各时间点表达均强于其他各组(P<0.05)。伤后3 d各组均未见Bax表达,7 d后开始见新生血管内皮细胞Bax表达,且随时间延长表达逐渐增强,其中E组表达强度均低于其余各组(P<0.05)。结论局部联合应用NGF和胰岛素可通过抑制创面血管内皮细胞凋亡、增加创面血管生成,促进糖尿病大鼠创面愈合。     

VEGF165-胰岛素复合凝胶对糖尿病大鼠局部深Ⅱ度烫伤创面MVD及Ⅰ型胶原蛋白表达的影响

目的:制备外用VEGF165-胰岛素复合凝胶并观察其对糖尿病大鼠深Ⅱ度烫伤创面愈合的作用。方法:以新型凝胶卡波姆980为基质,分别加入VEGF165、胰岛素,分别配制成胰岛素凝胶、VEGF165凝胶、VEGF165-胰岛素复合凝胶和空白对照凝胶。观察各组凝胶性状,检测其p H值。75只SPF级雄性Wistar大鼠,体重190~290g,随机分为正常对照组、糖尿病对照组、胰岛素凝胶组、VEGF165凝胶组、VEGF165-胰岛素复合凝胶组,每组15只。各组大鼠禁食12h后,除正常对照组外,其余各组一次性腹腔注射链脲佐菌素(55 mg/kg)制备1型糖尿病模型,正常对照组大鼠注射相同剂量柠檬酸钠缓冲液。1型糖尿病大鼠造模成功后,采用恒温水浴箱80℃,制备深Ⅱ度烫伤模型。正常对照组、糖尿病对照组创面外敷空白凝胶基质,其余各组外敷对应凝胶制剂,每天换药1次。深Ⅱ度烫伤造模成功后观察各组大鼠存活情况,于3、7、11、15、21天时点观察糖尿病大鼠创面愈合情况并计算创面愈合率,在各时点随机处死每组3只大鼠取烫伤皮肤全层留取标本,行组织学切片及免疫组化染色观察各时点炎性细胞,微血管形成及胶原蛋白。结果:制备的各组凝胶透明,保湿性极佳,黏附性良好,便于涂抹及清洗,无组织毒性。各组大鼠无感染及死亡发生。3天后烫伤各组创面无明显缩小,7、11、15、21天时,VEGF165-胰岛素复合凝胶组创面愈合率最高,糖尿病组最低。VEGF165-胰岛素复合凝胶组愈合率与其余各组比较,差异均有统计学意义(P0.05)。病理切片观察示各时点VEGF165-胰岛素复合凝胶组肉芽组织形成及上皮化均优于其余各组。免疫组化染色示各组微血管数量、Ⅰ型胶原蛋白阳性率均于3天开始表达,且随时间阳性细胞增多明显;各时间点VEGF165-胰岛素复合凝胶组微血管密度及Ⅰ型胶原蛋白最高,糖尿病组最低,与其余各组比较以及VEGF165-胰岛素复合凝胶组、糖尿病组间比较差异均有统计学意义(P0.05)。结论:糖尿病大鼠皮肤深Ⅱ度烫伤外用VEGF165-胰岛素复合凝胶对创面愈合有明显的促进作用     

Local arginine supplementation results in sustained wound nitric oxide production and reductions in vascular endothelial growth factor expression and granulation tissue formation

OBJECTIVE: The goal of this work was to test the functional role of L-arginine in promotion of nitric oxide (NO) production and the vigorous granulation tissue formation characteristic of this wound model. BACKGROUND: Therapeutic use of supplemental arginine has been proposed as a safe and efficacious method to produce NO from nitric oxide synthase (NOS) and to produce proline and polyamines from arginase to improve wound healing. Although NO appears to be necessary to promote wound healing, the preferential metabolism of arginine to NO via NOS 2 may be detrimental if maintained beyond the initial days of healing. METHODS: A ventral hernia, surgically created in the abdominal wall of 12 swine, was repaired with silicone sheeting and skin closure. Osmotic infusion pumps, inserted in remote subcutaneous pockets, continuously delivered saline (n = 6) or L-arginine (n = 6) into the wound environment. Granulation tissue thickness was determined by ultrasonography. Fluid was aspirated serially from the wound compartment for measurements of nitrite/nitrate (NOx), vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1), and amino acid concentrations. On day 14, the animals were sacrificed and the abdominal wall was harvested for immunohistochemical and molecular analysis. RESULTS: In animals receiving saline, a nearly linear four-fold increase in granulation tissue thickness was measured during the 14-day interval. In contrast, quantitative ultrasound analysis detected significant reductions in L-arginine infused granulation tissue thickness compared with controls between days 4 and 14 (P < 0.05). Wound vessel count and luminal vascular surface area estimates derived from image analysis of histological sections were two- to three-fold lower in the L-arginine animals compared with controls (P < 0.05). Significant and sustained increases in wound fluid NOx levels were noted in L-arginine animals compared to saline controls (230 microM versus 75 microM at day 14, P < 0.05). Conversely, late VEGF levels (days 11 to 14) were reduced in the L-arginine animals compared to controls (7500 pg/ml versus 10,000 pg/ml at day 11, P < 0.05; 7250 pg/ml versus 11,101 pg/ml at day 14, P < 0.05). Arginine concentrations remained two- to four-fold greater in L-arginine treated animals compared with controls over the entire time course (P < 0.05). There were no significant differences in concentrations of ornithine, citrulline, or proline noted between groups over the 14-day period. Finally, TGF-beta1 levels were unaffected by L-arginine treatment. CONCLUSION: Although NO appears to be necessary for granulation tissue formation, early supplemental arginine may disturb the reciprocal regulation of NOS 2 and arginase, leading to the preferential metabolism of arginine to excess NO rather than ornithine, with consequent reductions in angiogenesis and granulation tissue formation.     

Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats

Bone marrow stem cells participate in tissue repair processes and may have a role in wound healing. Diabetes is characterised by delayed and poor wound healing. We investigated the potential of bone marrow-derived mesenchymal stromal cells (BMSCs) to promote healing of fascial wounds in diabetic rats. After manifestation of streptozotocin (STZ)-induced diabetic state for 5 weeks in male adult Sprague-Dawley rats, healing of fascial wounds was severely compromised. Compromised wound healing in diabetic rats was characterised by excessive polymorphonuclear cell infiltration, lack of granulation tissue formation, deficit of collagen and growth factor [transforming growth factor (TGF-beta), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor PDGF-BB and keratinocyte growth factor (KGF)] expression in the wound tissue and significant decrease in biomechanical strength of wounds. Treatment with BMSC systemically or locally at the wound site improved the wound-breaking strength (WBS) of fascial wounds. The improvement in WBS was associated with an immediate and significant increase in collagen levels (types I-V) in the wound bed. In addition, treatment with BMSCs increased the expression of growth factors critical to proper repair and regeneration of the damaged tissue moderately (TGF-beta, KGF) to markedly (EGF, VEGF, PDGF-BB). These data suggest that cell therapy with BMSCs has the potential to augment healing of the diabetic wounds.     

Supplemental l-arginine enhances wound healing following trauma/hemorrhagic shock

To determine whether parenteral L-arginine supplementation enhances the impaired wound healing of rats subjected to trauma/hemorrhagic shock. Impaired wound healing after trauma and shock has been documented experimentally and clinically. L-arginine has been shown to enhance wound strength and collagen synthesis in rodents and humans. Its efficacy under conditions of impaired wound healing is less well defined. Forty-eight male Lewis rats were used in this study. Using a well-defined model, 24 rats underwent trauma/hemorrhagic shock before wounding. Twenty-four untreated rats served as controls. All animals underwent a dorsal skin incision with implantation of polyvinyl-alcohol sponges. Half of the animals in each group were assigned to receive 1 g/kg/day of L-arginine by intraperitoneal injection in three divided doses, while the other half received saline injections only. Animals were sacrificed 10 days postwounding, and wound-breaking strength (WBS) and wound sponge total hydroxyproline (OHP) and nitrite/nitrate (NO(x)) content were determined. Wound sponge RNA was collected and subjected to Northern blot analysis for procollagens I and III. Trauma/hemorrhage greatly decreased WBS with a concomitant diminution in collagen (OHP) deposition. L-arginine significantly enhanced WBS (19%) and increased OHP (21%) levels in control animals as well as in rats subjected to trauma/hemorrhage (WBS +29%, OHP 40%) compared with their saline-treated counterparts. Procollagen I and III mRNA levels were elevated by L-arginine treatment in both trauma/hemorrhage and control rats. Arginine treatment had no effect on wound fluid and plasma NO(x). The data demonstrate that the impaired healing subsequent to trauma/hemorrhage can be greatly alleviated by L-arginine supplementation.     

一种大鼠蒸气烫伤模型的建立

目的　建立一种可控深度及面积的大鼠烫伤模型。　方法　用高压蒸气消毒锅及自制烫伤支架制作高压蒸气烫伤装置 ,用压力为 0 .12MPa(1MPa=75 0 0mmHg)、直径 2 .6cm的致伤孔上分别在大鼠背部烫 3、4、5、6、7、8、9、10s,每时相点 5个创面。伤后 2 4h取标本行组织学观察 ,用Photoshop软件测量烫伤深度 ,并观察有无毛囊、汗腺附件受损、烫伤区被毛生长及创面愈合情况。　结果　烫伤深度与烫伤时间的变化呈正相关 (r =0.99)。浅Ⅱ、深Ⅱ、Ⅲ度烫伤模型的致伤时间分别为 3、5、7s。烫伤 7～ 10s创面深度虽逐渐加重 ,愈合时间却相近。　结论　该模型可以控制烫伤深度、面积 ,烧伤深度划分准确且操作简便 ,是研究创伤修复机制及评价创面用药的较好模型。     

硒化壳聚糖促创面愈合作用的体内研究

目的:研究硒化壳聚糖软膏促浅Ⅱ度烫伤小鼠创面愈合的作用。方法:147只昆明种小鼠随机分为硒化壳聚糖软膏治疗组、基质治疗组和京万红治疗组,每组49只,采用将脱毛区置于70℃恒温水浴中6s的方法制成10%体表面积浅Ⅱ度烫伤模型,伤情经病理切片证实。各组创面分别用硒化壳聚糖软膏纱布(1ml/cm2)、基质纱布(1ml/cm2)、京万红纱布(1ml/cm2)覆盖包扎固定后放回笼中饲养,换药1次/天,观察愈合时间,于伤后12h,第1、3、5、7和9天,分别处死各组小鼠7只,检测含水量、羟脯氨酸、TNF、NO、ALT,另取7只做为正常对照。结果:①创面愈合时间:硒化壳聚糖软膏组为(12.9±2.9)天、基质组为(16.3±2.1)天、京万红组为(11.8±2.4)天,硒化壳聚糖软膏组比基质组明显缩短(P<0.05);②创面含水量:伤后第1、3、5天硒化壳聚糖软膏组[(86.3±3.5)%、(77.8±3.9)%、(72.3±2.7)%]创面含水量显著低于基质组[(92.8±3.2)%、(84.9±4.2)%、(77.2±2.8)%,(P<0.05)],伤后7～9天,各组均基本恢复到正常水平;③创面TNF-α水平:伤后12h至1天达高峰,随后逐渐下降,至伤后第5天仍高于正常对照组(P<0.05);伤后12h、1天、3天、5天,硒化壳聚糖软膏组和京万红组TNF水平明显低于基质组(P<0.05);④创面组织NO含量:伤后12h各烫伤组NO含量达高峰,随后下降,至伤后第9天仍高于正常对照组(P<0.05);伤后12h、1天,硒化壳聚糖软膏组和京万红组NO含量明显低于基质组(P<0.05);⑤创面羟脯氨酸含量:伤后5、7、9天硒化壳聚糖软膏组羟脯氨酸含量显著高于基质组和京万红组(P<0.05)。结论:硒化壳聚糖软膏能有效减少烫伤后早期创面组织NO和TNF-α释放,减少渗出和水肿,晚期通过增强创面胶原合成来促进创面愈合。     

NO mediates antifibrotic actions of L-arginine supplementation following induction of anti-thy1 glomerulonephritis

NO mediates antifibrotic actions of L-arginine supplementation following induction of anti-thy1 glomerulonephritis. BACKGROUND: L-Arginine plays a complex role in renal matrix expansion, involving endogenous metabolism into nitric oxide (NO), polyamines, L-proline and agmatine. Supplementing dietary L-arginine intake has been shown to limit transforming growth factor (TGF)-beta 1 overproduction and matrix accumulation in rats with induced anti-thy1 glomerulonephritis (GN). The present study tests the hypothesis that this beneficial effect on in vivo TGF-beta overexpression is mediated via the generation of NO. METHODS: One day after induction of anti-thy1 GN, male Wistar rats fed a normal protein diet were assigned to the following groups: (1) normal controls; (2) GN; (3) GN-Arg (plus 500 mg L-arginine/day); (4) GN-Arg-NAME [plus 500 mg L-arginine/day and 75 mg/L of the NO synthase inhibitor nitro-L-arginine-methyl ester (L-NAME) in the drinking water]; and (5) GN-Molsi (10 mg/day of the NO donor molsidomine). In protocol 1, treatment lasted until day 7, and in protocol 2, until day 12 after disease induction, respectively. Analysis included systolic blood pressure, a glomerular histologic matrix score, and the glomerular mRNA and protein expression of the key fibrogen TGF-beta1, the matrix protein fibronectin, and the protease inhibitor plasminogen activator inhibitor type 1 (PAI-1). RESULTS: Blood pressure was normal in untreated anti-thy1 animals and not significantly affected by any of the treatments. Compared to untreated nephritic rats, administration of both L-arginine and molsidomine reduced glomerular TGF-beta 1 overexpression significantly and to a similar degree in both protocols, while the beneficial effect of L-arginine was abolished by concomitant NO synthesis inhibition. Glomerular matrix accumulation, fibronectin and PAI-1 mRNA and protein expression closely followed the expression of TGF-beta 1. CONCLUSION: The present study shows that L-arginine's antifibrotic action in normotensive anti-thy1 GN is mainly mediated by endogenous production of NO. The data suggest that NO limits in vivo TGF-beta overexpression in a pressure-independent manner and that NO donors may be of benefit in the treatment of human fibrotic renal disease.     

局部应用不同浓度硝普钠对创伤愈合影响的时效性研究

目的:应用组织化学及计算机辅助图像分析方法,观察外源性一氧化氮(nitricoxide,NO)在创伤愈合过程不同时间对肉芽组织生长及成纤维细胞增殖的影响,探讨其对促进创伤愈合和抑制病理性瘢痕形成的机制。方法:60只大鼠随机分为对照组及实验A、B、C、D组,每组12只,通过建立大鼠创伤模型,并分别在创面局部应用5%葡萄糖溶液、0.5mmol/L、1mmol/L、2mmol/L和4mmol/L硝普钠,观察及测量创伤后3天、7天、10天和14天的肉芽生长情况、成纤维细胞数密度、胶原纤维面密度和肉芽组织中羟脯氨酸含量。结果:实验A、B组相对于对照组和实验C、D组表现出更好的伤口愈合,且瘢痕形成最小。结论:局部应用外源性NO具有显著的促修复作用,主要体现在伤后第7～10天,小剂量的NO促进创面愈合的作用远远大于大剂量NO。局部过量的NO聚积可阻碍正常的伤口愈合,并呈现全身毒性反应。在伤后7～10天应用外源性NO可抑制病理性瘢痕形成。     

The effect of single administration of vascular endothelial growth factor or L-arginine on necrosis and vasculature of the epigastric flap in the rat model.

OBJECTIVES: Vascular endothelial growth factor (VEGF) and nitric oxide (NO) produce vasodilation, induce angiogenesis, and improve survival of surgical flaps. We used the rat epigastric skin flap to study the effect of a single intra-arterial dose of VEGF or L-arginine, a substrate for NO production, on flap regional necrosis and pedicle dependence of flap perfusion. METHODS: In 30 Sprague-Dawley rats an 8 x 8 cm2 skin flap, consisting of four vertical zones marked A through D (right to left), based on the proximal right inferior epigastric vessels was raised. Subsequently, 1 ml of either saline (control, n =10), 5 microg VEGF (VEGF, n = 10), or 50 mg of L-arginine (L-arginine, n = 10) was injected into the arterial pedicle by cannulating the right saphenous artery, and the flap was resutured in place. After 8 days, the animals were perfused systemically with 15 microm coloured fluorescent microspheres before (blue) and after (yellow-green) ligation of the right inferior epigastric vascular pedicle. After sacrifice, the area of flap necrosis was measured in each zone by templates and weight-to-surface ratio, and the flap zones were harvested and processed for determination of fluorescence and blood flow. RESULTS: Administration of VEGF or L-arginine resulted in decreased total and regional (zone D) flap necrosis (ANOVA <0.001). The total and regional flap shrinkage was greater in the experimental groups (ANOVA <0.02). While VEGF and L-arginine decreased the percentage of necrosis in the zone most distal to the pedicle (ANOVA <0.01) only L-arginine diminished percentage of total flap necrosis (p = 0.04). In the VEGF group, total and regional flap perfusion did not change after pedicle ligation, but perfusion decreased significantly in zones B through D in the L-arginine treated rats. CONCLUSION: Single intra-pedicle administration of VEGF or L-arginine decreased necrosis of the epigastric skin flap at 8 days postoperatively, but flap shrinkage also increased in the zone with the greatest degree of necrosis. Perfusion data suggest that beneficial effects of VEGF and L-arginine on flap survival may be based on different mechanisms.     

The JCR:LA-cp rat: a novel model for impaired wound healing.

JCR:LA-cp/cp obese rats and their lean controls were evaluated as a type 2 diabetic wound healing model and the healing quality was characterized. This model of insulin resistance has been used extensively to study atherosclerosis but has not previously been used to study wound healing. Six circular excisional wounds were made on the dorsum of each rat and followed to day 21.Tracings of the wounds were made and used to assess the rate of wound closure. Planimetry showed a significantly diminished contraction of wounds in obese rats, but no significant difference in reepithelialization was observed. Collagen content was determined from the hydroxyproline content in wounded and unwounded skin. There were significantly lower levels of hydroxyproline in the wounds of obese compared to lean animals at day 21. Histology showed adipose tissue in place of dermal tissue in the JCR:LA-cp/cp rat in both unwounded tissue and in the wound at day 21. Active transforming growth factor-beta 1 (TGF-beta 1) was measured in the serum using the plasminogen activator inhibitor-1/luciferase assay and serum total TGF-beta was measured using an enzyme-linked immunosorbent assay. Active TGF-beta was significantly higher in the serum of obese animals compared with lean animals, while total TGF-beta 1 was not significantly different between the groups. Both active and total TGF-beta was measured in tissue sections using the plasminogen activator inhibitor-1/luciferase assay. There was no significant difference in active TGF-beta between genotypes, while obese rats had significantly higher levels of total TGF-beta at day 21. These results indicate a deficiency in wound healing in obese animals characterized by decreased wound contraction, decreased collagen production, and changes in histology. The JCR:LA-cp rat develops insulin resistance, atherosclerosis and early type 2 diabetes and may be a good model for impairment of wound healing in humans with metabolic syndrome.     

Supplemental L-arginine enhances wound healing in diabetic rats

L-arginine has been shown to enhance wound strength and collagen deposition in rodents and humans. Diabetes mellitus, which impairs wound healing, is accompanied by a reduction in nitric oxide at the wound site. The amino acid L-arginine is the only substrate for nitric oxide synthesis. We sought to determine whether supplemental L-arginine can restore the impaired wound healing of diabetic rats. Fifty-six male Lewis rats were used in this study, of which twenty-nine rats were rendered diabetic 7 days prior to surgery with intraperitoneal streptozotocin. Twenty-seven untreated rats served as controls. Animals underwent a dorsal skin incision with implantation of polyvinyl-alcohol sponges. Sixteen diabetic and 14 normal rats received 1 g/kg/day of L-arginine by injection, while the remainder received saline injections only. Animals were euthanized 10 days postwounding, and their wounds were analyzed for breaking strength. The wound sponges were assayed for total hydroxyproline and nitrite/nitrate content. Plasma and wound fluid concentrations of L-arginine, ornithine, and citrulline were determined. Wound sponge RNA was extracted and subjected to Northern blot analysis for procollagen I and III. Diabetic wounds had greatly decreased breaking strengths compared with controls. L-arginine significantly enhanced wound breaking strengths in both control (+23%) and diabetic animals (+44%), and also increased wound hydroxyproline levels in both diabetic (+40%) and control animals (+24%) as compared to their saline-treated counterparts. mRNA for procollagen I and III were elevated by L-arginine treatment in both diabetic rats and controls. Treatment with L-arginine significantly increased wound fluid nitrite/nitrate levels in diabetic animals. The data show that the impaired healing of diabetic wounds can be partially corrected by L-arginine supplementation, and that this effect is accompanied by enhanced wound nitric oxide synthesis.