肠三叶因子和黏蛋白对烧伤血清所致肠上皮细胞增殖移行能力变化的影响

目的 观察烧伤血清所致肠上皮细胞增殖、移行能力的变化,以及联合应用肠三叶因子(ITF)和黏蛋白对其的影响.方法 将大鼠小肠上皮细胞株IEC-6传代培养,按照随机数字袁法分为正常对照组、烧伤血清组、ITF+烧伤血清组、黏蛋白+烧伤血清组和ITF+黏蛋白+烧伤血清组,均采用DMEM培养液培养,其内分别添加体积分数10％小牛血清、体积分数10％烧伤大鼠血清、25 μg/mL ITF和体积分数10％烧伤大鼠血清、250 μg/mL黏蛋白和体积分数10％烧伤大鼠血清以及联合使用上述剂量ITF、黏蛋白和烧伤大鼠血清.处理后0～4d观察细胞增殖能力;划痕实验后12、24、36、48、72 h观察细胞移行情况;Transwell法(细胞置于上室、培养液加入下室)培养4、6、8、10、12 h,观察细胞变形能力,以下室内细胞计数表示.对数据进行t检验.结果 (1)细胞增殖能力.处理后1～4d烧伤血清组细胞数量明显低于正常对照组(t值为-16.569 ～ - 2.613.P＜0.05或P＜0.01).ITF+烧伤血清组和黏蛋白+烧伤血清组细胞数量与烧伤血清组接近(t值分别为0.037～0 740、0.116～0.429,P值均大于0 05);处理后2d,ITF+黏蛋白+烧伤血清组细胞数量明显高于烧伤血清组(t =6.484,P＜0.01)、ITF+烧伤血清组(t =3.838,P＜0.01).(2)细胞移行能力.烧伤血清组划痕后各时相点细胞移行距离均远远短于正常对照组(t值为-37.594～ -6.727,P值均小于0.0I).与烧伤血清组比较,黏蛋白+烧伤血清组划痕后各时相点细胞移行距离无明显变化(t值为0.055 ～0.589,P值均大于0 05).ITF+烧伤血清组划痕后12、24、36 h细胞移行距离分别为(47±6)、(126±13)、(170±11) μm,明显长于烧伤血清组[(42±7)、(98±14)、(154±22) μm,t值为2.230～4.817,P＜0.05或P＜0.01].ITF+黏蛋白+烧伤血清组划痕后各时相点细胞移行距离明显长于烧伤血清组(t值为2.982 ～7.390,P＜0.05或P＜0.01),划痕后12 ～48 h明显长于ITF+烧伤血清组(t值为2.707 ～2.918,P＜0.05或P＜0.01).(3)细胞变形能力.与正常对照组比较,烧伤血清组穿孔细胞数大幅减少(t值为- 23.965 ～ -6.436,P值均小于0.01).与烧伤血清组比较,黏蛋白+烧伤血清组各时相点穿孔细胞数改变不明显(t值为0.199～ 0.797,P值均大于0 05);ITF+烧伤血清组各时相点穿孔细胞数均明显增加(t值为3.650 ～10.028,P值均小于0.01).ITF+黏蛋白+烧伤血清组各时相点穿孔细胞数明显多于烧伤血清组(t值为4.313～15.100,P值均小于0.01),培养10、12 h时穿孔细胞数分别为(328±47)、(465±37)个,明显多于ITF+烧伤血清组[(277±25)、(353±34)个,t值分别为3.051、6 945,P值均小于0.01].结论 ITF对肠上皮细胞增殖影响有限,但能明显改善细胞变形能力,促进细胞移行;单独使用黏蛋白对细胞无明显作用,与ITF联合应用能增强ITF的作用.ITF维护肠黏膜屏障的主要机制为促进细胞移行.     

烧伤后肠黏膜细胞外基质与细胞凋亡关系的实验研究

目的　探讨烧伤后大鼠肠黏膜细胞凋亡与细胞外基质的关系。　方法　 30只Wistar大鼠随机分为烧伤后 6、12h,1、3、5d组及正常对照组 ,每组 5只。检测肠黏膜上皮细胞凋亡数、cas pases 3酶活性、细胞外基质成分 (层粘连蛋白、Ⅳ型胶原 )含量 ,并作相关分析。　 结果　烧伤后大鼠肠上皮凋亡细胞数、caspases 3的活性较正常对照组明显升高 (P <0.0 5或 0 .0 1) ,大鼠肠黏膜层粘连蛋白、Ⅳ型胶原含量较正常对照组下降 ( P <0.0 5或 0 .0 1)。直线相关分析结果 :烧伤后肠黏膜层粘连蛋白、Ⅳ型胶原含量变化与细胞凋亡数呈显著的负相关 (r =- 0.5 75, - 0.6 13,P <0 0 5 )。　 结论　烧伤后大鼠肠黏膜细胞凋亡增加 ,且与细胞外基质的变化相关。     

大鼠烧伤后库普弗细胞在促炎细胞因子产生中的作用

目的　观察大鼠严重烧伤后早期 ,库普弗细胞在肿瘤坏死因子α(TNFα)、白细胞介素(IL) 1β、IL 6产生中的作用。方法　观察 (1)烧伤血清对体外培养的大鼠库普弗细胞分泌TNFα、IL 1β、IL 6的刺激作用 ;(2 )烧伤后大鼠库普弗细胞的细胞因子mRNA表达变化 ;(3)应用库普弗细胞特异性抑制剂三氯化钆后 ,烧伤大鼠血浆内细胞因子含量变化。　结果　烧伤血清能刺激库普弗细胞释放TNFα、IL 1β、IL 6 ;大鼠烧伤后库普弗细胞TNFα、IL 1β、IL 6mRNA表达量显著升高 ;预先抑制库普弗细胞的活性 ,烧伤后血浆TNFα、IL 1β、IL 6水平均显著降低 ,分别为烧伤组的 34.71%、36 99%、33.70 %。结论　库普弗细胞是大鼠烧伤后血浆中TNFα、IL 1β、IL 6的主要来源     

肠上皮细胞缺血缺氧凋亡模型的实验研究

我们建立了以体外人工基底膜为表衬的肠上皮细胞（IEC）原代培养，模拟临床缺血缺氧损伤后IEC的脱落凋亡变化的实验模型，报道如下，     

核因子κB在烧伤血清诱导血管内皮细胞损伤中的作用

目的:探讨核因子κB在烧伤血清诱导血管内皮细胞损伤中的作用。方法:体外培养人脐静脉内皮细胞(HUVECs),随机分为空白对照组、20%(V/V)人烧伤血清刺激组、PDTC预处理组,1h后采用MTT及流式细胞仪观察HUVECs损伤情况,蛋白印迹法检测HUVECs胞核NF-κB-p65的蛋白表达变化。结果:与空白对照组比较,烧伤血清诱导了HUVECs损伤和凋亡,胞核NF-κB-p65蛋白表达增多。相对于烧伤血清刺激组,PDTC预处理组HUVECs损伤减轻,细胞凋亡显著减少。结论:核因子κB参与烧伤血清致内皮细胞损伤,阻断核因子κB可能对防治严重烧伤所致的内皮细胞损伤具有一定潜在价值。     

大鼠烫伤后肠道免疫屏障损伤的实验研究

为探索烧伤后肠道免疫屏障损伤的规律及其与肠道细菌移位的关系,我们对烫伤大鼠肠道免疫屏障的损伤进行了比较全面的动态观察,结果表明,伤后早期动物肠道内容物 IgA(免疫球蛋白 A)含量明显降低,肠固有层及上皮内 CD3 (T 细胞总数)和 CD4 (辅助/诱导 T 细胞)T 淋巴细胞明显减少,上皮内 CD4 与 CD8 (抑制/杀伤 T 细胞)T 细胞数量的比值倒置;伤后肠道细菌 IgA 包被率亦明显降低,且在整个实验过程中均明显低于伤前组;与上述改变相对应,肠道细菌移位率在伤后早期明显升高。提示,伤后肠道免疫屏障损伤是多方面的,而这一损伤对烧伤后肠道细菌移位和脓毒症的发生、发展可能起着重要作用。     

CD3 AK细胞提高烧伤患者细胞免疫功能的研究

目的 探索治疗烧伤患免疫功能低下的新方法。方法 用无血清培养法培养CD3AK细胞,治疗大面积烧伤患,类似患另设对照组进行普通治疗,观察不同时相点两组患的细胞免疫指标变化。结果 同对照组相比,经体外培养的CD3AK细胞治疗后,患T细胞亚群中CD4细胞比例升高,CD8细胞比例下降,CD4／CD8细胞比值升高。血清可溶性白介素2受体水平下降,T淋巴细胞转化活性增强,NK细胞活性增强。结论 无血清培养法培养的CD3AK细胞,能改善烧伤患细胞免疫功能低下的状态,促进细胞免疫功能恢复。     

硫酸软骨素对牛磺胆酸钠诱导的大鼠离体胰腺细胞骨架损伤的影响

为探讨硫酸软骨素（CS）对牛磺胆酸钠诱导的大鼠离体胰腺腺泡细胞氧化应激损伤及细胞骨架蛋白F actin结构的影响。笔者将雄性Wistar大鼠36只，经分离提纯获取胰腺腺泡细胞悬液，牛磺胆酸钠处理离体胰腺细胞后，随机分3组即细胞模型（细胞骨架损伤）组，CS处理组和对照组。各组分别于30min，1h，3h进行MDA， GSH， SOD和ATP含量测定。离心细胞涂片后，以罗丹明-法罗丁F actin染色，在共聚焦激光显微镜下观察F actin结构的变化；用流式细胞术检测细胞的F actin蛋白含量。结果示,模型组GSH，SOD和ATP明显下降（P<0.05），MDA明显升高(P<0.05)。CS组GSH，SOD，ATP下降幅度小于模型组，差异有显著性（P<0.05）；MDA升高幅度小于模型组，差异有显著性（P<0.05）。模型组细胞骨架蛋白F actin解聚并弥漫分布于胞浆内，其蛋白含量持续下降（P<0.05）；CS组F actin结构较稳定，其蛋白水平明显高于模型组（P<0.05）。提示：牛磺胆酸钠诱导的离体胰腺细胞早期已存在内源性抗氧化物质的显著下降，脂质过氧化增加和ATP耗竭加重了细胞损伤。CS可通过减轻氧化应激损伤维持ATP含量，缓解F actin的降解，稳定细胞骨架结构从而减轻细胞损伤。     

重组人生长激素对烧伤大鼠肠粘膜结构及细胞凋亡的影响

目的　探讨重组人生长激素 (rhGH)的早期应用对严重烧伤大鼠小肠粘膜的保护作用。　方法　 30只成年雄性Wistar大鼠随机分为对照组、烧伤组和rhGH组 ,每组 10只。后两组造成2 5 %TBSAⅢ度烧伤创面 ,立即腹腔注射地塞米松 80mg/kg ,从伤后 2h开始分别给予等渗盐水和rhGH(1.33U·kg-1·d-1)。于伤后 30、96h ,观察回肠末端粘膜组织形态、肠上皮细胞超微结构和肠粘膜细胞增殖活性与凋亡率变化。　结果　烧伤组肠粘膜形态结构及上皮细胞损伤严重 ,rhGH组明显减轻 ,基本接近对照组 ;伤后 30h烧伤组和rhGH组肠粘膜细胞增殖指数 (proliferativeindex ,PI)均明显高于对照组 (P <0 .0 5～ 0 .0 1) ,但两组之间差异无显著性意义 ;伤后 96h ,rhGH组PI较烧伤组和对照组均明显升高 (P <0 .0 1)。烧伤组肠粘膜细胞凋亡率较对照组明显升高 (P <0 .0 1) ,rhGH组凋亡率较烧伤组 (P <0 .0 1)和对照组 (P <0 .0 5 )明显下降。　结论　rhGH能促进严重烧伤后肠粘膜细胞增殖 ,但作用缓慢 ;能抑制肠粘膜细胞凋亡 ,而且作用迅速、效果明显 ,并可能通过抑制作用减轻肠粘膜损伤 ,维护形态结构的基本正常。     

烧伤血清对血管内皮细胞损伤及抗凝特性的影响

采用血管内皮细胞培养模型,动态观察了家兔烧伤早期(30%Ⅲ度)血清对内皮细胞结构和功能的影响,通过相差显微镜动态观察、台盼兰染色、HE 染色光镜检查及扫描电镜检查,同时检测孵育不同时相点培养液中 LDH、6-keto-PGF_(1α)及 tPA 含量及内皮细胞抗血小板粘附、聚集能力的变化。结果显示:①烧伤早期烧伤血清对内皮细胞有明显的损伤作用,功能改变早于结构损害,而结构损害又以细胞间连接及其表面结构的改变为早;②烧伤血清可明显降低内皮细胞抗血小板粘附、聚集能力,这可能是局部微血栓形成的促发因素;③烧伤血清还降低了内皮细胞的纤溶能力,主要是由于表面结构严重破坏,表面的各种纤溶成份丢失或破坏;同时释放的 tPA 虽然增多,但其活性可能减弱。     

Rho激酶在烧伤大鼠血清诱导的血管内皮细胞骨架变化中的作用

目的　观察烧伤血清刺激下血管内皮细胞骨架的变化及Rho信号转导通路所起的作用。　方法　常规培养人脐静脉血管内皮细胞系ECV304,随机分为空白对照组、实验对照组、烧伤组、Y -27632组、烧伤 Y -27632组、Y- 27632 烧伤组、溶血磷脂酸(LPA)组和LPA Y- 27632组,分别用正常大鼠血清、烧伤大鼠血清、30μmol/LRho激酶抑制剂Y -27632、13μmol/LRhoA激动剂LPA单独刺激或联合刺激。采用HE染色于光镜下观察各组内皮细胞形态。于荧光倒置相差显微镜下观察内皮细胞骨架变化,除Y -27632组外,各组均在刺激后6、7、8h3个时相点进行观察。用流式细胞仪检测实验对照组、烧伤组、Y -27632组、烧伤 Y- 27632组、LPA组和LPA Y 27632组刺激6h的肌动蛋白含量。　结果　空白对照组、实验对照组细胞呈梭形或多边形,生长良好;丝状肌动蛋白主要分布在细胞周边,形成周边肌动蛋白丝带,细胞生长融合为单层后呈网状结构;球状肌动蛋白集中在细胞中央。烧伤组烧伤血清刺激6h,细胞贴壁差,丝状肌动蛋白重组,应力纤维形成,周边肌动蛋白丝带模糊;球状肌动蛋白松散,胞浆中可见散在分布的絮状球状肌动蛋白,且这些反应随着刺激时间延长而增强。烧伤 Y- 27632组或Y -27632 烧伤组细胞生长、贴壁良好,丝状肌动蛋白的分布与空白对照组、实验对     

重视烧伤后肠道紧密连接屏障功能障碍的研究

Severe burn injury is often accompanied by intestinal epithelial tight junction barrier dysfunction, which is believed to be closely associated with postburn shock, inflammation, hypermetabolism, infection, organ dysfunction etc. Recent studies have documented the critical role of tight junction-associated protein regulation in intestinal epithelial barrier dysfunction induced by severe burn injury. Myosin light chain (MLC) phosphorylation regulated by both myosin light chain kinase, which can phosphorylate MLC directly, and Rho-associated kinase,which can inhibit MLC phosphatase and then induce MLC phosphorylation indirectly, play a critical role in intestinal epithelial tight junction barrier dysfunction which occurs in severe burn injury. Recent advances have provided new insights into the mechanisms and the therapeutic strategies of intestinal epithelial tight junction barrier dysfunction following severe burn injury.     

Alteration in cellular morphology, density and distribution in rat vocal fold mucosa following injury

The vocal fold mucosa plays an important role in voice production. Its cellular composition and density frequently change under various pathological conditions, often contributing to altered extracellular matrix production, tissue viscoelasticity, and voice quality. In this study, cellular changes in the rat mucosa following a unilateral stripping injury were investigated and analyzed semi-quantitatively. Distinctive and sequential changes in cellular morphology, composition, and density were observed in the mucosa post-injury. Cellular recruitment was a major event during the early stage of injury and reached its peak level by day 5 post-injury. Several types of cells, including neutrophil-like cells, epithelial cells, and fibroblast-like cells, were sequentially recruited. The sequential emergence of reactive cell populations following injury and subsequent reconstruction of the mucosa suggests their involvement in vocal fold tissue repair and scar formation processes.     

Induction of heat shock protein 70 by sodium arsenite attenuates burn-induced intestinal injury in severe burned rats

The present study was designed to assess the effects of induced heat shock protein 70 (HSP70) on intestinal injury after severe burn. Wistar rats were randomly divided into four groups: control group, burn group (B group), sodium arsenite pretreatment group (SA group), and sodium arsenite+quercetin pretreatment group (SA+Qu group). Plasma endotoxin and d-lactic acid content were determined at 3, 6, 12, 24, and 48h after severe burn. Samples of small intestine were obtained for histologic assessment of intestinal mucosal injury and the expression of HSP70 was assayed by Western blot. Apoptosis of the intestinal epithelial cells was examined by the TUNEL method. Results showed that SA pretreatment significantly increased expression of HSP70 in the small intestine. SA pretreatment attenuated the burn-induced increase in plasma endotoxin and d-lactic acid content, intestinal injury scores and the percentage of apoptotic intestinal epithelial cells. Co-administration of quercetin with SA abolished the SA-induced HSP70 over-expression and the beneficial effects of SA. Our findings suggest increasing expression of HSP70 induced by SA pretreatment attenuates burn-induced intestinal injury apparently by preventing apoptosis.     

c-jun反义基因重组体转染对缺氧复合烧伤血清刺激下大鼠心肌细胞的保护作用

目的 观察c-jun反义基因重组体转染对缺氧复合烧伤血清刺激下大鼠心肌细胞的保护作用。方法 培养大鼠心肌细胞，分为：(1)正常对照组；(2)转染组：构建c-jun反义基因重组体并转染人心肌细胞，随后进行缺氧复合烧伤血清刺激；(3)非转染组：仅进行缺氧复合烧伤血清刺激，不作其他处理。于刺激后l、3、7h，采用逆转录聚合酶链式反应(RT-PCR)检测c-jun mRNA的表达变化；用Western blot检测c-jun蛋白、肌钙蛋白T(TnT)和β-微管蛋白的表达变化；在光镜和电镜下观察心肌细胞形态结构改变。结果 (1)与正常对照组比较，非转染组c-jun mRNA与蛋白表达显，转染组较非转染组显下降。(2)与正常对照组比较，非转染组TnT与β-微管蛋白表达显下降，转染组较非转染组明显回升。(3)转染组心肌细胞骨架结构基本保持完好，偶见断裂与溶解。非转染组骨架网状结构紊乱、溶解与断裂，呈颗粒状。结论 缺氧复合烧伤血清刺激可使大鼠心肌细胞c-jun表达上调，进而引起心肌细胞损伤，c-jun反义基因重组体转染对该刺激条件下的心肌细胞具有保护作用。     

The effect of hepatocyte growth factor on gut mucosal apoptosis and proliferation, and cellular mediators after severe trauma

BACKGROUND: A severe burn injury is associated with an impairment of gut mucosal integrity and function, which is due to increases in small-bowel epithelial cell apoptosis and decreases in cell proliferation. Hepatocyte growth factor (HGF) was shown to improve regeneration in the liver, mesentery, and skin. The purpose of this study was to determine whether HGF can improve small-bowel homeostasis after injury and the cellular mechanisms by which these changes occur. METHODS: Rats were pair-fed, underwent thermal trauma, and received saline (0.9% NaCl; n = 28) or HGF (200 microg/kg iv every 12 hours, n = 28). Small intestine and serum were taken at 1, 2, 5, and 7 days after injury. Measures were mucosal apoptosis, proliferation, villous morphology, and apoptotic and proliferative mediators, such as caspase-3 and caspase-7, Fas and Fas-ligand, Bcl-2, and Bax. In addition, serum cytokines were determined. RESULTS: Gut epithelial cell apoptosis was increased in the saline and HGF groups after the thermal injury. Despite an increase in serum tumor necrosis factor-alpha and interleukin-1beta, HGF did not affect small-bowel cell apoptosis, but it improved proliferation at days 1 and 2 after injury, which was associated with increased villous height and cell per villous, compared with saline controls, P < .05. Increased mucosal cell proliferation was associated with increased Bcl-2 in the HGF group, P < .05. HGF had no effect on apoptotic mediators, such as Fas, Fas-L, or caspase-3 and caspase-7. CONCLUSIONS: HGF improves small-bowel morphology after a severe burn by increasing mucosal Bcl-2 and, concomitantly, small-bowel epithelial cell proliferation.     

Inhibition of p38 MAP kinase improves survival of cardiac myocytes with hypoxia and burn serum challenge

This study was aimed to investigate the effects of SB203580, the specific p38 mitogen-activated protein (MAP) kinase inhibitor, on cardiac myocyte survival and secretion of cytokines in an in vitro model of hypoxia and burn serum challenge. Results demonstrated that hypoxia and burn serum induced a persistent activation of p38 MAP kinase in primary cultured neonatal rat cardiomyocytes during the 12h period of stimulation, concomitant with a time-dependent increased expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide (iNOS), a progressively developed oxidative stress reflected by malondialdehyde (MDA) production, and myocytes injury evidenced by the increased levels of released lactate dehydrogenase (LDH) and the decreased myocyte viability. Furthermore, hypoxia and burn serum resulted in a significant increase in myocyte apoptosis, which may account for the impairment of myocyte viability as observed. SB203580 abolished p38 MAP kinase activation, blunted the upregulation of TNF-alpha, iNOS and the subsequent nitric oxide (NO) production, reduced oxidative stress, and alleviated hypoxia and burn serum-induced myocytes injury or apoptosis. These results demonstrated for the first time that inhibition of p38 MAP kinase improves survival of cardiac myocytes with hypoxia and burn serum challenge possibly via reducing the production of cytokines, such as TNF-alpha and NO, and the subsequent oxidative stress, providing strong evidence that the excessive inflammatory cytokines produced by cardiomyocytes themselves may be sufficient to cause myocardial injury after burn.     

烧伤大鼠小肠肌间神经丛中一氧化氮合酶的组织化学研究

目的　探讨烧伤后大鼠小肠壁内一氧化氮合酶 (NOS)的活性及分布的变化规律。　方法　采用NADPH -黄递酶 (NDP)组织化学法和整装铺片技术对烧伤大鼠小肠壁内NOS的活性及分布进行定量和定位研究。　结果　NOS广泛分布于大鼠小肠壁内 ,主要定位于肌间神经丛。NOS阳性神经元多为圆形和卵圆形 ,神经纤维中多含有膨体 ,形成“串珠”样结构 ,且多与血管和肌纤维伴行。烧伤后肌间神经丛中NOS阳性神经元密度变化不显著 (P >0 .0 5 ) ,但神经元内NOS活性则显著下降(P <0 .0 5～ 0 .0 1) ,同时发现烧伤后NOS阳性神经元结构模糊 ,神经纤维断裂较多 ,未能形成完整的神经纤维网 ,神经纤维中NOS阳性膨体亦明显减少。结论　烧伤后大鼠小肠肌间神经丛中NOS阳性神经结构受损 ,神经元内NOS活性下降 ,同时一氧化氮 (NO)的释放途径亦存在障碍 ,这些变化可能与烧伤后肠道结构受损 ,功能障碍密切相关     

烧伤血清导致大鼠离体心室肌细胞钙超载的研究

目的观察烫伤血清对分离的成年大鼠心室肌细胞游离钙浓度(［Ca