去甲肾上腺素/烧伤血清诱导大鼠神经胶质细胞VEGF基因表达

目的： 观察血管内皮细胞生长因子（VEGF）在去甲肾上腺素（NE）/烧伤血清诱导神经胶质细胞时基因表达变化。 方法： 用免疫荧光法观察NE/烧伤血清刺激神经胶质细胞后24 h VEGF在细胞内的分布。用免疫蛋白印迹法检测细胞VEGF蛋白表达。用荧光定量聚合酶联反应（PCR）法检测细胞VEGF mRNA表达。 结果： ①高剂量NE（50 μmol/L）刺激时，神经胶质细胞胞浆内绿色荧光增强，烧伤血清刺激后绿色荧光明显增强，当烧伤血清加高剂量NE刺激时绿色荧光显著增强。②烧伤血清组细胞VEGF蛋白表达增加，烧伤血清加中剂量（20 μmol/L）、高剂量NE组VEGF的蛋白表达明显增加。③烧伤血清刺激后，细胞VEGF mRNA表达增加，烧伤血清与NE同时刺激时，随着NE剂量的增加，细胞中VEGF mRNA表达明显增加。 结论： NE/烧伤血清能够诱导神经胶质细胞VEGF的基因表达增强，提示烧伤应激因素诱导神经胶质细胞分泌VEGF可能是烧伤后脑水肿形成的一个重要因素。     

去甲肾上腺素诱导大鼠神经胶质细胞cAMP生成与VEGF mRNA变化的相关性

目的： 探讨去甲肾上腺素(NE)诱导大鼠神经胶质细胞环磷酸腺苷（cAMP）变化及与血管内皮细胞生长因子（VEGF）mRNA的关系。方法: 用放射免疫法检测细胞cAMP的变化。用实时荧光定量PCR法，检测细胞VEGFmRNA表达，并分析VEGF mRNA和细胞内cAMP含量的相关性。 结果: ①随着20 μmol/L NE刺激细胞时间逐渐延长，细胞中cAMP含量逐渐增加，有明显的时间依赖性关系（P<0.01）。②不同浓度(10-50 μmol/L)的NE刺激细胞2 h后，随着NE浓度的逐渐增高，细胞中cAMP含量逐渐增加，有明显的剂量依赖性关系，与对照组相比，有显著差异（P<0.01），VEGFmRNA与cAMP的含量随NE浓度变化2者呈显著正相关（r=0.94，P<0.01）。结论: NE能够诱导神经胶质细胞分泌cAMP，与NE诱导细胞VEGFmRNA变化有相关性。     

重度烧伤大鼠血清对巨噬细胞Notch1的激活及分泌功能的影响

目的研究重度烧伤大鼠血清刺激后巨噬细胞Notch1蛋白表达变化,及其细胞分泌因子白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的水平变化。方法选取成年雄性SD大鼠24只,随机分为假伤组、烧伤24 h组和烧伤7 d组,每组8只。烧伤组SD大鼠造成约30%总体表面积(TBSA)Ⅲ度烧伤,分别于伤后24 h、7 d收集血清;假伤组大鼠用37℃水浴,水浴后24 h取其血清作为对照。用上述各组血清制成20%培养液,刺激小鼠源巨噬细胞系RAW264.7,分别于加入血清刺激后即刻和刺激4、8、12、24、48 h后收取细胞及其上清液。另外用含20%假伤血清+脂多糖(100 ng/m L)培养液刺激巨噬细胞,于相同时间点收样。Western Blot检测各时间点巨噬细胞中Notch1蛋白表达变化,酶联免疫吸咐试验检测刺激24 h后培养上清液中IL-6及TNF-α的含量变化。结果 (1)假伤组血清刺激后即刻和刺激4、8、12、24、48 h后,Notch1蛋白表达无明显变化;(2)烧伤24 h组血清刺激后,Notch1蛋白表达明显升高,并随时间延长而达高峰;(3)烧伤7 d组血清刺激后,Notch1蛋白表达无明显变化,与假伤组血清刺激结果类似;(4)假伤组血清+脂多糖混合刺激后,Notch1蛋白表达明显增加;(5)烧伤24 h组血清及假伤组血清+脂多糖,上清液中IL-6和TNF-α含量均明显高于假伤组血清及烧伤7 d组血清。结论烧伤血清刺激下的巨噬细胞Notch1信号被激活,IL-6及TNF-α分泌能力增强,且这种激活可被脂多糖刺激所模拟。     

电烧伤大鼠创面组织中VEGF及血清ET-1表达水平的变化

目的 探讨电烧伤大鼠血管内皮生长因子(VEGF)和内皮素1(ET-1)的动态变化.方法 将大鼠64只随机分成2组:电烧伤组(56只)和对照组(8只).采用免疫组织化学方法测定伤后1d,3d,7d和14d的创周组织中VEGF的表达.采用ELISA方法测定烧伤后2h,6h,12h,24h血清ET-1的浓度.结果 伤后各个时间点创周组织VEGF蛋白表达均高于正常,VEGF表达在伤后第7天达到峰值.血清ET-1浓度显著升高,2h即明显高于正常值,6h达到峰值,此后逐渐下降,伤后24h仍明显高于正常.结论 电烧伤后VEGF和ET-1的表达均显著升高,这二种变化可能参与烧伤后微血管通透性的调节.     

电针结合经颅脉冲电刺激通过miRNA-150调控VEGF促进脑卒中大鼠血管新生

目的 探讨电针结合经颅脉冲电刺激促进脑卒中大鼠血管新生的作用机制。方法 60只SD大鼠被随机分为假手术组(sham)、模型组(model)、电针组(EA)、经颅脉冲电刺激组(tPCS)、电针结合经颅脉冲电刺激组(EA+tPCS)及EA+tPCS+miRNA-150 inhibitor组(miRNA-150 inhibitor),每组10只。采用线栓法制备慢性脑卒中模型。对各组大鼠神经行为学进行评分；HE染色观察大鼠脑皮质区病理损伤；免疫荧光染色测定大鼠脑组织微血管密度；qRT-PCR检测脑组织中血管内皮生长因子(VEGF)、血管生成素-2(Ang-2)mRNA及miRNA-150表达水平；Western blot检测VEGF蛋白表达。结果 与模型组相比，EA、tPCS及EA+tPCS组大鼠神经行为学评分显著降低，大鼠脑皮质病理损伤明显减轻，脑组织微血管密度显著增加，VEGF和Ang-2 mRNA表达水平显著升高，miRNA-150表达水平显著升高，且VEGF蛋白表达水平亦显著增加，上述结果尤以EA+tPCS组的效果最为显著。然而，当大鼠注射miRNA-150 inhibitor后，EA...     

腺苷A3受体(A3AR)在失血性休克血管反应性调节中的作用

目的： 阻力血管对血管活性物质反应性的降低是决定创伤休克发生、发展及预后的重要因素。腺苷是机体遭受创伤、缺氧时大量释放的重要内源性调质,并通过相应受体发挥作用。本研究拟观察腺苷A3受体(A3AR)在失血性休克大鼠肠系膜上动脉的表达变化情况及其与休克血管反应性变化间的关系,初步探讨A3AR是否参与对休克血管反应性的调节。方法：参照以往的工作基础,建立大鼠失血性休克(40 mmHg)模型;大鼠肠系膜上动脉对缩血管物质去甲肾上腺素(NE)诱导的收缩反应性采用离体小血管张力测定仪检测;A3AR的蛋白及mRNA表达变化情况分别采用Western blotting及RT-PCR进行检测。结果：结果显示,在大鼠失血性休克后0-4 h,其肠系膜上动脉1-2级分支血管对由NE诱导的收缩反应性呈现“双向性”变化;A3AR mRNA表达随休克时间的延长呈逐渐降低的趋势,但无显著差异;而肠系膜上动脉血管平滑肌A3AR的蛋白表达在休克后即刻呈增加趋势,随着休克时间的延长,其表达逐渐下降,尤其以休克后4h的表达下降最为明显;此外,A3AR激动剂可部分恢复休克2 h大鼠肠系膜上动脉对NE的收缩反应性,且该作用可被A3AR阻断剂MRS1523所拮抗。结论：A3AR参与失血性休克血管低反应性的形成,在失血性休克后激动A3AR受体可保护血管功能,部分恢复失血性休克后大鼠阻力血管对NE的收缩反应性。     

亚低温对脑出血大鼠血肿周围神经营养因子和血管新生的影响

目的:研究亚低温治疗对脑出血大鼠血肿周围脑组织中胶质细胞源性神经营养因子(GDNF)、血管内皮生长因子(VEGF)的影响。方法:雄性SD大鼠随机分为3组:假手术组(Sham组)、脑出血组(ICH组)和亚低温组。采用立体定向注射自体血复制脑出血大鼠模型,造模后15 min给予持续4 h的亚低温治疗。应用修正神经功能缺损评分(mNSS)对各组大鼠进行神经行为学评分;干湿重法检测各组大鼠脑组织含水量;Real-time PCR检测各组大鼠血肿周围脑组织内G-DNF、VEGF的mRNA表达水平;免疫印迹检测各组大鼠血肿周围脑组织内GDNF、VEGF的蛋白表达含量。结果:与假手术组相应时问点相比,脑出血组大鼠神经功能评分明显升高,脑组织含水量显著增多,血肿周围脑组织GDNF、VEGF的mRNA和蛋白表达明显上调;与脑出血组对应时间点相比,亚低温组神经功能评分明显降低,脑组织含水量显著减少,GDNF、VEGF的mRNA和蛋白表达显著上调。结论:亚低温治疗可增加脑出血大鼠脑内神经营养因子和血管生长因子的表达,促进神经修复和新生血管的形成,改善神经功能缺损和脑水肿。     

TNF-α对小鼠脑微血管内皮细胞RhoA活性的影响

目的: 观察肿瘤坏死因子-α(TNF-α)对小鼠脑微血管内皮细胞RhoA活性和表达的影响,探讨p115RhoGEF是否参与其调控。方法: 以小鼠脑微血管内皮细胞株bEnd.3为实验对象,TNF-α(10 μg/L)刺激后pull-down检测RhoA活性,Western blotting检测RhoA总蛋白表达;再将bEnd.3分为siRNA组(转染p115RhoGEF siRNA)和对照组(转染无活性的nsRNA),Western blotting检测p115RhoGEF蛋白的表达,pull-down检测TNF-α预处理后RhoA活性的变化。结果: RhoA活性在TNF-α刺激30 min后明显增高达到高峰,RhoA总蛋白表达在TNF-α刺激12 h和24 h显著上调;TNF-α刺激siRNA组bEnd.3细胞30 min后,RhoA活性较对照组明显降低。结论: TNF-α可导致小鼠脑微血管内皮细胞RhoA活性增加和表达上调,p115RhoGEF可能参与了TNF-α诱导RhoA活化的调控过程。     

热应激时大鼠肺中肾上腺素能受体的变化及其调节的研究

研究了大鼠经急性热应激致肛温达42℃及热应激后在室温下恢复4h时肺肾上腺素能受体(α,β)的改变变相应配基去甲肾上腺素(NE),肾上腺素(E)含量的改变。同时测定了肺组织中磷酯酶A_2(PLA_2)的活性改     

miR-30a抑制Ang2诱导的大鼠心肌细胞系H9c2自噬

目的 探索miR-30a对血管紧张素2(Ang2)诱导的大鼠心肌细胞自噬的影响.方法 1)将H9c2细胞分为对照组(control)、Ang2处理组(Ang22×10-6 mol/L)、去甲肾上腺素阳性对照组(NE 5×10-6 mol/L).处理细胞48 h,RT-qPCR检测miR-30a表达;Western bl...     

核因子-κB对大鼠烧伤早期中性粒细胞在肺组织聚集中的作用

目的： 探讨大鼠烧伤早期肺组织核因子-κB（ NF-κB）活化对中性粒细胞(PMN)在肺组织中聚集和发生损害作用的影响。方法： 用Wistar大鼠 Ⅲ°35%TBSA烧伤模型。实验分正常大鼠对照组、烧伤组、烧伤后吡咯烷二硫代氨基甲酸盐（ PDTC）干预组。凝胶电泳迁移率分析法检测肺组织NF-κB活性；逆转录-聚合酶链式反应（RT-PCR）检测白细胞介素8（IL-8）和 细胞间黏附分子-1（ICAM-1） mRNA的表达；并测定肺组织髓过氧化物酶（MPO）活性和肺微血管von Willebrand因子（vWF)含量。结果： 大鼠烧伤后肺组织NF-κB活性在伤后1 h内即迅速增高，并持续增高到伤后24 h。伤后肺组织ICAM-1 和 IL-8 mRNA表达、MPO活性均明显高于、肺微血管vWF含量低于对照组。 PDTC处理显著缓解上述变化。结论： 严重烧伤后肺组织NF-κB活化，从而启动细胞黏附因子和趋化因子的合成和释放，导致PMN在肺组织中聚集，引起肺血管组织细胞损伤。     

Growth factors in porcine full and partial thickness burn repair. Differing targets and effects of keratinocyte growth factor, platelet-derived growth factor-BB, epidermal growth factor, and neu differentiation factor.

The topical application of recombinant growth factors such as epidermal growth factor, platelet-derived growth factor-BB homodimer (rPDGF-BB), keratinocyte growth factor (rKGF), and neu differentiation factor has resulted in significant acceleration of healing in several animal models of wound repair. In this study, we established highly reproducible and quantifiable full and deep partial thickness porcine burn models in which burns were escharectomized 4 or 5 days postburn and covered with an occlusive dressing to replicate the standard treatment in human burn patients. We then applied these growth factors to assess their efficacy on several parameters of wound repair: extracellular matrix and granulation tissue production, percent reepithelialization, and new epithelial area. In full thickness burns, only rPDGF-BB and the combination of rPDGF-BB and rKGF induced significant changes in burn repair. rPDGF-BB induced marked extracellular matrix and granulation tissue production (P = 0.013) such that the burn defect was filled within several days of escharectomy, but had no effect on new epithelial area or reepithelialization. The combination of rPDGF-BB and rKGF in full thickness burns resulted in a highly significant increase in extracellular matrix and granulation tissue area (P = 0.0009) and a significant increase in new epithelial area (P = 0.007), but had no effect on reepithelialization. In deep partial thickness burns, rKGF induced the most consistent changes. Daily application of rKGF induced a highly significant increase in new epithelial area (P < 0.0001) but induced only a modest increase in reepithelialization (83.7% rKGF-treated versus 70.2% control; P = 0.016) 12 days postburn. rKGF also doubled the number of fully reepithelialized burns (P = 0.02) at 13 days postburn, at least partially because of marked stimulation of both epidermal and follicular proliferation as assessed by proliferating cell nuclear antigen expression. In situ hybridization for KGFR in porcine burns revealed strong expression of KGFR on hair follicles and basal epidermis, confirming direct rKGF action on follicular as well as epidermal keratinocytes. Although the epithelial proliferation induced by rKGF resulted in marked neoepidermal psoriasiform hyperplasia with exaggerated rete ridges and neoepidermal and follicular maturation as assessed by expression of cytokeratin 10, a marker of keratinocyte terminal differentiation was not delayed and appeared to be accelerated in some rKGF-treated burns. Recombinant epidermal growth factor induced a trend toward increased new epithelial area in deep partial thickness burns, but had no effect on reepithelialization. The recombinant neu differentiation factor-alpha 2 isoform had no significant biological effects in either full or deep partial thickness burns.(ABSTRACT TRUNCATED AT 250 WORDS)     

大鼠烧伤早期心肌营养性血流的变化与心肌损伤的关系

目的：探讨心肌血液灌流变化在烧伤后心肌损伤中的作用。方法：大鼠随机分为对照组（ｎ＝１０，不致伤）和烧伤组（ｎ＝５０，致３０％ＴＢＳＡⅢ度烧伤）。＾８６Ｒｂ示踪摄取法测定大鼠烧伤前（对照组）和烧伤后１、３、６、１２、２４ｈ心肌营养性血流（ＮＢＦ）变化，同时监测血清心肌肌球蛋白轻链１ＣＭＬＣ１含量变化。结果：大鼠烧伤后心肌ＮＢＦ迅速大幅度下降，心肌损伤特异性标志物ＣＭＬＣ１明显升高。心肌ＮＢＦ与ＣＭＬ     

一氧化氮合酶激活参与烧伤后Ca~(2 )介导的心肌线粒体损伤

目的 :探讨线粒体一氧化氮合酶 (mtNOS)在严重烧伤早期心肌线粒体损害中的作用。方法 :复制30 %TBSAⅢ°烧伤大鼠模型 ,取正常及伤后 1、3、6、12、2 4h大鼠心肌分离线粒体 ,测其呼吸功能、Ca2 浓度 ([Ca2 ]m)及细胞色素c氧化酶、mtNOS活性。结果 :①伤后 1h心肌线粒体呼吸控制率 (RCR)显著高于正常组 ,但 3、6、12、2 4h明显低于正常组 ,Ⅲ态呼吸速率 (ST3 )变化与RCR平行 ,ST4 仅于伤后 3h明显升高 ;②伤后各时点 [Ca2 ]m 均明显高于正常组 ,尤以 3、6h为甚 ,而mtNOS活性于伤后 3、6、12、2 4h显著高于对照组 ,细胞色素c氧化酶活性于伤后 3、6、12、2 4h显著低于正常组 ;③伤后mtNOS活性与 [Ca2 ]m 呈明显正相关 ,相关系数为 0 8945 (P <0 0 5 ) ,RCR与mtNOS活性呈显著负相关 ,相关系数为 - 0 934 7(P <0 0 5 )。结论 :伤后 [Ca2 ]m 升高激活mtNOS ,可能参与严重烧伤早期心肌线粒体损害。     

电烧伤大鼠血清诱导单核细胞分泌VEGF对单核-内皮细胞黏附作用的影响

目的:观察电烧伤大鼠血清诱导的单核细胞分泌血管内皮生长因子(VEGF)的水平,探讨其对单核细胞与血管内皮细胞黏附作用的影响。方法:制做电烧伤大鼠模型,制备电烧伤大鼠血清,同时制备正常大鼠血清作为对照。采用夹心ELISA法检测正常大鼠血清和电烧伤大鼠血清中VEGF及其可溶性受体s Flt-1含量。按照随机数字表法将人单核细胞株THP-1细胞分为正常血清组和电烧伤血清组,ELISA法检测2组细胞上清液中VEGF和s Flt-1含量。按照随机数字表法将人单核细胞株THP-1细胞分为正常血清组、烧伤血清组、正常血清+阻断剂组和烧伤血清+阻断剂组。取培养3 h、6 h的THP-1细胞,加入单层血管内皮细胞株EA.hy926细胞,行单核-内皮细胞黏附检测。结果:大鼠电烧伤后血清VEGF水平较正常大鼠显著增加,s Flt-1水平较正常大鼠明显减少。电烧伤血清诱导THP-1细胞分泌VEGF,s Flt-1水平随之减少。电烧伤血清可促进单核细胞与内皮细胞的黏附作用,s Flt-1可抑制电烧伤血清诱导的单核细胞与内皮细胞的黏附作用。结论:电烧伤大鼠血清诱导单核细胞分泌VEGF,从而促进单核-内皮细胞黏附。阻断VEGF的生物学效应,可有效抑制单核-内皮细胞的黏附。     

Cardiovascular and metabolic responses during burn shock in the guinea pig.

The hemodynamic and metabolic responses of fatally burned, nonfatally burned, and unburned control guinea pigs were compared. The burns were induced in temporarily anesthetized animals by immersion to either the xyphoid process (70% fatal) or the midabdomen (100%survival) in boiling water for 3 s. Although cardiac output was reduced in all animals postburn, the survivors (MAG) has higher cardiac outputs at lower arterial pressures than the nonsurvivors (XPN). The postburn lactate levels in the XPN were higher than in the MAG, and the postburn values for pH, oxygen consumption, and core temperature were lower in the XPN. In each group, hyperglycemia was evident for 8 h postburn and terminal plasma glucose concentrations were usually elevated or similar to the prevalue. It was concluded that fatal and nonfatal burn shock were distinguished primarily by differences in tissue perfusion.     

Bumetanide protects focal cerebral ischemia-reperfusion injury in rat

Objective: Bumetanide has been reported to attenuate ischemia-evoked cerebral edema. However, whether bumetanide can protect cerebral ischemia-reperfusion injury (IRI) in vivo is unclear. In the present study, we aim to determine whether intravenously injection bumetanide can attenuate cerebral IRI and if its protection effect might be related to the modification of cerebral NKCC1 and KCC2 protein expression. Methods: Focal cerebral ischemia was induced by occluding the right middle cerebral artery (MCAO) for 2-h, followed by 3-h, 24-h or 48-h of reperfusion respectively. Brain edema, neurological deficits, and infarction volume were determined by (wet weights - dry weights)/dry weights ×100, 5-point neurological function score evaluation system, and TTC staining, respectively. The expression levels of NKCC1 and KCC2 were determined by immunohistochemical staining. Results: Reperfusion increased brain edema, neurological deficits, and infarction volume. Bumetanide decreased brain edema, attenuated the neurological defects and reduced post-ischemic cerebral infarction. Cerebral ischemia-reperfusion injury increased NKCC1 expression level and decreased KCC2 expression level. Interestingly, bumetanide down-regulated the NKCC1 protein expression level without changing the KCC2 protein expression level in rat brain cortex. Conclusion: These results suggest that bumetanide protects focal cerebral ischemia-reperfusion injury in rat, which might through the inhibition of NKCC1.