TAT-血红素氧合酶-1融合蛋白对原位肝移植术大鼠肝细胞凋亡的影响

目的 评价TAT-血红素氧合酶-1(TAT-HO-1)融合蛋白对原位肝移植术大鼠肝细胞凋亡的影响.方法 健康成年雄性SD大鼠,体重250 ～ 280 g,作为肝移植术的供体和受体.参照文献构建TAT-HO-1融合蛋白(1 mg/ml).采用二袖套法建立大鼠原位肝移植术模型.将受体大鼠随机分为2组(n=24):对照组(C组)和融合蛋白组(TAT-HO-1组).C组和TAT-HO-1组分别于肝移植术毕静脉注射生理盐水、TAT-HO-1融合蛋白10 ml/kg.于无肝期前即刻(T0)、肝脏移植术后1h、6h和12 h(T1～3)时,分别取动脉血样,检测血清谷丙转氨酶(ALT)活性及透明质酸(HA)浓度;取肝组织标本,采用免疫组化法检测HO-1表达水平,采用TUNEL法检测凋亡细胞,计算凋亡指数.结果 与C组比较,TAT-HO-1组T1-3时HO-21表达上调,血清ALT、HA水平及肝实质细胞和肝窦内皮细胞凋亡指数降低(P＜0.05).结论 大鼠原位肝移植术后静脉注射TAT-HO-1融合蛋白可转导进入移植肝脏细胞,通过减轻肝细胞和肝窦内皮细胞凋亡,对肝移植术后的肝脏功能发挥保护作用.     

热休克蛋白70和血红素加氧酶-1表达在肾缺血后处理减轻肾缺血再灌注损伤中的作用

目的 评价热休克蛋白70(HSP70)和血红素加氧酶-1(HO-1)表达在肾缺血后处理减轻肾缺血再灌注损伤中的作用.方法健康雄性SD大鼠140只,体重250～280 g,采用随机数字表法,将大鼠随机分为4组(n=35):假手术组(S组)仅开腹,游离双侧肾脏,分离双侧肾蒂不夹团;肾缺血再灌注组(I/R组)夹闭双侧肾蒂缺血45 min,恢复灌注;缺血后处理组(IPo组)夹闭双侧肾蒂45 min,再灌注10 s,缺血10 s,反复3次,恢复灌注;HSP抑制剂槲皮黄酮+缺血后处理组(Q+IPo组)缺血前1 h 腹腔注射槲皮黄酮100 mg/kg,余操作同IPo组.于再灌注即刻(T0)、1、3、6、12、24、48 h(T1～6)时各组随机取5只大鼠抽心脏血后取肾,检测肾组织HSP70、HO-1的mRNA和蛋白表达,T3时抽心脏血,测定血清肌酐(Cr)和尿素氮(BUN)浓度、caspase-3 mRNA的表达,TUNNEL法检测肾组织凋亡细胞,计算凋亡指数(AI),光镜下观察肾组织病理学结果.结果 与S组比较,其余组T3时血清Cr和BUN浓度和AJ升高,caspase-3 mRNA表达上调,各时点HSF70、BO-1的mRNA和蛋白表达上调(P＜0.05);与I/R组比较,IPo组T3时血清Cr和BUN浓度和AI降低,caspase-3 mRNA表达下调,T1～5时HSP70、HO-1的mRNA和蛋白表达上调(P＜0.05);与IPo组比较,Q+IPo组T3时血清Cr和BUN浓度和AJ升高,caspase-3mRNA表达上调,T1～5时HSP70、HO-1的mRNA和蛋白表达下调(P＜0.05).IPo组肾组织病理学损伤较I/R组减轻,Q+IPo组肾组织病理学损伤程度与I/R组相似.结论 HSP70和H0-1表达参与了肾缺血后处理减轻肾缺血再灌注损伤的过程.

Abstract：

Objective To evaluate the role of the expression of heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1) in the reduction of renal ischemia-reperfusion (I/R) injury by ischemic postconditioning in tats.Methods One hundred and forty healthy male SD rats weighing 250-280 g were randomized into 4 groups ( n = 35 each) : sham operation group (S group) ; I/R group; ischemic postconditioning group (IPo group); quercetin (an inhibitor of HSP) + ischemic postconditioning group (Q + IPo group). Renal I/R was produced by clamping bilateral renal pedicels for 45 min followed by reperfusion. In group S, bilateral kidneys were only exposed through a midline incision but their- pedicels were not clamped. In IPo and Q + IPo groups, 45 min ischemia was followed by three 10 s episodes of ischemia at 10 s intervals for reperfusion and in addition intraperitoneal quercetin 100 mg/kg was injected at 1 h before ischemia in group Q + IPo. Blood samples from hearts were obtained at 0, 1, 3, 6, 12, 24 and 48 h of reperfusion (T0-6) and the rats were then sacrificed and kidneys removed to detect the expression of HSP70 and HO-1 mRNA and protein in renal tissues. The blood samples obtained at T3 were used to determine serum creatinine (Cr) and urea nitrogen (BUN) concentrations and the expression of caspase-3 mRNA . The apoptosis in the renal tissues was detected using TUNEL and apoptotic index ( AI) was calculated. Microscopic examination was performed with light microscope. Results Compared with group S, the serum Cr and BUN concentrations and AI were significantly increased at T3,the expression of caspase-3 mRNA was up-regulated at T3, and the expression of HSP70 and HO-1 mRNA and protein was up-regulated at T0-6 in the other groups (P ＜ 0.05) . Compared with group I/R, the serum Cr and BUN concentrations and AI were significantly decreased at T3, the expression of caspase-3 mRNA was down-regulated at T3, and the expression of HSP70 and HO-1 mRNA and protein was up-regulated at T1-5 in group IPo ( P ＜ 0.05) . Compared with group IPo, the serum Cr and BUN concentrations and AI were significantly increased at T3, the expression of caspase-3 mRNA was up-regulated at T3, and the expression of HSP70 and HO-1 mRNA and protein was down-regulated at T1-5, in group Q + IPo ( P ＜ 0.05) . The microscopic examination showed that the renal I/R injury was significantly attenuated by ischemic postconditioning and the degree of injury in group IPo was similar to that in group I/R. Conclusion The expression of HSP70 and HO-1 is involved in the reduction of renal I/R injury by ischemic postconditioning in rats.     

妊娠合并法洛四联症患者清宫术麻醉管理1例

<正>1 病例资料患者女性,19岁,孕13周,因复杂先天性心脏病要求终止妊娠于2021年4月13日就诊于河北省人民医院。患者自出生时诊断为法洛四联症,未行手术治疗,自述平时行走时间长及爬楼梯出现喘息、呼吸困难,休息后好转。查体：神志清醒,口唇紫绀,指甲青紫;血压99/73 mm Hg, 心率88次/min, 呼吸22次/min, 血氧饱和度(SpO₂)76.60%(鼻管吸氧,氧流量2 L/min);心前区可闻及3/6级收缩期杂音。     

不同氧合器对体外循环心脏手术患儿中性粒细胞凋亡的影响

Objective To evaluate the effect of two kind oxygenators on polymorphonuclear neutrophil (PMN) apoptosis, and to give a method of inhibting the systemic inflammatory response syndrome(SIRS) to cardiopulmonary bypass(CPB). Methods Sixty VSD patients undergoing open heart surgery with CPB were randomly divided into two groups(n = 30): group B(bubble oxygenator), group M(membrane oxygenator). Blood samples were drawn from the central venous line before starting CPB, at the end of CPB, 4, 8 and 24 h after CPB. The PMN counts were performed by blood cell counter. PMN apoptosis and the expression of CD11b were evaluated by flow eytometry. The plasma concentration of elastase was determined by enzyme linked immunosorbent assays. Results The rates of PMN apoptosis were significantly reduced (P<0.05) and the PMN counts, the expression of CD11b and the plasma con-centration of elastase were remarkably increased after CPB(P<0.05), but at the end of CPB, 4 h and 8 h after CPB the rate of PMN apoptosis was higher in group M than that in group B. PMN counts, the expression of CDI lb and the plasma concentration of elastase were higher in group B than in group M (P<0.05). The PMN counts were higher in group B than in group M 24 h after CPB (P< 0.05). Conclusion XiJian-Ⅱ membrane oxygenator can increase PMN apoptosis and depress systemic inflammatory response better than XIJing-90 bubble oxygenator.     

不同氧合器对体外循环心脏手术患儿中性粒细胞凋亡的影响

目的 研究小儿体外循环(cardiopulmonary bypass,CPB)中不同氧合器对中性粒细胞(PMN)凋亡的影响,为减轻体外循环伞身炎症反应提供新的思路.方法 将60例室间隔缺损患儿随机分为两组(n=30):西京-90鼓泡式氧合器组(B组)和希健-Ⅱ膜式氧合器组(M组).分别于CPB前、CPB结束时、CPB结束后4、8、24 h 5个时点采取静脉血,以伞血细胞计数仪测定PMN数量,流式细胞仪测定PMN凋亡率和PMN表面黏附分子CD11b表达变化,ELISA法测定血浆弹性蛋白酶浓度.结果 两组患者CPB结束后PMN凋亡率明显降低(P<0.05),PMN数量、CD11b表达、血浆弹性蛋白酶浓度明显升高(P<0.05),在CPB结束时及CPB结束后4、8 h PMN凋亡率M组均高于B组(P<0.05);而PMN数量、CD11b表达、血浆弹性蛋白酶水平B组均高于M组(P<0.05).CPB结束后24 h PMN数量B组高于M组(P<0.05).结论 与西京-90鼓泡氧合器相比,应用希健-Ⅱ膜式氧合器可以减轻CPB对PMN凋亡的抑制,进而减轻全身炎症反应.     

不同氧合器对体外循环心脏手术患儿中性粒细胞凋亡的影响

Objective To evaluate the effect of two kind oxygenators on polymorphonuclear neutrophil (PMN) apoptosis, and to give a method of inhibting the systemic inflammatory response syndrome(SIRS) to cardiopulmonary bypass(CPB). Methods Sixty VSD patients undergoing open heart surgery with CPB were randomly divided into two groups(n = 30): group B(bubble oxygenator), group M(membrane oxygenator). Blood samples were drawn from the central venous line before starting CPB, at the end of CPB, 4, 8 and 24 h after CPB. The PMN counts were performed by blood cell counter. PMN apoptosis and the expression of CD11b were evaluated by flow eytometry. The plasma concentration of elastase was determined by enzyme linked immunosorbent assays. Results The rates of PMN apoptosis were significantly reduced (P<0.05) and the PMN counts, the expression of CD11b and the plasma con-centration of elastase were remarkably increased after CPB(P<0.05), but at the end of CPB, 4 h and 8 h after CPB the rate of PMN apoptosis was higher in group M than that in group B. PMN counts, the expression of CDI lb and the plasma concentration of elastase were higher in group B than in group M (P<0.05). The PMN counts were higher in group B than in group M 24 h after CPB (P< 0.05). Conclusion XiJian-Ⅱ membrane oxygenator can increase PMN apoptosis and depress systemic inflammatory response better than XIJing-90 bubble oxygenator.     

体外循环对小儿血浆细胞因子水平及中性粒细胞凋亡的影响

目的通过检测小儿体外循环(CPB)引起的致炎细胞因子肿瘤坏死因子-α(TNF-α)、白介素-8(IL-8)和抗炎细胞因子白介素-10(IL-10)的水平及中性粒细胞(PMN)凋亡率的动态变化,探讨CPB前后PMN凋亡及致炎和抗炎细胞因子水平的变化与规律。方法选择于CPB时行室间隔缺损修补术的患儿30例,分别于麻醉诱导前(T0)、麻醉诱导后(T1)、复温前(T2)、停CPB即刻(T3)、术后24h(T4)及48h(T5)6个时间点取外周血,ELISA法检测血浆TNF-α、IL-8和IL-10水平,流式细胞仪测定PMN凋亡率,进行比较分析。结果血浆TNF-α水平、IL-8水平及PMN凋亡率在T2与T0间差异有统计学意义(P<0.05);T3与T0间差异亦有统计学意义(P<0.01);T4、T5与T0间差异均无统计学意义(P>0.05)。血浆IL-10水平在T2、T3、T4与T0间差异均有统计学意义(P<0.01);T5与T0间差异无统计学意义(P>0.05)。CPB结束时,血浆TNF-α水平与PMN凋亡率呈负相关(r=-0.578,P<0.05);血浆IL-8水平与PMN凋亡率呈负相关(r=-0.455,P<0.05);血浆IL-10水平与PMN凋亡率呈正相关(r=0.374,P<0.05)。结论CPB引起致炎细胞因子TNF-α、IL-8水平升高,抗炎细胞因子IL-10水平升高,PMN凋亡率下降;致炎细胞因子TNF-α、IL-8可能抑制PMN的凋亡;抗炎细胞因子IL-10可能促进PMN的凋亡。     

山莨菪碱对过度训练大鼠心肌细胞凋亡及炎性反应的影响

目的 评价山莨菪碱对过度训练大鼠心肌细胞凋亡及炎性反应的影响.方法 雄性Wistar大鼠24只,体重200～220 g,按随机数字表法分为3组(n=8):对照组(C组)、过度训练组(E组)和山莨菪碱组(A组).采用游泳至力竭法建立过度训练致大鼠心肌损伤模型.A组于过度训练前20 min腹腔注射山莨菪碱10 m∥ks.于过度训练后6 h时采集下腔静脉血后处死,取心肌组织,应用全自动生化仪检测血清CK-MB活性,免疫组化法检测心肌组织TNF-α含量及NF-κB活性,流式细胞仪检测心肌细胞凋亡率.结果 与C组比较,E组和A组过度训练后6 h时血清CK-MB活性、心肌细胞凋亡率、心肌组织TNF-α含量及NF-κB活性升高(P＜0.05);与E组比较,A组过度训练后6 h时血清CK-MB活性、心肌细胞凋亡率、心肌组织TNF-α含量及NF-κB活性降低(P＜0.05).结论 山莨菪碱可抑制过度训练大鼠心肌细胞凋亡,其机制与减轻心肌组织炎性反应有关.

Abstract：

Objective To evaluate the effects of anisodamine on apoptosis in cardiomyocytes and inflammatory response in overtrained rats. Methods Twenty-four male Wistar rats, weighing 200-220 g, were randomly divided into 3 groups ( n = 8 each) : control group (group C) , overtraining group (group O) , anisodamine group (group A) . The model of overtraining-induced acute heart injury was established by exhausting swimming. Anisodamine 10 mg/kg was given intraperitoneally 20 min before overtraining in group A. Blood samples were taken at 6 h after overtraining for measurement of serum CK-MB activity. The rats were then sacrificed and myocardial tissues taken for determination of TNF-α content and NF-κB activity (by immunohistochemistry) . The apoptosis rate was detected by flow cytometry. Results The CK-MB activity, apoptosis rate, TNF-α content and NF-κB activity were significantly higher at 6 h after overtraining in groups O and A than in group C, while lower at 6 h after overtraining in group A than in group O ( P < 0.05) . Conclusion Anisodamine can inhibit apoptosis in cardiomyocytes by reducing inflammatory response in overtrained rats.     

TAT蛋白转导结构域的研究进展

目前,应用基因重组技术人工合成治疗性蛋白质分子已经成为可能.但如何有效地将合成的蛋白质转运进入目标组织和细胞仍是当前需要解决的重大难题.蛋白转导结构域HIV-TAT的发现为体内外转运蛋白质进入细胞提供了新思路.近年来蛋白转导结构域转运蛋白质进入细胞的有效性以及其在疾病治疗中的价值已经得到不断证实.现主要对蛋白转导结构域HIV-TAT的研究进展作一综述.     

HIV-1反式激活蛋白-血红素氧合酶-1融合蛋白对大鼠供肝冷保存期肝细胞凋亡的影响

Objective To study the effects of TAT-HO-1 fusion protein,HIV-1 transactiviting protein (TAT) and heme oxygenase-1 (HO-1),on hepatic cell apoptosis of rat donors in cold storage stage.Methods Forty-eight male SD rats were randomly divided into two groups.Rat livers were flushed and preserved with 4℃ HTK solution containing(group P) or uncontaining(group C) 50 mg/L of TAT-HO-1.The preserved solution and hepatic tissue were collected at 0,6,12,18 h of cold storage stage.TAT-HO-1 transducing into liver,alanine aminotransferase(ALT) level in preserved solution,hyaluronic acid(HA) level and the expression of caspase-3 in hepatic tissue,and the apoptotic index (AI) of hepatocytes and sinusoidal endothelial cells (SECs) were measured or detected.Results ALT level in preserved solution,HA level and the expression of caspase-3 in hepatic tissue,and the AI of hepatocytes and SECs increased time-dependently in cold storage stage in both groups (P＜0.05),with lower increasing extent in group P than that in group C (P＜0.05) at 6h,12h and 18h of cold storage stage.A stronger accumulation of HO-1 staining was also detected at the same time-points in group P than that in group C(P＜0.05).Conclusion TAT-HO-1 may transduce efficiently into rat livers,exerting protective effects on both hepatocytes and SECs during cold storage stage.Protein transduction technology may be a novel therapeutic means to reduce donor liver injury in preservation period for transplantion.