雷公藤甲素预处理诱导Tregs细胞减轻小鼠肝脏缺血再灌注损伤的实验研究

Objective To investigate the effect and related mechanism of triptolide pretreatment to prevent from ischemia/reperfusion (I/R) injury in mice liver. Methods Sixty male C57BL/6 mouse were randomized into four groups (15/group): A:sham group with saline , B: sham group with triptolide, C: saline I/R group, D: triptolide I/R group. The mice were pretreated with either saline or triptolide (0. 1 mg/kg/d) through intraperitoneal (ip) injection for one week. The mouse partial liver model of I/R injury was established, and samples were collected at 24 h after the I/R injury. Results Serum ALT and AST levels were significantly decreased and histological damage was significantly alleviated in the triptolide I/R group as compared with the saline I/R group (P＜0.05), the concentration of MDA in the triptolide groups was significantly decreased, while SOD activity was significantly increased compared with that of the saline I/R group (P＜0.05). The percentages of CD4+ CD25+ regulatory T cells (Tregs) cells among CD4+ T cells in groups A, B, C, and D were(7. 55 ± 1.87)%, (12. 59±3. 87)%,(7. 85±1.07)%, and(12. 02±3. 16)% in liver tissue, respectively. The expression levels of Foxp3 mRNA were significantly higher in the triptolide I/R group than those of saline I/R group (P＜0. 05). ELISA showed that triptolide could significantly inhibit the levels of IL-6, IL-Iβ and TNF-αand promoted the level of IL-10 in the serum (P＜0.05). Conclusion Pretreatment with triptolide could effectively prevent from liver I/R injury, which may be related to the induction of Treg cells by triptolide, the increase in the level of IL-10 in serum, and the inhibition of IL-6, IL-1β and TNF-α production in serum.     

高渗氯化钠羟乙基淀粉40注射液高容量血液稀释对大鼠肝脏缺血再灌注损伤的影响

Objective To investigate the effects of hypervolemic hemodilution (HH) with hypertonic saline plus hetastarch solution 40 injectio on hepatic ischemia-reperfusion (I/R) injury in rats. Methods Thirty male Wistar rats weighing 300-350 g were randomly divided into 3 groups ( n = 10 each): group I sham operation (group S); group II I/R and group Ⅲ HH. Partial liver ischemia was produced by clamping hepatic portal vein and left arteria hepatica for 30 min with atraumatic mini-clamp, followed by 2 h of reperfusion in I/R group and HH group. In HH group the animals were infused hypertonic saline plus hetastarch solution 40 injectio 10 ml/kg through vena caudalis over 30 min and then hepatic I/R was performed IS min after the infusion.The animals were killed at 2 h of reperfusion. The left liver was removed and blood sample was taken from inferior caval vein for determination of (1) serum alanine amino transferase (ALT) and aspartate amino transferase (AST) activities; (2) superoxide dismutase ( SOD) activity and malondialdehyde ( MDA) content in the liver; ( 3 ) microscopic examination. Results The serum ALT and AST activities and MDA content in the liver were significantly higher, SOD activity in the liver significantly lower after hepatic I/R and pathological changes in the liver severer in group I/R and HH than in group S. The serum ALT and AST activities and MDA content in the liver were significantly lower, SOD activity in the liver significantly higher after hepatic I/R and pathological changes in the liver milder in group HH than in group I/R. Conclusion Hypervolemic hemodilution with hypertonic saline plus hetastarch solution 40 injectio can ameliorate hepatic I/R injury by decreasing oxygen free radical production in rats.     

高渗氯化钠羟乙基淀粉40注射液高容量血液稀释对大鼠肝脏缺血再灌注损伤的影响

Objective To investigate the effects of hypervolemic hemodilution (HH) with hypertonic saline plus hetastarch solution 40 injectio on hepatic ischemia-reperfusion (I/R) injury in rats. Methods Thirty male Wistar rats weighing 300-350 g were randomly divided into 3 groups ( n = 10 each): group I sham operation (group S); group II I/R and group Ⅲ HH. Partial liver ischemia was produced by clamping hepatic portal vein and left arteria hepatica for 30 min with atraumatic mini-clamp, followed by 2 h of reperfusion in I/R group and HH group. In HH group the animals were infused hypertonic saline plus hetastarch solution 40 injectio 10 ml/kg through vena caudalis over 30 min and then hepatic I/R was performed IS min after the infusion.The animals were killed at 2 h of reperfusion. The left liver was removed and blood sample was taken from inferior caval vein for determination of (1) serum alanine amino transferase (ALT) and aspartate amino transferase (AST) activities; (2) superoxide dismutase ( SOD) activity and malondialdehyde ( MDA) content in the liver; ( 3 ) microscopic examination. Results The serum ALT and AST activities and MDA content in the liver were significantly higher, SOD activity in the liver significantly lower after hepatic I/R and pathological changes in the liver severer in group I/R and HH than in group S. The serum ALT and AST activities and MDA content in the liver were significantly lower, SOD activity in the liver significantly higher after hepatic I/R and pathological changes in the liver milder in group HH than in group I/R. Conclusion Hypervolemic hemodilution with hypertonic saline plus hetastarch solution 40 injectio can ameliorate hepatic I/R injury by decreasing oxygen free radical production in rats.     

高渗氯化钠羟乙基淀粉40注射液高容量血液稀释对大鼠肝脏缺血再灌注损伤的影响

Objective To investigate the effects of hypervolemic hemodilution (HH) with hypertonic saline plus hetastarch solution 40 injectio on hepatic ischemia-reperfusion (I/R) injury in rats. Methods Thirty male Wistar rats weighing 300-350 g were randomly divided into 3 groups ( n = 10 each): group I sham operation (group S); group II I/R and group Ⅲ HH. Partial liver ischemia was produced by clamping hepatic portal vein and left arteria hepatica for 30 min with atraumatic mini-clamp, followed by 2 h of reperfusion in I/R group and HH group. In HH group the animals were infused hypertonic saline plus hetastarch solution 40 injectio 10 ml/kg through vena caudalis over 30 min and then hepatic I/R was performed IS min after the infusion.The animals were killed at 2 h of reperfusion. The left liver was removed and blood sample was taken from inferior caval vein for determination of (1) serum alanine amino transferase (ALT) and aspartate amino transferase (AST) activities; (2) superoxide dismutase ( SOD) activity and malondialdehyde ( MDA) content in the liver; ( 3 ) microscopic examination. Results The serum ALT and AST activities and MDA content in the liver were significantly higher, SOD activity in the liver significantly lower after hepatic I/R and pathological changes in the liver severer in group I/R and HH than in group S. The serum ALT and AST activities and MDA content in the liver were significantly lower, SOD activity in the liver significantly higher after hepatic I/R and pathological changes in the liver milder in group HH than in group I/R. Conclusion Hypervolemic hemodilution with hypertonic saline plus hetastarch solution 40 injectio can ameliorate hepatic I/R injury by decreasing oxygen free radical production in rats.     

高渗氯化钠羟乙基淀粉40注射液高容量血液稀释对大鼠肝脏缺血再灌注损伤的影响

Objective To investigate the effects of hypervolemic hemodilution (HH) with hypertonic saline plus hetastarch solution 40 injectio on hepatic ischemia-reperfusion (I/R) injury in rats. Methods Thirty male Wistar rats weighing 300-350 g were randomly divided into 3 groups ( n = 10 each): group I sham operation (group S); group II I/R and group Ⅲ HH. Partial liver ischemia was produced by clamping hepatic portal vein and left arteria hepatica for 30 min with atraumatic mini-clamp, followed by 2 h of reperfusion in I/R group and HH group. In HH group the animals were infused hypertonic saline plus hetastarch solution 40 injectio 10 ml/kg through vena caudalis over 30 min and then hepatic I/R was performed IS min after the infusion.The animals were killed at 2 h of reperfusion. The left liver was removed and blood sample was taken from inferior caval vein for determination of (1) serum alanine amino transferase (ALT) and aspartate amino transferase (AST) activities; (2) superoxide dismutase ( SOD) activity and malondialdehyde ( MDA) content in the liver; ( 3 ) microscopic examination. Results The serum ALT and AST activities and MDA content in the liver were significantly higher, SOD activity in the liver significantly lower after hepatic I/R and pathological changes in the liver severer in group I/R and HH than in group S. The serum ALT and AST activities and MDA content in the liver were significantly lower, SOD activity in the liver significantly higher after hepatic I/R and pathological changes in the liver milder in group HH than in group I/R. Conclusion Hypervolemic hemodilution with hypertonic saline plus hetastarch solution 40 injectio can ameliorate hepatic I/R injury by decreasing oxygen free radical production in rats.     

高渗氯化钠羟乙基淀粉40注射液高容量血液稀释对大鼠肝脏缺血再灌注损伤的影响

Objective To investigate the effects of hypervolemic hemodilution (HH) with hypertonic saline plus hetastarch solution 40 injectio on hepatic ischemia-reperfusion (I/R) injury in rats. Methods Thirty male Wistar rats weighing 300-350 g were randomly divided into 3 groups ( n = 10 each): group I sham operation (group S); group II I/R and group Ⅲ HH. Partial liver ischemia was produced by clamping hepatic portal vein and left arteria hepatica for 30 min with atraumatic mini-clamp, followed by 2 h of reperfusion in I/R group and HH group. In HH group the animals were infused hypertonic saline plus hetastarch solution 40 injectio 10 ml/kg through vena caudalis over 30 min and then hepatic I/R was performed IS min after the infusion.The animals were killed at 2 h of reperfusion. The left liver was removed and blood sample was taken from inferior caval vein for determination of (1) serum alanine amino transferase (ALT) and aspartate amino transferase (AST) activities; (2) superoxide dismutase ( SOD) activity and malondialdehyde ( MDA) content in the liver; ( 3 ) microscopic examination. Results The serum ALT and AST activities and MDA content in the liver were significantly higher, SOD activity in the liver significantly lower after hepatic I/R and pathological changes in the liver severer in group I/R and HH than in group S. The serum ALT and AST activities and MDA content in the liver were significantly lower, SOD activity in the liver significantly higher after hepatic I/R and pathological changes in the liver milder in group HH than in group I/R. Conclusion Hypervolemic hemodilution with hypertonic saline plus hetastarch solution 40 injectio can ameliorate hepatic I/R injury by decreasing oxygen free radical production in rats.     

硼替佐米对小鼠急性移植物抗宿主病的抑制作用

目的 探讨硼替佐米对小鼠急性移植物抗宿主病(aGVHD)的抑制作用及其可能机制.方法 以C57BL/6小鼠为供鼠,获取骨髓细胞及脾细胞.以Balb/c小鼠为受鼠,共分为5组:空白对照组(n=17)小鼠不予任何处理;单纯照射组(n=17)小鼠仅接受7.0 Gy X射线全身照射(TBI);药物对照组(n=17)小鼠也接受TBI,并且由尾静脉注射硼替佐米;aGVHD组(n=8)小鼠TBI后注射供鼠骨髓细胞及脾细胞;实验组(n=8)小鼠TBI后输注供鼠骨髓细胞及脾细胞,并予以硼替佐米.观察各组小鼠aGVHD的发生情况、存活时间及嵌合状态,蛋白印迹法测定空白对照组、单纯照射组和药物对照组小鼠肝脏及小肠组织细胞核中核因子κB(NF-κB)p65的表达.结果 aGVHD组和实验组的临床aGVHD评分分别为7.37±0.32和5.85±0.40,实验组明显低于aGVHD组(P＜0.05).aGVHD组受鼠肝脏、小肠及皮肤组织为Thomas GVHD病理分级Ⅲ～Ⅳ级改变.实验组受鼠肝脏、小肠及皮肤组织为Ⅰ～Ⅲ级GVHD改变,较aGVHD组有所减轻.实验组存活时间长于aGVHD组(P＜0.05).aGVHD组和实验组小鼠移植后12 d时外周血细胞中H-2Kb分子阳性细胞的百分率均＞90%.药物对照组肝脏及小肠组织细胞核内NF-κB p65表达均高于单纯照射组(P＜0.05).结论 硼替佐米可能通过在一定程度上抑制照射预处理损伤所致肝脏及小肠组织中NF-κB的激活起到减轻aGVHD的作用.

Abstract：

Objective To observe the effect of bortezomib on acute graft-versus-host disease (aGVHD) in an aGVHD model of mice and investigate the related mechanism. Methods Male C57BL/6( H-2Kb)mice were used as donors and female Balb/c (H-2Kd) mice used as recipients. Balb/c mice received total body irradiation (TBI) by 7.0 Gy X-radiation, and randomly divided into five groups. normal (group A), TBI (group B), TBI + bortezomib (group C), TBI + bone marrow cells (BMC) + spleen cells (SC) (group D) and TBI + bortezomib + BMC + SC (group E). The physical signs and the pathological damage of aGVHD, mean survival time, and chimerism were observed in recipients. The NF-κB p65 levels in nuclei of the liver and small intestine tissues of groups A,B and C were analyzed by Western blot. Results ( 1 ) The clinical aGVHD score in group D was (7.37±0. 32), significantly higher than in group E (5.85 ± 0.40) (P＜0. 05). Histopathology of the gut, liver and skin illuminated that the Ⅲ-Ⅳ degree GVHD occurred in group D. The occurrence of aGVHD in group E was later than in group D. The symptoms and the pathological damage of aGVHD in group E were milder than in group D. The average survival time in group E was significantly longer than that in group D (P＜0.05). The percentage of donor-derived cells in recipient mice was above 90% at day 12 after transplantation; (2) NF-κB p65 levels in nuclei of the liver and small intestine tissues in group B was significantly higher than in group C on the day 1,3 and 5 (P＜0. 05). Conclusion Bortezomib can inhibit the activation and expression of NF-κB,which may be the underlying mechanism for it to relieve aGVHD.     

联合内皮祖细胞移植对小鼠骨髓移植预处理中肝脏内皮损伤的修复作用

目的 研究联合内皮祖细胞(EPC)移植对小鼠骨髓移植预处理中肝脏内皮损伤的修复作用.方法 将C57BL/6小鼠分为4组,每组10只.(1)正常对照组:小鼠不做任何处理,仅作为正常对照;(2)单纯照射组:单次给予全身照射(TBI)预处理,不进行骨髓移植;(3)单纯移植组:给予单纯照射组相同的TBI预处理,TBI后4 h内经小鼠尾静脉输注C57BL/6小鼠骨髓单个核细胞5×106/只;(4)联合移植组,小鼠的处理方式与单纯移植组相同,仅在骨髓移植的同时经尾静脉输注C57BL/6小鼠EPC 5×105/只.TBI后第2、4、7、14、21天,检测各组小鼠肝脏重量的变化情况,并于TBI后第4、7、14、21天对各组小鼠肝脏进行组织病理学检查.结果 单纯照射组、单纯移植组和联合移植组小鼠肝脏重量均于TBI后第2天开始明显增加,于第14天达到高峰,峰值分别为正常对照组的(1.65±0.15)倍(P＜0.05)、(1.61±0.06)倍(P＜0.05)和(1.11±0.4(0)倍(P＜0.05);以后均呈下降趋势,第21天时单纯照射组和单纯移植组肝脏重量仍明显高于正常对照组(P＜0.05),但联合移植组小鼠肝脏重量已完全恢复正常.组织病理学检查显示,单纯照射组小鼠肝窦内皮损伤明显,肝细胞水肿及严重的炎症细胞浸润,第7天时肝细胞水肿、坏死较前明显加重,几乎无存活的肝血窦内皮细胞;第14天时单纯移植组小鼠肝窦内皮损伤较前有所减轻,但到第21天时仍未恢复正常;联合移植组小鼠第7天时肝窦内皮及肝细胞水肿、坏死程度均较轻,到第14天时已基本恢复正常.结论 造血干细胞移植前的预处理会造成受者肝脏内皮损伤,且此损伤持续存在;移植时联合输注EPC能修复肝窦内皮的损伤.

Abstract：

Objective To study the repair function of united endothelial progenitor cells (EPC)transplantation on injured liver endothelium by bone marrow transplantation (BMT) conditioning.Methods C57BL/6 mice were divided into four groups randomly: normal control group, without any treatment; irradiation alone group, administered a total body irradiation(TBI) pretreatment, without BMT; (3) BMT alone group: C57BL/6 mice were infused with bone marrow mononuclearcells (MNC) 5 × 106/only through caudal vein not more than 4 h after the same TBI pretreatment as the irradiation alone group; united transplantation group: receiving the same way as the BMT alone group, but C57BL/6 mice were infused with EPC 5 × 105/only at the same time. Two, 4, 7, 14, and 21 days after the TBI, the changes of the liver weight were observed regularly. The histopathological examination of liver was done at the 4th, 7th, 14th, and 21st day after the TBI. Results In irradiation alone group, BMT alone group and united transplantation group the liver weight began to increase significantly on the day 2 and peaked at 14th day after the TBI, and the peaks were respectively (1.65±0. 15) times (P＜0. 05), (1.61 ±0.06) times (P＜0.05), and (1.11 ±0.40)times (P＜0. 05) of those in normal control group. At the day 14, the liver weight in irradiation alone group, BMT alone group and united transplantation group began to decrease, and on the day 21 the liver weight in united transplantation group had been completely restored to normal level, however the liver weight in irradiation alone group and BMT alone group were still significantly heavier than that in normal control group (P＜0. 05). Liver histopathological examination revealed that there were obvious sinusoidal endothelial cells (SEC) injury, hepatocyte edema and severe inflammatory cell infiltration in irradiation alone group, and on the day 7 the hepatocyte edema and necrosis were significantly worse than before, and almost no alive SEC were found. On the day 14 the injury of SEC in BMT alone group was lighter than before, but on the day 21 the injury had not returned to normal. On the day 7 the injury of SEC, hepatocyte edema and necrosis were alleviated in united transplantation group as compared with irradiation alone group and BMT alone group, and on the day 14 the injury had returned to normal basically. Conclusion The transplantation conditioning could damage recipient liver endothelium and the injury would persist, and united EPC infusion could repair the injured SEC following BMT.     

雌激素对大鼠肝切除肝缺血再灌注损伤中核因子-κB/IκB传导通路的影响及保护作用

目的 观察雌激素对大鼠肝切除肝缺血再灌注损伤中核因子-κB(NF-κB)/抑制蛋白(IκB)传导通路影响.方法 制作肝切除肝缺血再灌注损伤动物模型,雄性SD大鼠随机分为3组:假手术组(Sham组);肝切除肝缺血再灌注组(I/R组);肝切除肝缺血再灌注+雌激素组(I/R+ E2 组).分别在缺血再灌注后1、3、6h光镜下观察肝组织病理学改变,检测血清天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)的水平和肝组织丙二醛(MDA)的含量及超氧化物岐化酶(SOD)的活性,免疫组织化学法测定肝组织NF-κB的表达,Western blot检测NF-κB抑制蛋白(IκB-α)和细胞间黏附分子-1(ICAM-1)表达,流式细胞仪检测细胞凋亡率.结果 再灌注后I/R组在各时相血清ALT、AST均显著高于I/R +E2组,并于6h达到峰值(P＜0.05).与I/R+E2组和Sham比较,I/R组肝细胞凋亡率显著升高(P＜0.01);肝组织中IκB-α表达降低,而NF-κB表达增高(P＜0.05);ICAM-1 和MDA的结果变化和NF-κB表达水平变化类似,SOD呈相反变化.在光镜下观察,I/R组肝小叶结构紊乱,肝窦淤血,肝细胞水肿变性,肝细胞片状坏死,在Sham组和I/R +E2组上述病理学变化明显改善.结论 雌激素对肝切除肝脏缺血再灌注损伤有显著保护作用,其作用机制可能与雌激素影响NF-κB/IκB传导通路、减轻脂质过氧化反应、减少炎症介质释放及抑制细胞凋亡有关.     

再灌注期间给予富氢液对大鼠全脑缺血再灌注损伤的影响

目的 评价再灌注期间给予富氢液对大鼠全脑缺血再灌注损伤的影响.方法 成年雄性SD大鼠72只,月龄2.0～2.5个月,体重260～300 g,采用随机数字表法,将其随机分为3组(n＝24):假手术组(S组)、脑缺血再灌注组(I/R组)和富氢液组(H组).I/R组和H组采用四血管阻塞法(缺血15 min)制备全脑缺血再灌注损伤模型.H组于再灌注6h时腹腔注射0.6 mmol/L富氢液5ml/kg,I/R组给予等容量生理盐水.再灌注24h时,每组处死18只大鼠,取海马组织,测定丙二醛(MDA)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)含量、NF-κB活性、活化的caspase-3表达;再灌注72 h时,处死6只大鼠,取脑组织,HE染色,光镜下观察海马CA1区病理学结果,进行海马CA1区正常锥体细胞计数.结果 与S组比较,I/R组海马组织MDA、TNF-α、IL-6含量和NF-κB活性升高,活化caspase-3表达上调,正常锥体细胞计数减少(P<0.05);与I/R组比较,H组海马组织MDA、TNF-α、IL-6含量和NF-κB活性降低,活化caspase-3表达下调,正常锥体细胞计数增多(P<0.05).H组脑组织海马CA1区病理学损伤程度轻于I/R组.结论 再灌注期间给予富氢液可减轻大鼠全脑缺血再灌注损伤,其机制与抑制脂质过氧化反应、炎性反应和细胞凋亡有关.     

富氢液对大鼠骨癌痛行为学及脊髓低密度脂蛋白受体1/核因子-κB通路的影响

目的 探讨富氢液对大鼠骨癌痛行为学及脊髓低密度脂蛋白受体1(lectin-like oxidized low-density lipoproteinreceptor-1,LOX-1)/NF-κB通路的影响. 方法 清洁级健康雌性SD大鼠32只,体重180～200 9,按照随机数字表法分为4组(每组8只):假手术组(S组)、骨癌痛组(BCP组)、骨癌痛+富氢液组(BH2组)和骨癌痛+生理盐水组(BS组).BH2组在造模后1、3、5、7、9、11、13 d腹腔内注射富氢液(5 mg/kg),BS组注射等量的生理盐水(5 mg/kg).分别在造模前1d,造模后1、3、5、7、10、12、14 d测定大鼠的机械缩足反射阈值(paw mechanical withdrawal threshold,PMWT)和热缩足反射潜伏期(paw thermal withdrawal latency,PTWL).术后14 d行为学测定后处死大鼠,取脊髓腰膨大节段,利用Western blot技术检测脊髓LOX-1、磷酸化NF-κB(p-NF-κB)的表达. 结果 与S组比较,BCP组造模后3～14 d大鼠PMWT和PTWL明显下降(P＜0.05);与BS组比较,BH2组术后7～14 d PMWT和PTWL明显升高(P＜0.05).与S组比较,BCP组和BS组LOX-1、p-NF-κB表达明显升高(P＜0.05);与BS组比较,BH2组LOX-1、p-NF-κB表达明显下降(P＜0.05). 结论 腹腔内注射富氢液明显抑制大鼠骨癌痛,其机制可能与抑制LOX-1/NF-κB通路活化有关.     

治疗性高碳酸血症对大鼠移植肝脏缺血再灌注损伤的影响

目的 探讨治疗性高碳酸血症对大鼠移植肝脏缺血再灌注损伤的影响.方法 清洁级雄性Wistar大鼠32只,6周龄,体重220～280 g,采用完全随机设计的方法两两配对实施肝脏原位移植16只.采用随机数字表法,将受体鼠随机分为2组(n=8):肝移植组(LT组)和治疗性高碳酸血症组(TH组).LT组再灌注即刻吸入50％ O2-50％N2混合气体2 h;TH组再灌注即刻吸入O2-N2-CO2混合气体lh,调节气体浓度和呼吸频率,维持FiO2 50％、PaCO2 80～ 100 mm Hg,而后继续吸入50％O2-50％N2混合气体lh.再灌注期间记录MAP、PaO2和PaCO2.再灌注2h时,取动脉血样,测定血清ALT、AST、TNF-α、IL-1和IL-6的水平;取肝组织,测定NF-κB活性,计算凋亡指数,观察肝组织病理学结果.结果 与LT组比较,TH组血清ALT、AST、TNF-α、IL-1和IL-6水平、肝组织NF-κB活性和凋亡指数降低,MAP、PaO2和PaCO2升高(P＜0.05).TH组肝组织病理学损伤较LT组减轻.结论 治疗性高碳酸血症可减轻大鼠移植肝脏缺血再灌注损伤,机制与抑制炎性反应和细胞凋亡有关.     

脾切除对大鼠小体积肝脏模型的小肝综合征的预防作用

目的 探讨脾切除对大鼠小体积肝脏模型的小肝综合征(SFSS)的预防作用及其可能机制.方法 建立大鼠小体积肝脏模型,切除80%肝组织,即规则切除左外叶、左内叶、中叶和乳头叶,保留肝右叶和锥体叶,脾切除组在切除80%肝组织的同时行脾切除,对照组仅切除80%肝组织,另设仅游离肝脏而不行肝切除的假手术组.各组分别于术后0、3、6、12、24和72 h等6个时点各处死大鼠6只,取血清和肝组织,检测肝功能指标、肝组织中核因子Κb(NF-Κb)p65含量以及肝组织中肿瘤坏死冈子α(TNF-α)、细胞间黏附分子-1(ICAM-1)、增殖细胞核抗原(PCNA)Mrna和蛋白表达、髓过氧化物酶(MPO)活性,测量门静脉压力,计算SFSS发生率及动物术后1周存活率.结果 对照组术后门静脉压力明显升高,为(12.14±0.90)cm H2O,明显高于假手术组(P＜0.05),而脾切除组门静脉压力为(11.11±0.80)cm H2O,明显低于对照组(P＜0.05).对照组术后血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、乳酸脱氧酶(LDH)和胆红素总量(Tbil)明显升高,血清白蛋白(Alb)和胆碱酯酶(CHE)明显下降,脾切除组的ALT、AST、LDH和Tbil水平明显低于对照组,差异均有统计学意义(P＜0.05).对照组术后NF-Κb p65含量升高,TNF-α和ICAM-1 Mrna及其蛋白表达增加,MPO活性亦增高,PCNA Mrna及其蛋白表达亦增强,与对照组比较,脾切除组的NF-κBp65含量明显减少,TNF-α和ICAM-1 Mrna及其蛋白表达显著降低,MPO活性减弱,但PCNAmRNA和蛋白的表达更明显(P＜0.05).假手术组、对照组与脾切除组术后1周内的SFSS发生率分别为0、75%和25%,术后1周存活率分别为100%、50%和83.3%,后二者比较,差异均有统计学意义(P＜0.05).结论 脾切除能降低大鼠小体积肝脏模型的SFSS发生率,可能与脾切除后减少门静脉灌流、抑制NF-Κb活化、减轻细胞损害和促进肝细胞再生有关.     

激活腺苷2A受体对大鼠小体积移植肝核因子-κB活性和炎性反应的影响

目的 观察激活腺苷2A受体(A2AR)对大鼠小体积移植肝核因子(NF)-κB活性和炎性反应的影响.方法 建立35%～45%大鼠小体积肝移植模型,通过激活/拮抗A2AR,检测NF-κB活性,磷酸化IκpB(pIκB)、p65亚基蛋白表达及其下游炎症因子肿瘤坏死因子(TNF)-α、巨噬细胞炎性因子(MIP)-2、细胞间黏附分子(ICAM)-1表达水平,组织中髓过氧化物酶(MPO)活性变化.结果与对照组比较,激动组大鼠肝脏组织NF-κB活性及pIκB蛋白表达明显下调,再灌注后1、2、6 h MPO活性(U/g)(6.30±0.09比7.30±0.15、7.30±0.20比8.30±0.20、8.80±1.35比9.60±1.20)及炎性因子TNF-α(pg/mg)(700.0±57.1比967.0±145.0、800.0±57.2比1067.0±145.0、283.0±60.5比350.0±76.5)、MIP-2(pg/mg)(90.0±5.7比105.0±2.1、113.0±8.8比120.0±1.2、120.0±2.9比145.0±8.7)、ICAM-1(pg/mg)(393.0±23.3比500.0±28.9、450.0±28.9比767.0±44.1、380.0±23.1比460.0±28.8)的表达均显著下降(P均<0.05);而拮抗组NF-κB活性及pIκB蛋白显著升高,MPO活性及炎性因子TNF-α、MIP-2、ICAM-1的表达也显著升高(P均<0.05);激动、拮抗A2AR组p65差异均无统计学意义(P>0.05).结论 激活A2AR可明显下调大鼠肝组织NF-κB活性,减轻肝移植炎性损伤.     

饱和氢盐水对大鼠下肢缺血再灌注及远隔肺组织损伤的保护作用

目的 观察饱和氢盐水对大鼠下肢组织缺血再灌注及远隔肺组织损伤的保护作用.方法 将30只健康SD雄性大鼠随机分为3组:假手术组(S组)、生理盐水组(C组)、氢水治疗组(H组).建立大鼠下肢缺血再灌注损伤模型(阻断腹主动脉4 h,再灌注4 h),在再灌注前10min分别注入饱和氢盐水1 ml/100 g(H组),生理盐水1 ml/100 g(C组).再灌注4 h后比较肺及腓肠肌湿/干重(W/D)比值、观察病理切片的改变,测定血清、肺及腓肠肌组织中丙二醛(MDA)、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6含量变化.结果 H组肌(4.04±0.22)、肺(4.47±0.23)组织湿/干重比低于C组肌(4.67±0.27),肺(4.67±0.27)、组织(P＜0.05),MDA、TNF-α及IL-6血浆[分别为(2.08±1.27)μmol/L、(528.22±221.17)、(41.14±13.01)ng/L]及组织[肺组织分别为(3.58±1.62)μmol/L、(1.17±0.15)、(0.79±0.29)ng/L;肌组织分别为(8.91±3.13)μmol/L、(3.13±0.41)、(3.07±1.69)ng/L)含量均低于C组(P＜0.05),腓肠肌及肺病理学损害明显减轻,与S组比较差异无统计学意义(P＞0.05).结论 缺血再灌注前输注饱和氢盐水可有效保护大鼠下肢缺血再灌注及远隔肺组织的损伤.

Abstract：

Objective To explore the protective effects of hydrogen-rich saline against hind limb ischemia reperfusion injury and associated remote lung injury in rats. Methods Thirty Sprague-Dawley rats were randomly divided into three groups: sham group ( group S) ; saline control group (group C) , undergoing intravenous infusion of saline (1 ml/100 g) at 10 min before reperfusion; hydrogen-treated group ( group H) , undergoing intravenous infusion of hydrogen-rich saline (1 ml/100 g) at 10 min before reperfusion. Hind limb ischemia reperfusion model was established by 4 h of the abdominal aorta ligation followed by a 4 h of reperfusion. Rats were killed at the end of reperfusion, and blood samples were collected for determination of serum levels of tumor necrosis factor-α ( TNF-α) , interleukin-6 (IL-6) and malonyldialdehyde ( MDA). Specimens of lung and gastrocnemius muscle were collected for measurement of wet-todry weight (W/D) ratio, light microscopic examination and detection of plasma TNF-α, IL-6 and MDA.Results The pathological changes were milder in group H than in group C. The lung (4. 47 ±0. 23) and gastrocnemius muscle (4. 04 ±0. 22) W/D ratio in group H was significantly lower than in group C (4. 67 ±0.27 vs4. 67 ±0.27, P＜0.05). The serum [(2.08 ± 1. 27) μmol/L, (528. 22 ±221. 17) , (41.14± 13. 01) ng/L, respectively] and homogenate levels [(3. 58 ± 1. 62) μmol/L, ( 1. 17 ±0. 15) , (0. 79±0.29) ng/L in the lung; (8.91 ±3. 13) μmol/L, (3. 13 ±0. 41) ,(3. 07 ± 1. 69) ng/L in the gastrocnemius muscle, respectively] of MDA, IL-6 and TNF-a were also significantly lower in group H than in group C (P ＜0. 05) , but there was no significant difference from group S. Conclusion Infusion of hydrogen-rich saline before reperfsion significantly alleviates the hind limb ischemia reperfusion injury and associated remote lung injury.     

静脉注射含饱和氢气生理盐水减轻小鼠肾脏缺血再灌注损伤

目的 研究静脉注射含饱和氢气生理盐水对小鼠肾脏缺血再灌注(IR)损伤的保护作用及其机制.方法 健康、雄性的C57BL/6小鼠随机分为3组,每组10只.假手术组(SO组)小鼠仅接受中线开腹、双侧肾蒂游离及关腹操作;缺血再灌注组(IR组)小鼠用无损伤动脉夹同时钳夹双侧肾蒂,阻断45 min,制成肾脏IR损伤模型,并于肾脏缺血同时经尾静脉注射生理盐水,5 ml/kg;实验组小鼠制成肾脏IR损伤模型,并于肾脏缺血同时经尾静脉注射含饱和氢气生理盐水,5 ml/kg.各组小鼠于肾脏再灌注6 h时检测血清尿素氮(BUN)和肌酐(Scr);检测肾组织中丙二醛(MDA)和髓过氧化物酶(MPO)的含量;观察肾脏组织形态学变化并检测肾小管上皮细胞的凋亡情况;观察肾组织中巨噬细胞的浸润情况;检测各组小鼠肾组织中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-1β和IL-17 mRNA的水平.结果 实验组血清BUN和Scr水平明显低于IR组(P<0.05).实验组肾组织病理改变较IR组明显减轻,其肾小管损伤评分明显低于IR组(P<0.01),肾小管上皮细胞凋亡明显轻于IR组(P<0.05).实验组肾组织内MDA含量低于IR组(P<0.05).实验组小鼠肾组织内中性粒细胞和巨噬细胞的浸润较IR组减少(P<0.05).实验组TNF-α、IL-6、IL-1β和IL-17mRNA的水平均低于IR组(P<0.05).结论 静脉注射含饱和氢气生理盐水能够在一定程度上减轻肾脏IR损伤,其机制可能与抑制肾脏IR后炎症反应有关.     

核因子κB、表皮生长因子受体及黏蛋白1在肝内胆管结石合并肝内胆管癌组织的表达及其意义

目的 探讨肝内胆管结石合并肝内胆管癌组织核因子κB(NF-κB)、表皮生长因子受体(EGFR)和黏蛋白1(MUCl)的表达及其临床意义。方法取中山大学附属第二医院1989年8月至2009年6月间肝切除组织石蜡标本90例,其中肝内胆管结石合并肝内胆管癌33例为实验组,肝内胆管结石32例为对照组,肝良性肿瘤旁1～2 cm正常肝内胆管组织25例为空白组。组织切片分别进行NF-κB、EGFR及MUC1免疫组化染色,分析其表达差异的意义。结果 3组NF-κB的表达阳性率分别为51.5％(17/33)、25％ (8/32)和4％(1/25),P＜0.01;3组EGFR的表达阳性率分别为57.6％(19/33)、31.3％ (10/32)和0(o/25),P＜0.01; MUC1表达阳性率分别为54.5％ (18/33)、28.1％(9/32)和0(0/25),P＜0.01。EGFR、MUC1在病理组织学分级、肿瘤局部浸润及淋巴结转移的表达差异有统计学意义。肿瘤患者EGFR、MUC1阳性表达者的累积生存率比阴性表达者低(P＜0.01)。结论NF-κB是肝内胆管癌发生的早期事件;NF-κB、EGFR及MUC1过表达在肝内胆管癌发展过程起协同的重要作用,EGFR及MUC1高表达与肿瘤恶性程度以及预后有关。