目的:构建携带人白细胞介素(human interleukin,hIL)-10基因的腺病毒载体,细胞学及动物模型实验鉴定其介导的目的基因表达,并验证其对大鼠肝细胞损伤的保护作用。方法:以pORF-hIL10质粒为模板,PCR扩增hIL-10基因,插入腺病毒质粒pDC315,...目的:构建携带人白细胞介素(human interleukin,hIL)-10基因的腺病毒载体,细胞学及动物模型实验鉴定其介导的目的基因表达,并验证其对大鼠肝细胞损伤的保护作用。方法:以pORF-hIL10质粒为模板,PCR扩增hIL-10基因,插入腺病毒质粒pDC315,构建pDC315-hIL10。将质粒pDC315-hIL10与腺病毒包装质粒共转染至293细胞,产生复制缺陷型重组腺病毒Ad5-hIL10;感染大鼠肝细胞,免疫学方法鉴定目的基因的表达;建立体内外大鼠肝细胞损伤模型,验证Ad5-hIL10对肝细胞损伤的保护作用。结果:经PCR扩增、鉴定,证明已正确构建重组腺病毒Ad5-hIL10。大鼠肝细胞感染Ad5-hIL10后,随着病毒感染强度的升高,hIL-10表达明显上升。大鼠肝细胞体外培养模型预先感染病毒Ad5-hIL10,经四氯化碳诱导损伤,则细胞存活率随感染强度(multiplicity of infection,MOI)值升高而增加,至MOI=50时,细胞存活率恢复至92%;大鼠体内肝损伤模型显示,与损伤对照组和空白病毒对照组比较,Ad5-hIL10处理的大鼠肝细胞水样变性和间质炎症反应均减轻,肝功能的各项指标均有明显改善(P<0.05)。结论:成功构建了重组腺病毒质粒Ad5-hIL10,且能明显提高肝细胞的存活能力,对肝功能有明显保护作用。展开更多
AIM:To investigate the correlation of hyperlipemia(HL) and acute cerebral ischemia/reperfusion(I/R) injury on liver damage and its mechanism.METHODS:Rats were divided into 4 groups:control,HL,I/R and HL+I/R.After the ...AIM:To investigate the correlation of hyperlipemia(HL) and acute cerebral ischemia/reperfusion(I/R) injury on liver damage and its mechanism.METHODS:Rats were divided into 4 groups:control,HL,I/R and HL+I/R.After the induction of HL via a high-fat diet for 18 wk,middle cerebral artery occlusion was followed by 24 h of reperfusion to capture I/R.Serum alanine transaminase(ALT) and aspartate aminotransferase(AST) were analyzed as part of liver function tests and liver damage was further assessed by histological examination.Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL) assay.The expression of genes related to apoptosis(caspase-3,bcl-2) was assayed by immunohistochemistry and Western blotting.Serum tumor necrosis factor-(TNF-),interleukin-1(IL-1) and liver mitochondrial superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),malondialdehyde(MDA) and Ca 2+ levels were measured to determine inflammatory and oxidative/antioxidative status respectively.Microsomal hydroxylase activity of the cytochrome P450 2E1(CYP2E1)-containing enzyme was measured with aniline as the substrate,and CYP2E1 expression in the liver tissue and microsome was determined by immunohistochemistry and Western blotting respectively.RESULTS:HL alone induced by high-fat diet for 18 wk resulted in liver damage,indicated by histopathological analysis,and a considerable increase in serum ALT(25.13 ± 16.90 vs 9.56 ± 1.99,P < 0.01) and AST levels(18.01 ± 10.00 vs 11.33 ± 4.17,P < 0.05) compared with control.Moreover,HL alone induced hepatocyte apoptosis,which was determined by increased TUNEL-positive cells(4.47 ± 0.45 vs 1.5 ± 0.22,P < 0.01),higher caspase-3 and lower bcl-2 expression.Interestingly,compared with those in control,HL or I/R groups,massive increases of serum ALT(93.62 ± 24.00 vs 9.56 ± 1.99,25.13 ± 16.90 or 12.93 ± 6.14,P < 0.01) and AST(82.32 ± 26.92 vs 11.33 ± 4.17,18.01 ± 10.00 or 14.00 ± 6.19,P < 0.01) levels in HL+I/R group were observed suggesting severe liver damage,which was confirmed by liver histology.In addition,HL combined with I/R also caused significantly increased hepatocyte apoptosis,as evidenced by increased TUNEL-positive cells(6.20 ± 0.29 vs 1.5 ± 0.22,4.47 ± 0.45 or 1.97 ± 0.47,P < 0.01),elevated expression of caspase-3 and lower expression of bcl-2.Furthermore,when compared to HL or I/R alone,HL plus I/R enhanced serum TNF-,IL-1,liver mitochondrial MDA and Ca 2+ levels,suppressed SOD and GSH-Px in liver mitochondria,and markedly up-regulated the activity(11.76 ± 2.36 vs 4.77 ± 2.31 or 3.11 ± 1.35,P < 0.01) and expression(3.24 ± 0.38 vs 1.98 ± 0.88 or 1.72 ± 0.58,P < 0.01) of CYP2E1 in liver.CONCLUSION:The coexistence of HL and acute cerebral I/R induces severe liver damage,suggesting that cerebral ischemic stroke would exaggerate the damage of liver caused by HL.This effect is possibly due to en-hanced CYP2E1 induction which further promotes oxidative damage,inflammation and hepatocyte apoptosis.展开更多
AIM: To investigate the effects of a novel Leukotriene B4 receptor antagonist and/or tacrolimus on ischemia-reperfusion in a rat liver model. METHODS: Male Lewis rats were pretreated with ONO-4057 (100 mg/kg) and/or t...AIM: To investigate the effects of a novel Leukotriene B4 receptor antagonist and/or tacrolimus on ischemia-reperfusion in a rat liver model. METHODS: Male Lewis rats were pretreated with ONO-4057 (100 mg/kg) and/or tacrolimus (1 mg/kg) orally, and divided into four experimental groups; group 1 (control), group 2 (ONO-4057), group 3 (tacrolimus), group 4 (ONO-4057 + tacrolimus). RESULTS: There was a tendency for long survival in the groups treated with tacrolimus alone and ONO-4057 plus tacrolimus. Post-reperfusion serum aspartate aminotransferase levels decreased more signif icantly in ONO-4057 plus tacrolimus group (P < 0.01), than in the tacrolimus alone group (P < 0.05), compared to controls. CONCLUSION: This study demonstrated that pretreat-ment with ONO-4057 in combination with tacrolimus produced additive effects in a rat model of liver isch-emia-reperfusion injury.展开更多
文摘目的:构建携带人白细胞介素(human interleukin,hIL)-10基因的腺病毒载体,细胞学及动物模型实验鉴定其介导的目的基因表达,并验证其对大鼠肝细胞损伤的保护作用。方法:以pORF-hIL10质粒为模板,PCR扩增hIL-10基因,插入腺病毒质粒pDC315,构建pDC315-hIL10。将质粒pDC315-hIL10与腺病毒包装质粒共转染至293细胞,产生复制缺陷型重组腺病毒Ad5-hIL10;感染大鼠肝细胞,免疫学方法鉴定目的基因的表达;建立体内外大鼠肝细胞损伤模型,验证Ad5-hIL10对肝细胞损伤的保护作用。结果:经PCR扩增、鉴定,证明已正确构建重组腺病毒Ad5-hIL10。大鼠肝细胞感染Ad5-hIL10后,随着病毒感染强度的升高,hIL-10表达明显上升。大鼠肝细胞体外培养模型预先感染病毒Ad5-hIL10,经四氯化碳诱导损伤,则细胞存活率随感染强度(multiplicity of infection,MOI)值升高而增加,至MOI=50时,细胞存活率恢复至92%;大鼠体内肝损伤模型显示,与损伤对照组和空白病毒对照组比较,Ad5-hIL10处理的大鼠肝细胞水样变性和间质炎症反应均减轻,肝功能的各项指标均有明显改善(P<0.05)。结论:成功构建了重组腺病毒质粒Ad5-hIL10,且能明显提高肝细胞的存活能力,对肝功能有明显保护作用。
文摘AIM:To investigate the correlation of hyperlipemia(HL) and acute cerebral ischemia/reperfusion(I/R) injury on liver damage and its mechanism.METHODS:Rats were divided into 4 groups:control,HL,I/R and HL+I/R.After the induction of HL via a high-fat diet for 18 wk,middle cerebral artery occlusion was followed by 24 h of reperfusion to capture I/R.Serum alanine transaminase(ALT) and aspartate aminotransferase(AST) were analyzed as part of liver function tests and liver damage was further assessed by histological examination.Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL) assay.The expression of genes related to apoptosis(caspase-3,bcl-2) was assayed by immunohistochemistry and Western blotting.Serum tumor necrosis factor-(TNF-),interleukin-1(IL-1) and liver mitochondrial superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),malondialdehyde(MDA) and Ca 2+ levels were measured to determine inflammatory and oxidative/antioxidative status respectively.Microsomal hydroxylase activity of the cytochrome P450 2E1(CYP2E1)-containing enzyme was measured with aniline as the substrate,and CYP2E1 expression in the liver tissue and microsome was determined by immunohistochemistry and Western blotting respectively.RESULTS:HL alone induced by high-fat diet for 18 wk resulted in liver damage,indicated by histopathological analysis,and a considerable increase in serum ALT(25.13 ± 16.90 vs 9.56 ± 1.99,P < 0.01) and AST levels(18.01 ± 10.00 vs 11.33 ± 4.17,P < 0.05) compared with control.Moreover,HL alone induced hepatocyte apoptosis,which was determined by increased TUNEL-positive cells(4.47 ± 0.45 vs 1.5 ± 0.22,P < 0.01),higher caspase-3 and lower bcl-2 expression.Interestingly,compared with those in control,HL or I/R groups,massive increases of serum ALT(93.62 ± 24.00 vs 9.56 ± 1.99,25.13 ± 16.90 or 12.93 ± 6.14,P < 0.01) and AST(82.32 ± 26.92 vs 11.33 ± 4.17,18.01 ± 10.00 or 14.00 ± 6.19,P < 0.01) levels in HL+I/R group were observed suggesting severe liver damage,which was confirmed by liver histology.In addition,HL combined with I/R also caused significantly increased hepatocyte apoptosis,as evidenced by increased TUNEL-positive cells(6.20 ± 0.29 vs 1.5 ± 0.22,4.47 ± 0.45 or 1.97 ± 0.47,P < 0.01),elevated expression of caspase-3 and lower expression of bcl-2.Furthermore,when compared to HL or I/R alone,HL plus I/R enhanced serum TNF-,IL-1,liver mitochondrial MDA and Ca 2+ levels,suppressed SOD and GSH-Px in liver mitochondria,and markedly up-regulated the activity(11.76 ± 2.36 vs 4.77 ± 2.31 or 3.11 ± 1.35,P < 0.01) and expression(3.24 ± 0.38 vs 1.98 ± 0.88 or 1.72 ± 0.58,P < 0.01) of CYP2E1 in liver.CONCLUSION:The coexistence of HL and acute cerebral I/R induces severe liver damage,suggesting that cerebral ischemic stroke would exaggerate the damage of liver caused by HL.This effect is possibly due to en-hanced CYP2E1 induction which further promotes oxidative damage,inflammation and hepatocyte apoptosis.
文摘AIM: To investigate the effects of a novel Leukotriene B4 receptor antagonist and/or tacrolimus on ischemia-reperfusion in a rat liver model. METHODS: Male Lewis rats were pretreated with ONO-4057 (100 mg/kg) and/or tacrolimus (1 mg/kg) orally, and divided into four experimental groups; group 1 (control), group 2 (ONO-4057), group 3 (tacrolimus), group 4 (ONO-4057 + tacrolimus). RESULTS: There was a tendency for long survival in the groups treated with tacrolimus alone and ONO-4057 plus tacrolimus. Post-reperfusion serum aspartate aminotransferase levels decreased more signif icantly in ONO-4057 plus tacrolimus group (P < 0.01), than in the tacrolimus alone group (P < 0.05), compared to controls. CONCLUSION: This study demonstrated that pretreat-ment with ONO-4057 in combination with tacrolimus produced additive effects in a rat model of liver isch-emia-reperfusion injury.
