【作者】 刘悦晖；

【导师】 范学工；

【作者基本信息】 中南大学 ， 内科学， 2008， 博士

【摘要】 第一章刀豆球蛋白所致实验性肝损伤模型的构建目的:构建Con A诱导的小鼠实验性肝损伤模型。方法:Balb/C小鼠40只,分别尾静脉注射Con A,根据注射剂量的大小,小鼠被随机分为4组(每组10只):A组为10mg/kg,B组为20mg/kg,C组为30mg/kg,另D组作为实验对照组尾静脉仅注射生理盐水。给药后8h时观察小鼠血清丙氨酸氨基转移酶(ALT)活性、死亡率和C组心、肺、肾、脑、肝组织的病理改变。选择Con A剂量,并观察此剂量下不同时间点ALT活性、死亡率和肝组织的病理改变特点。结果:A组和D组未见小鼠死亡,B组死亡3只小鼠,C组全部死亡。A组和B组ALT活性显著高于D组。光镜下A组可见肝细胞变性,B和C组可见肝组织炎症细胞浸润和肝细胞变性、坏死,且以C组为甚。C组肺可见轻度充血、肾小管上皮细胞出现轻度空泡变性、而心、脑未见明显异常。20mg/kg Con A尾静脉注射,小鼠死亡率、ALT活性、肝组织炎症细胞浸润和肝细胞变性、坏死在一定时间范围内(24h)随着时间增加而加剧。结论:1.成功构建了Con A小鼠实验性肝损伤模型。2.Con A小鼠实验性肝损伤模型Con A最佳剂量为20mg/kg。第二章抑制性寡脱氧核苷酸对刀豆球蛋白所致肝损伤保护作用的观察第一节造模前12小时注射抑制性寡脱氧核苷酸对刀豆球蛋白所致肝损伤保护作用的观察目的:探讨造模前12小时(h)注射抑制性寡脱氧核苷酸(suppressive oligonucleotides,Sup ODN)对刀豆球蛋白A(ConA)所致小鼠实验性肝损伤的保护作用。方法:雌性Balb/C小鼠80只随机分为生理盐水(NS)组、环孢霉素(CsA)组、对照寡脱氧核苷酸(Control oligonucleotides,Con ODN)组、Sup ODN组,每小组又分8h和24h两个时间点,每个时间点观察10只小鼠。Con A(20 mg/kg)尾静脉注射小鼠造模。造模前12h时每组分别给予NS(0.3 mL/只)、Con ODN(20mg/kg)、CsA(130mg/kg)、Sup ODN(20mg/kg)腹腔给药。给药8h和24h时分别观察小鼠的死亡率;采血检测血清中TNF-α、IL-4、IFN-γ含量及丙氨酸氨基转氨酶(ALT)活性。RT-PCR检测肝组织的TNF-α、IFN-γmRNA的表达,同时观察肝组织的病理改变。结果:与NS、Con ODN组相比,Sup ODN组ALT活性和IFN-γ含量和IFN-γmRNA表达明显降低,IL-4含量明显升高(P＜0.01)。与NS和Con ODN组相比Sup ODN组肝细胞坏死程度和炎症细胞浸润显著减轻(P＜0.05)。第二节造模同时注射抑制性寡脱氧核苷酸对Con A所致肝损伤保护作用的观察目的:探讨造模同时注射Sup ODN对Con A所致小鼠实验性肝损伤的保护作用。方法:雌性Balb/C小鼠80只随机分为NS组、CsA组、Con ODN组、Sup ODN组,每小组又分8h和24h两个时间点,每个时间点观察10只小鼠。Con A(20 mg/kg)尾静脉注射小鼠造模。造模同时每组分别给予NS(0.3 mL/只)、Con ODN(20mg/kg)、CsA(130 mg/kg)、Sup ODN(20mg/kg)腹腔给药。给药8h和24h时分别观察小鼠的死亡率;采血检测血清TNF-α、IL-4、IFN-γ含量及ALT活性。RT-PCR检测肝组织的TNF-α、IFN-γmRNA的表达,同时观察肝组织的病理改变。结果:与NS、Con ODN组相比,Sup ODN组ALT活性和IFN-γ含量和IFN-γmRNA表达明显降低,而IL-4含量明显升高(P＜0.01)。与NS和Con ODN组相比Sup ODN组肝细胞坏死程度和炎症细胞浸润显著减轻(P＜0.05)。第三节造模后2小时注射抑制性寡脱氧核苷酸对Con A所致肝损伤保护作用的观察目的:探讨造模后2h注射Sup ODN对Con A所致小鼠实验性肝损伤的保护作用。方法:雌性Balb/C小鼠80只随机分为NS、,CsA组、Con ODN组、Sup ODN组,每小组又分8h和24h两个时间点,每个时间点观察10只小鼠。Con A(20 mg/kg)尾静脉注射小鼠造模。造模后2h时每组分别给予NS(0.3 mL/只),Con ODN(20mg/kg),CsA(130mg/kg),Sup ODN(20mg/kg)。给药8h和24h时观察小鼠的死亡率;采血检测血清TNF-α、IL-4、IFR-γ含量及ALT活性。RT-PCR检测肝组织的TNF-α、IFN-γmRNA的表达,同时观察肝组织的病理改变。结果:Sup ODN与CsA组相比,死亡率明显降低(P＜0.01)。与NS、Con ODN组相比,Sup ODN组ALT活性和IFN-γ含量和IFN-γmRNA表达明显降低,而IL-4含量明显升高(P＜0.01)。与NS和Con ODN组相比Sup ODN组肝细胞坏死程度和炎症细胞浸润显著减轻(P＜0.05)。结论:Sup ODN对Con A诱导的小鼠实验性肝损伤有明显保护作用。第三章抑制性寡脱氧核苷酸作用机制的初步探目的:初步探讨抑制性寡脱氧核苷酸(suppressiveoligonucleotides,Sup ODN)对刀豆球蛋白A(Con A)所致肝损保护作用的机制。方法:实验动物分组、肝损伤模型构造及实验动物给药同第二章。Western blot测定脾组织中磷酸化的STAT4(phosphorylated STAT6,pSTAT4)、pSTAT6、T-bet蛋白表达,余指标检测同第二章。结果:血清TNF-α、IL-4、IFN-γ水平,RT-PCR检测肝组织的TNF-α、IFN-γmRNA的表达及肝组织的病理改变结果同第二章。SupODN抑制pSTAT4和T-bet表达,而对pSTAT6表达无影响。CsA对pSTAT6、pSTAT4和T-bet表达都无影响。结论:Sup ODN通过下调pSTAT4和T-bet表达,从而抑制IFN-γ产生,达到保护效应。Sup ODN对Con A诱导的肝损伤保护机制与CsA不同。更多还原

【Abstract】 Chapter One Development of the model about concanavalin A-induced experimental liver injury in miceObjective:To establish the model about concanavalin A-induced experimental liver injury in mice.Methods:Forty Balb/C mice were randomly divided into 4 groups based on the dose of Con A(ten for each group),A,B,C and D.Con A 10mg/kg,20mg/kg or 30mg/kg was intravenous(tail vein) injected into the mouse group A,B or C respectively while NS was used for the group D(control group).Survival rate of each group at 8 hours after via was calculated.The survived mice were killed in order to get blood,heart, liver,lung,kidney and brain samples.Activity of aminnotransferase(ALT) was tested.Histopathological examination for heart,liver,lung,kidney and brain were also performed.The optimal Con A dose(20mg/kg) and treated different times were selected.The ALT level,death rate and histopathological results were observed under the optimal Con A dose.Results:The activity of ALT in both group A and group B was significantly higher than that in group D(P＜0.01).No dead mouse was found in group A and D,but 3 and 10 mice were dead in group B and C respectively.Hepatic histopathology showed only degeneration of liver cells was present in group A,while degeneration,necrosis and inflammatory cells were found infiltration in group B and C.There were no pathological changes in heart and brain,while congestion was found in lung and little vacuolar degeneration in renal cells respectively.Within 24 hours of injection of 20mg/kg of Con A,the death rate,activity of ALT, liver inflammatory cells infiltration,degeneration,hepatocyte degeneration and necrosis exacerbated with the prolongation of time.Conclusion:The Con A-induced liver injury in mice was established successfully.The optimal dose for Con A-induced experimental liver injury in mice is 20mg/kg. Chapter Two The protective effect of suppressive oligonucleotides on Con A-induced liver injury in miceSection one:The protection effect of suppressive oligonucleotides injected at 12 hours before Con A-induced liver injury in miceObjective:To investigate the protective effect of suppressive oligonucleotides(Sup ODN) injected at 12 hours before concanavalin A (Con A)-induced liver injury in mice.Methods:Eighty Balb/C mice were randomly divided into 4 groups, normal saline(NS) group,control oligonucleotides(Con ODN) group, cyclosporin A(CsA) group and Sup ODN group.Mice in each group were injected with Con A(20mg/kg) via the tail vein.NS(0.3mL per mouse),Con ODN(20mg/kg),CsA(130 mg/kg) or Sup ODN(20mg/kg) was injected intraperitoneally at 12 hours before Con A injection.Death rate of each group at 8 and 24 hours after intraperitoneal injection was calculated and survived mice were killed in order to get blood and liver samples.Activity of aminnotransferase(ALT) was tested and levels of TNF-α,IFN-γand IL-4 were detected by ELISA.Expressions of TNF-αand IFN-γmRNA in liver tissue were detected by RT-PCR. Histopathological examination for liver was also Performed.Results:The ALT activity in Sup ODN group was found significantly lower than that in NS and Con ODN group respectively(P＜0.01).Level of IFN-γ,and its mRNA expression were significantly decreased,while the level of IL-4 was significantly increased in Sup ODN group compared with NS,Con ODN and CsA group(P＜0.01). Compared to NS and Con ODN group,the liver necrosis and infiltration of inflammatory cells significantly reduced.Section two:The protective effect of Sup ODN injected simulativsly with the start of Con A-induced liver injury in miceObjective:To investigate the protective effect of Sup ODN injected simulativsly with the start of Con A-induced liver injury in mice.Methods:Eighty Balb/C mice were randomly divided into 4 groups, NS group,Con ODN group,CsA group and Sup ODN group.Mice in each group were injected with Con A(20mg/kg) via the tail vein. NS(0.3mL per mouse),Con ODN(20mg/kg),CsA(130 mg/kg) or Sup ODN(20mg/kg) was injected intraperitoneally at the same time point Con A injection.Death rate of each group at 8 and 24 hours after intraperitoneal injection was calculated and survived mice were killed in order to get blood and liver samples.Activity of ALT was tested and levels of TNF-α,IFN-γ,and IL-4 were detected by ELISA.Expressions of TNF-αand IFN-γ,mRNA in liver tissue were detected by RT-PCR. Histopathological examination for liver was also performed.Results:The ALT activity in Sup ODN group was found significantly lower than that in NS and Con ODN group respectively(P＜0.01).Level of IFN-γ,and its mRNA expression were significantly decreased,while the level of IL-4 was significantly increased in Sup ODN group compared with NS,Con ODN and CsA group(P＜0.01).Compared to NS and Con ODN group,the liver necrosis and infiltration of inflammatory cells significantly reduced.Section Three:The protective effect of Sup ODN injected at 2 hours after Con A-induced liver injury in miceObjective:To investigate the protective effect of Sup ODN injected at 2 hours after Con A-induced liver injury in mice.Methods:Eighty Balb/C mice were randomly divided into 4 groups, NS group,control Con ODN group,CsA group and Sup ODN group. Mice in each group were injected with Con A(20mg/kg) via the tail vein. NS(0.3 mL per mouse),Con ODN(20mg/kg),CsA(130 mg/kg) or Sup ODN(20mg/kg) was injected intraperitoneally at 2 hours after Con A injection.Death rate of each group at 8 and 24 hours after intraperitoneal injection was calculated and survived mice were killed in order to get blood and liver samples.Activity of ALT was tested and levels of TNF-α, IFN-γ,and IL-4 were detected by ELISA.Expressions of TNF-αand IFN-γ,mRNA in liver tissue were detected by RT-PCR.Histopathological examination for liver was also performed.Results:The death rate in group Sup ODN significantly lower than that in group CsA.The ALT activity in Sup ODN group was found significantlylower than that in NS and Con ODN group respectively(P＜0.01).Level of IFN-γ,and its mRNA expression were significantly decreased,while the level of IL-4 was significantly increased in Sup-ODN group compared with NS,Con ODN and CsA group(P＜0.01).Compared to NS and Con ODN group,the liver necrosis and infiltration of inflammatory cells significantly reduced.Conclusion:The research indicated Sup ODN had a obviously protective effect on Con A-induced mice liver injury. Chapter Three The preiminaly study of mechanism and the protective effect of suppressive oligonucleotidesObjective:To investigate mechanism of suppressive oligonucleotides (Sup ODN) on concanavalin A(Con A )-induced liver injury in mice.Methods:The levels of TNF-α,IFN-γ,and IL-4 were detected by ELISA and the other methods is the same to chapter two.Expression of pSTAT4(phosphorylated pSTAT4),pSTAT6,T-bet in spleen tissue were detected by Western blot.Results:Sup ODN obviously suppressed the expression of pSTAT4 and T-bet in spleen tissue,but had no influence on pSTAT6,cyclosporin A(CsA) had no influence on pSTAT6,pSTAT4 and T-bet.The other results were the same to chapter two.Conclusion:The protective effect of Sup ODN may suppressed the expressions of IFN-γ,via down regulatory the expressions of pSTAT4 and T-bet in spleen tissue,but had no influence on pSTAT6.The mechanism of Sup ODN differently from CsA on Con A-induced liver injury in mice.更多还原