摘要
目的探讨Pim-3基因对暴发性肝细胞凋亡的抑制机制。方法32只大鼠随机分成4组（8只／组）。A组为正常对照组，B、C组和D组采用流体力学注射方法，分别以林格氏液、空载体质粒pEGFP-N2和重组质粒pEGFP-N2／Pim-3溶液预处理大鼠；1d后，予脂多糖（LPS）／D-半乳糖胺（D-GaIN）腹腔注射诱导暴发性肝细胞凋亡。8h后处死大鼠，采集肝组织标本。用Caspase-3活性测定法检测细胞凋亡情况；RT-PCR检测肝组织诱导型一氧化氮合酶（iNOS）、p53基因表达水平；Westernblot检测肝组织Bcl-2、Bax蛋白表达水平。组间数据比较采用非参数Kruskai-Wallis检验。结果LPS／D—GalN联合攻击造成了大鼠肝内Caspase-3活性显著增高，而D组Caspase-3活性较B组和C组均显著降低[（141．7±13．7）RFU比（508．1±32．0）、（493．5±33．1）RFU，P值均〈0．0.01）。LPS／D-GalN的应用也诱导了肝组织iNOSmRNA、p53mRNA、Bax蛋白表达，与B组和C组相比，D组iNOSmRNA和p53mRNA的表达水平显著降低（0．06±0．01比0．42±0．08、0．35±0．06；0．73±0．11比1．17±0．25、1．23±0．31，P值均〈0．01），而Bax蛋白表达水平差异无统计学意义（1．19±0．09比1．13±0．08、1．25±0．11，P〉0．05）；另外，D组肝内Bcl-2蛋白表达水平较A、B组和C组均显著升高（3．05±0．29比1．03±0．05、0．98±0．06、1．10±0．08，P值均〈0．01）。结论Pim-3基因通过对损伤基因和凋亡相关基因的影响抑制了暴发性肝细胞凋亡的发生。
Objective To investigate the mechanisms of serine/threonine kinase Pim-3 inhibition of fulminant hepatic apoptosis. Methods Thirty-two rats were randomly divided into four groups （n = 8 each）： normal controls （A）; pretreatment with Ringer＇s solution （B）, vector plasmid （C）, or Pim-3 recombinant plasmid （D） by hydrodynamics-based procedure followed by intraperitoneal injections of lipopolysaccharide （LPS） and D-galactosamine （D-GaIN） after one day. At 8 h after the LPS/D-GalN injections, liver tissues were collected from all groups of mice and analyzed for cell apoptosis by detecting caspase-3 activity （measured in relative fluorescence units, RFU）. Changes in expression of relevant genes were determined by RT-PCR and Western blotting. Results Caspase-3 activity was induced in response to LPS/D-GalN injection. Pim-3-pretreated rats showed a lower level of caspase-3 activity than the Ringer＇s-pretreatod or vector plasmid-pretreated rats [（141.7 -4- 13.7）RFU vs. （508.1 ＋ 32.0） or （493.5 ~ 33.1） RFU; all P 〈 0.01 ]. High expressions of the liver injury marker gene, iNOS, and the apoptosis-induced genes, p53 and Bax, were found after LPS/D-GalN challenge, and were suppressed by exogenous Pim-3 gene injection. In addition, exogenous Pim-3 gene injection induced high expression of the liver anti-apoptosis protein, Bcl-2, but had no effect on Bax protein expression. Conclusion The Pim-3 gene can block fulminant hepatic apoptosis by affecting the expression of the iNOS liver injury gene and the p53, Bax and Bcl-2 apoptosis-related genes.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2012年第9期688-692,共5页
Chinese Journal of Hepatology
基金
国家自然科学基金（30660066,81070357）
