中国全科医学
中國全科醫學
중국전과의학
CHINESE GENERAL PRACTICE
2015年
19期
2310-2316
,共7页
史志勤%卞红磊%魏艳静%尹晓琳%张荣%梁文杰%刘瑞春%王维平%于江华
史誌勤%卞紅磊%魏豔靜%尹曉琳%張榮%樑文傑%劉瑞春%王維平%于江華
사지근%변홍뢰%위염정%윤효림%장영%량문걸%류서춘%왕유평%우강화
癫痫持续状态%重组人红细胞生成素%磷酸肌醇3-激酶类%蛋白激酶类%Bcl-2相关X蛋白质%X连锁凋亡抑制蛋白质
癲癇持續狀態%重組人紅細胞生成素%燐痠肌醇3-激酶類%蛋白激酶類%Bcl-2相關X蛋白質%X連鎖凋亡抑製蛋白質
전간지속상태%중조인홍세포생성소%린산기순3-격매류%단백격매류%Bcl-2상관X단백질%X련쇄조망억제단백질
Status epilepticus%Recombinant human erythropoietin%Phosphatidylinositol 3 - kinases%Protein kinases%Bcl-2-associated X protein%X-linked inhibitor of apoptosis protein
目的:观察重组人红细胞生成素( rHuEPO)对戊四氮( PTZ)点燃的癫痫持续状态( SE)的成年雄性( SD)大鼠海马神经元磷酸化蛋白激酶B( p-PKB/p-Akt)、Bcl-2相关X蛋白( Bax)和X-连锁凋亡抑制蛋白( XIAP)表达的影响,并进一步探讨rHuEPO作用的可能机制。方法于2010年3月,由河北省实验动物中心提供的健康清洁级成年SD大鼠。采用PTZ点燃大鼠SE模型,将大鼠随机分为B组( PTZ+0.9%氯化钠溶液)、C组( PTZ+rHuEPO)、D组(PTZ+LY294002+rHuEPO)、E组(PTZ+二甲基亚砜+rHuEPO),同时另选取不进行SE制模的SD大鼠作为A组(0.9%氯化钠溶液),检测大鼠行为学和脑电图的改变;用原位末端转移酶标记技术( TUNEL)检测海马神经元的凋亡情况;免疫组织化学法观察p-Akt、Bax、XIAP的阳性细胞数;反转录多聚酶链反应( RT-PCR)方法检测各组大鼠海马Bax信使RNA( BaxmRNA)的表达;Western blot方法检测各组大鼠海马蛋白激酶 B( Akt)、p-Akt、XIAP蛋白的表达。结果与B组比较,C组p-Akt、XIAP阳性细胞数和p-Akt、XIAP蛋白表达水平均增多, Bax阳性细胞数及BaxmRNA表达水平均减少,差异有统计学意义( P<0.05);与C组比较,D组p-Akt、XIAP阳性细胞数和p-Akt、XIAP蛋白表达水平均减少,Bax阳性细胞数及BaxmRNA表达水平均增多,差异有统计学意义( P<0.05)。结论 rHuEPO可以提高p-Akt、XIAP阳性细胞数及蛋白表达水平,降低Bax阳性细胞数及BaxmRNA表达水平,减少海马神经元的凋亡,发挥神经保护作用;加入磷脂酰肌醇3激酶( PI3K)抑制剂LY294002后,海马p-Akt、XIAP阳性细胞数及蛋白表达减少、Bax阳性细胞数及BaxmRNA表达增加,海马神经元的凋亡增加,减弱了rHuEPO的保护作用。因此,PI3K/Akt信号通路是rHuEPO发挥神经保护作用的通路之一,其作用机制可能是rHuEPO活化PI3K/Akt后,提高p-Akt蛋白及线粒体凋亡途径相关调控因子XIAP的表达,下调了Bax的表达,从而介导线粒体凋亡途经,发挥抗凋亡、促存活的神经保护作用。
目的:觀察重組人紅細胞生成素( rHuEPO)對戊四氮( PTZ)點燃的癲癇持續狀態( SE)的成年雄性( SD)大鼠海馬神經元燐痠化蛋白激酶B( p-PKB/p-Akt)、Bcl-2相關X蛋白( Bax)和X-連鎖凋亡抑製蛋白( XIAP)錶達的影響,併進一步探討rHuEPO作用的可能機製。方法于2010年3月,由河北省實驗動物中心提供的健康清潔級成年SD大鼠。採用PTZ點燃大鼠SE模型,將大鼠隨機分為B組( PTZ+0.9%氯化鈉溶液)、C組( PTZ+rHuEPO)、D組(PTZ+LY294002+rHuEPO)、E組(PTZ+二甲基亞砜+rHuEPO),同時另選取不進行SE製模的SD大鼠作為A組(0.9%氯化鈉溶液),檢測大鼠行為學和腦電圖的改變;用原位末耑轉移酶標記技術( TUNEL)檢測海馬神經元的凋亡情況;免疫組織化學法觀察p-Akt、Bax、XIAP的暘性細胞數;反轉錄多聚酶鏈反應( RT-PCR)方法檢測各組大鼠海馬Bax信使RNA( BaxmRNA)的錶達;Western blot方法檢測各組大鼠海馬蛋白激酶 B( Akt)、p-Akt、XIAP蛋白的錶達。結果與B組比較,C組p-Akt、XIAP暘性細胞數和p-Akt、XIAP蛋白錶達水平均增多, Bax暘性細胞數及BaxmRNA錶達水平均減少,差異有統計學意義( P<0.05);與C組比較,D組p-Akt、XIAP暘性細胞數和p-Akt、XIAP蛋白錶達水平均減少,Bax暘性細胞數及BaxmRNA錶達水平均增多,差異有統計學意義( P<0.05)。結論 rHuEPO可以提高p-Akt、XIAP暘性細胞數及蛋白錶達水平,降低Bax暘性細胞數及BaxmRNA錶達水平,減少海馬神經元的凋亡,髮揮神經保護作用;加入燐脂酰肌醇3激酶( PI3K)抑製劑LY294002後,海馬p-Akt、XIAP暘性細胞數及蛋白錶達減少、Bax暘性細胞數及BaxmRNA錶達增加,海馬神經元的凋亡增加,減弱瞭rHuEPO的保護作用。因此,PI3K/Akt信號通路是rHuEPO髮揮神經保護作用的通路之一,其作用機製可能是rHuEPO活化PI3K/Akt後,提高p-Akt蛋白及線粒體凋亡途徑相關調控因子XIAP的錶達,下調瞭Bax的錶達,從而介導線粒體凋亡途經,髮揮抗凋亡、促存活的神經保護作用。
목적:관찰중조인홍세포생성소( rHuEPO)대무사담( PTZ)점연적전간지속상태( SE)적성년웅성( SD)대서해마신경원린산화단백격매B( p-PKB/p-Akt)、Bcl-2상관X단백( Bax)화X-련쇄조망억제단백( XIAP)표체적영향,병진일보탐토rHuEPO작용적가능궤제。방법우2010년3월,유하북성실험동물중심제공적건강청길급성년SD대서。채용PTZ점연대서SE모형,장대서수궤분위B조( PTZ+0.9%록화납용액)、C조( PTZ+rHuEPO)、D조(PTZ+LY294002+rHuEPO)、E조(PTZ+이갑기아풍+rHuEPO),동시령선취불진행SE제모적SD대서작위A조(0.9%록화납용액),검측대서행위학화뇌전도적개변;용원위말단전이매표기기술( TUNEL)검측해마신경원적조망정황;면역조직화학법관찰p-Akt、Bax、XIAP적양성세포수;반전록다취매련반응( RT-PCR)방법검측각조대서해마Bax신사RNA( BaxmRNA)적표체;Western blot방법검측각조대서해마단백격매 B( Akt)、p-Akt、XIAP단백적표체。결과여B조비교,C조p-Akt、XIAP양성세포수화p-Akt、XIAP단백표체수평균증다, Bax양성세포수급BaxmRNA표체수평균감소,차이유통계학의의( P<0.05);여C조비교,D조p-Akt、XIAP양성세포수화p-Akt、XIAP단백표체수평균감소,Bax양성세포수급BaxmRNA표체수평균증다,차이유통계학의의( P<0.05)。결론 rHuEPO가이제고p-Akt、XIAP양성세포수급단백표체수평,강저Bax양성세포수급BaxmRNA표체수평,감소해마신경원적조망,발휘신경보호작용;가입린지선기순3격매( PI3K)억제제LY294002후,해마p-Akt、XIAP양성세포수급단백표체감소、Bax양성세포수급BaxmRNA표체증가,해마신경원적조망증가,감약료rHuEPO적보호작용。인차,PI3K/Akt신호통로시rHuEPO발휘신경보호작용적통로지일,기작용궤제가능시rHuEPO활화PI3K/Akt후,제고p-Akt단백급선립체조망도경상관조공인자XIAP적표체,하조료Bax적표체,종이개도선립체조망도경,발휘항조망、촉존활적신경보호작용。
Objective To observe the effect of recombinant human erythropoietin( rHuEPO)on the expression levels of p-PKB/p-Akt,Bax and XIAP in hippocampal neurons of male mature SD rats with pentylenetetrazol( PTZ)induced status epilepticus( SE),and to further investigate the possible mechanism. Methods The included male mature rats of clean grade were provided by Laboratory Animal Center of Hebei Province in March 2010. By using the PTZ kindling epileptic rat SE model, the SD rats were divided into four groups:group B( PTZ+0. 9% sodium chloride solution),group C( PTZ+rHuEPO),group D(PTZ+LY294002+rHuEPO)and group E(PTZ+dimethyl sulfoxide+rHuEPO). Meanwhile,we assigned SD rats without SE model building into group A(0. 9% sodium chloride solution). The behavioral and EEG changes were detected;TUNEL was used to check the apoptosis status of hippocampal neurons;immunohistochemistry was used to observe the number of cells with positive p-Akt, Bax and XIAP;the RT - PCR method was used to detect the expression level of BaxmRNA in hippocampus;Western blot method was employed to detect the protein expression levels of Akt,p-Akt and XIAP. Results Compared with group B,group C was higher(P<0. 05)in the number of cells with positive p-Akt and XIAP,the protein expression levels of p-Akt and XIAP and was lower in the number of cells with positive Bax and the protein expression level of BaxmRNA. Compared with group C,group D was lower in the number of cells with positive p-Akt and XIAP and the protein expression levels of p-Akta and XIAP and was higher(P <0. 05)in the number of positive cells with positive Bax and the expression level of BaxmRNA. Conclusion rHuEPO can increase the number of cells with positive p-Akt and XIAP and the protein expression of p-Akt and XIAP. It can also decrease the number of cells with positive Bax and the expression level of BaxmRNA,reduce the apoptosis of hippocampal neurons,and has a neuroprotective effect. When PI3K inhibitor LY294002 is added,the number of cells with positive p-Akt and XIAP and the protein expression of p-Akt and XIAP decrease,the number of cells with positive Bax and the expression of BaxmRNA increase,the apoptosis of hippocampal neurons increases,and the neuroprotective effect of rHuEPO is weakened. Therefore,the signal path of PI3K/Akt may be one of the neuroprotective ways of rHuEPO. The possible mechanism may be that rHuEPO activating the PI3K/Akt increases the expression levels of p-Akt and XIAP,decreases the expression level of Bax,thus mediating the path of mitochondrial apoptosis and playing a neuroprotective role of anti-apoptosis and survival promotion.