中华麻醉学杂志
中華痳醉學雜誌
중화마취학잡지
Chinese Journal of Anesthesiology
2015年
8期
1020-1024
,共5页
张立民%李睿%孙文波%王琦
張立民%李睿%孫文波%王琦
장립민%리예%손문파%왕기
线粒体膜转运蛋白类%麻醉药,吸入%再灌注损伤%神经元%细胞凋亡
線粒體膜轉運蛋白類%痳醉藥,吸入%再灌註損傷%神經元%細胞凋亡
선립체막전운단백류%마취약,흡입%재관주손상%신경원%세포조망
Mitochondrial membrane transport proteins%Anesthetics,inhalation%Reperfusion injury%Neurons%Apoptosis
目的 评价线粒体通透性转换孔(mPTP)在七氟醚抑制氧糖剥夺-复糖复氧诱发大鼠神经元凋亡中的作用.方法 出生24 h内的SD大鼠,体外分离原代皮质神经元,以1×106个/ml的密度接种于6孔板(2 ml/孔)培养皿,采用随机数字表法分为5组(n=72):对照组(C组)神经元正常培养;氧糖剥夺-复糖复氧组(O组)神经元行氧糖剥夺90 min;mPTP开放剂组(A组)和mPTP开放剂+七氟醚组(AS组)神经元在行氧糖剥夺后即刻加入mPTP开放剂苍术苷20 μmol/L;七氟醚组(Sev组)和AS组神经元在氧糖剥夺后即刻给予2％七氟醚处理1h;O组和A组复糖复氧24 h,Sev组和AS组复糖复氧23 h后收集神经元.采用Annexin V-FITC/PI双染色法计数凋亡神经元,计算神经元凋亡率,采用MTT法测定神经元存活率;采用荧光探针JC-1法检测线粒体膜电位(MMP)水平;采用酶标仪测定mPTP开放程度;采用RT-PCR和免疫印迹法测定促凋亡因子Bid、Bim、Puma及其mRNA的表达.结果 与C组比较,其余各组神经元凋亡率升高,神经元存活率和MMP降低,mPTP开放程度增加,Bid、Bim和Puma及其mRNA表达上调(P＜0.05);与O组比较,Sev组神经元凋亡率降低,神经元存活率和MMP升高,mPTP开放程度降低,Bid、Bim和Puma及其mRNA表达下调(P＜0.05);与Sev组比较,A组和AS组凋亡率升高,神经元存活率和MMP降低,mPTP开放程度增加,Bid、Bim和Puma及其mRNA表达上调(P＜0.05);与A组比较,AS组神经元凋亡率降低,神经元存活率和MMP升高,mPTP开放程度降低,Bid、Bim和Puma及其mRNA表达下调(P＜0.05).结论 七氟醚通过抑制mPTP开放减轻氧糖剥夺-复糖复氧诱发的大鼠神经元凋亡.
目的 評價線粒體通透性轉換孔(mPTP)在七氟醚抑製氧糖剝奪-複糖複氧誘髮大鼠神經元凋亡中的作用.方法 齣生24 h內的SD大鼠,體外分離原代皮質神經元,以1×106箇/ml的密度接種于6孔闆(2 ml/孔)培養皿,採用隨機數字錶法分為5組(n=72):對照組(C組)神經元正常培養;氧糖剝奪-複糖複氧組(O組)神經元行氧糖剝奪90 min;mPTP開放劑組(A組)和mPTP開放劑+七氟醚組(AS組)神經元在行氧糖剝奪後即刻加入mPTP開放劑蒼術苷20 μmol/L;七氟醚組(Sev組)和AS組神經元在氧糖剝奪後即刻給予2％七氟醚處理1h;O組和A組複糖複氧24 h,Sev組和AS組複糖複氧23 h後收集神經元.採用Annexin V-FITC/PI雙染色法計數凋亡神經元,計算神經元凋亡率,採用MTT法測定神經元存活率;採用熒光探針JC-1法檢測線粒體膜電位(MMP)水平;採用酶標儀測定mPTP開放程度;採用RT-PCR和免疫印跡法測定促凋亡因子Bid、Bim、Puma及其mRNA的錶達.結果 與C組比較,其餘各組神經元凋亡率升高,神經元存活率和MMP降低,mPTP開放程度增加,Bid、Bim和Puma及其mRNA錶達上調(P＜0.05);與O組比較,Sev組神經元凋亡率降低,神經元存活率和MMP升高,mPTP開放程度降低,Bid、Bim和Puma及其mRNA錶達下調(P＜0.05);與Sev組比較,A組和AS組凋亡率升高,神經元存活率和MMP降低,mPTP開放程度增加,Bid、Bim和Puma及其mRNA錶達上調(P＜0.05);與A組比較,AS組神經元凋亡率降低,神經元存活率和MMP升高,mPTP開放程度降低,Bid、Bim和Puma及其mRNA錶達下調(P＜0.05).結論 七氟醚通過抑製mPTP開放減輕氧糖剝奪-複糖複氧誘髮的大鼠神經元凋亡.
목적 평개선립체통투성전환공(mPTP)재칠불미억제양당박탈-복당복양유발대서신경원조망중적작용.방법 출생24 h내적SD대서,체외분리원대피질신경원,이1×106개/ml적밀도접충우6공판(2 ml/공)배양명,채용수궤수자표법분위5조(n=72):대조조(C조)신경원정상배양;양당박탈-복당복양조(O조)신경원행양당박탈90 min;mPTP개방제조(A조)화mPTP개방제+칠불미조(AS조)신경원재행양당박탈후즉각가입mPTP개방제창술감20 μmol/L;칠불미조(Sev조)화AS조신경원재양당박탈후즉각급여2％칠불미처리1h;O조화A조복당복양24 h,Sev조화AS조복당복양23 h후수집신경원.채용Annexin V-FITC/PI쌍염색법계수조망신경원,계산신경원조망솔,채용MTT법측정신경원존활솔;채용형광탐침JC-1법검측선립체막전위(MMP)수평;채용매표의측정mPTP개방정도;채용RT-PCR화면역인적법측정촉조망인자Bid、Bim、Puma급기mRNA적표체.결과 여C조비교,기여각조신경원조망솔승고,신경원존활솔화MMP강저,mPTP개방정도증가,Bid、Bim화Puma급기mRNA표체상조(P＜0.05);여O조비교,Sev조신경원조망솔강저,신경원존활솔화MMP승고,mPTP개방정도강저,Bid、Bim화Puma급기mRNA표체하조(P＜0.05);여Sev조비교,A조화AS조조망솔승고,신경원존활솔화MMP강저,mPTP개방정도증가,Bid、Bim화Puma급기mRNA표체상조(P＜0.05);여A조비교,AS조신경원조망솔강저,신경원존활솔화MMP승고,mPTP개방정도강저,Bid、Bim화Puma급기mRNA표체하조(P＜0.05).결론 칠불미통과억제mPTP개방감경양당박탈-복당복양유발적대서신경원조망.
Objective To evaluate the role of mitochondrial permeability transition pore (mPTP) in sevoflurane-induced inhibition of apoptosis induced by oxygen-glucose deprivation and restoration (OGD/ R) in rat neurons.Methods Cortical neurons isolated from neonatal Sprague-Dawley rats born within 24 h, were cultured primarily and seeded in 6-well plates (2 ml/well) at a density of 1 × 106 cells/ml.The neurons were randomly divided into 5 groups (n =72 each) using a random number table: control group (group C), OGD/R group (group O), mPTP opener group (group A), mPTP opener + sevoflurane group (group AS) , and sevoflurane group (group Sev).The cells were cultured in normal culture medium in group C, and the cells were subjected to OGD for 90 min followed by restoration of O2-glucose supply for 24 h in the other groups.In A and AS groups, mPTP opener atractyloside 20 μmol/L was added immediately after OGD.In Sev and AS groups, the cells were post-conditioned with 2％ sevoflurane for 1 h immediately after OGD.The neurons were collected at 24 h of OGD in O and A groups, or at 23 h of OGD in Sev and AS groups.Annexin V-FITC/PI double staining was performed to count apoptotic neurons, apoptotic rate was calculated, and cell survival rate was measured using MTT assay.The mitochondrial membrane potential (MMP) was measured by using JC-1 fluorescence probe as indicator.The opening of mPTP was determined through assessing the changes of mitochondrial optical density (△OD540 of mPTP).The expression of pro-apoptotic factors Bid, Bim, Puma protein and mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction and Western blot.Results Compared with group C, apoptotic rate was significantly increased, cell survival rate and MMP were decreased, the opening of mPTP was increased, and the expression of Bid, Bim, Puma protein and mRNA was up-regulated in the other groups.Compared with group O, apoptotic rate was significantly decreased, cell survival rate and MMP were increased, the opening of mPTP was decreased, and the expression of Bid, Bim, Puma protein and mRNA was down-regulated in Sev group.Compared with group Sev, apoptotic rate was significantly increased,cell survival rate and MMP were decreased, the opening of mPTP was increased, and the expression of Bid, Bim, Puma protein and mRNA was up-regulated in A and As groups.Compared with group A, apoptotic rate was significantly decreased, cell survival rate and MMP were increased, the opening of mPTP was decreased, and the expression of Bid, Bim, Puma protein and mRNA was down-regulated in group AS.Conclusion Sevoflurane mitigates OGD/R-induced apoptosis in rat neurons through inhibiting mPTP opening.
