肝糖原貯備對熱缺血再灌注大鼠肝細胞凋亡的影響

1、肝糖原貯備對熱缺血再灌注大鼠肝細胞凋亡的影響         08-07-21 10:43:00     編輯：studa20          作者：陳文斌 陳永亮 盧燦榮 梁斌    【摘要】  目的 探討肝糖原貯備對熱缺血再灌注肝細胞凋亡及肝損傷的影響。方法 建立大鼠肝熱缺血模型。實驗分組：術前24 h靜脈注射25%葡萄糖組，2 ml/只，每

2、6 h 1次，高糖飲食(H組)；術前禁食24 h，飲水不限(L組)；正常飲食對照組(N組)和假手術組(S組)。缺血45 min，再灌注2、24 h取材。流式細胞術檢測細胞凋亡及Bcl2、Bax蛋白。同時進行肝酶學檢測、肝組織形態(tài)學觀察。結果 1.細胞凋亡及蛋白表達：(1)24 h細胞凋亡百分率。S組<H組<N組<L組(P<0.01)，L組<N組(PN組(P<0.01)。2.肝酶學指標：各時相點4組間比較差異有統(tǒng)計學意義(P<0.05)，S組<H組<N組< p> 【關鍵詞】  肝細胞凋亡 肝糖原 熱缺血再灌注損傷 流式細

3、胞術 大鼠    【Abstract】  Objective  To study the role of hepatic glycogen storage in affecting hepatocyte apoptosis and liver injury in rats with liver warm ischemia/reperfusion injury. Methods  Models of liver warm ischemia/reperfusion injury (WIRI) in rats were set up and

4、 divided into Group H (injected with 25% glucose into tail vein for 2 ml, once per 6 and 24 hours before operation), Group L (fasting 24 hours before operation), Group N (normal diet control group) and Group S (sham operation group). After 45 minutes of ischemia and 2 and 24 hours of reperfusion, bl

5、ood was taken to detect hepatocyte apoptosis, Bcl2 and Bax by flow cytometry and observe the changes of pathological morphology. Results  (1) Apoptosis rate at 24 hours in a increase order was from Group S, Group H, Group N to Group L (P<0.01), with apoptosis rate at 24 hours higher than tha

6、t at 2 hours in all groups (P<0.01, except for Group S). Expression of Bcl2 in Group H at 24 hours was higher than that in other three groups (P<0.01), with expression of Bcl2 in Group L less than that in Group N (P<0.05). Expression of Bax in Group S at 24 hours was lower than that in othe

7、r groups (P<0.01), with expression of Bax in Group L more than that in Group N (P<0.01). Expression of Bax at 24 hours was higher than that at 2 hours in all groups (P<0.01, except for Group S). (2) There was difference upon enzyme level in the same period among four groups (P<0.05), wit

8、h level from least to highest in groups from Group S, Group H, Group N to Group L. (3) Pathological morphological changes after 24 hours of reperfusion in Group H was obviously slighter than those in Groups N and L, with the most significant change in Group L. Conclusion  Preventive increase of

9、 glycogen storage can relieve hepatocyte apoptosis and injury in the process of liver warm ischemia/reperfusion by modifying expressions of Bcl2 and Bax.    【Key words】  Hepatocyte apoptosis; Liver glycogen; Warm ischemia/reperfusion injury; Flow cytometry; Rats   

10、   肝臟熱缺血再灌注損傷(warm ischemia and reperfusion injury,WIRI)過程中，細胞凋亡起重要作用。有研究1-2表明提高肝糖原貯備對肝臟WIRI過程中肝損害具有拮抗效應，但該機制尚不明確。本實驗主要觀察其中肝糖原對肝細胞凋亡的影響。    1  材料與方法    1.1  材料    Wistar雄性大鼠，體重250300 g，由解放軍總醫(yī)院動物實驗中心提供。AST、ALT、AKP檢測試劑盒購自南京建成生物工程所。鼠抗鼠Bcl

11、2、Bax單克隆抗體購自Santa Cruz公司。羊抗鼠FITCIgG由軍事醫(yī)學科學院提供。流式細胞儀FACS420，美國BD公司產(chǎn)品。    1.2  方法    1.2.1  動物分組    Wistar雄性大鼠64只(體重250300 g)，隨機分組：高糖原組(H組)：術前24 h開始尾靜脈注射25%葡萄糖2 ml/只，每6 h 1次，共4次，自由飲食。正常糖原組(N組)：自由飲食。低糖原組(L組)：術前禁食24 h，飲水不限。以上各組進行實驗時，動物麻醉后打開腹腔，無創(chuàng)血管夾夾

12、閉肝蒂，45 min后打開血管夾并關腹，再灌注2、24 h后取材。假手術組(S組)：開腹騷擾腹腔，但不夾閉肝蒂，自由飲食，余同前組。每組16只(按缺血再灌注時相2、24 h隨機分為2個亞組，每組8只)。另取18只(6只尾靜脈注射25%葡萄糖2 ml/次，每6 h 1次，飲食不限；6只禁食24 h，飲水不限；6只正常飲食)24 h后剖殺取肝組織，苦味酸純酒精飽和甲醛溶液固定，行糖原染色。    陳文斌，等. 肝糖原貯備對熱缺血再灌注大鼠肝細胞凋亡的影響  1.2.2  動物模型的制備    乙醚吸入維持麻醉，建立尾靜

13、脈輸液通道，2 ml肝素(100 U/ml)尾靜脈注射，生理鹽水10 ml皮下注射。打開腹腔，無創(chuàng)血管夾夾閉肝蒂(S組除外)，并開始計時。肝蒂夾閉后肝臟顏色逐漸變深、腫脹、質地變硬，腸壁淤血。45 min后打開血管夾并關腹，再灌注2、24 h。    1.2.3  標本采集    按缺血再灌注時相點心腔取血，肝素抗凝，3000 r/min離心10 min，留取血清置于20 冰箱中保存待檢，用于測AKP、ALT、AST。各組取血后立即切取肝組織2份，分別以10%中性甲醛、70%酒精固定備檢。    1.3  指標檢測    1.3.1  細胞凋亡及Bcl2、Bax蛋白檢測    采用流式細胞技術進行。細胞凋亡采用溴化己啶染色法檢測。Bcl2、Bax蛋白表達量采用細胞免疫熒光標記法檢測。    1.3.2  肝臟酶學檢測