肝糖原貯備對(duì)熱缺血再灌注大鼠肝細(xì)胞凋亡的影響

1、肝糖原貯備對(duì)熱缺血再灌注大鼠肝細(xì)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(duì)熱缺血再灌注肝細(xì)胞凋亡及肝損傷的影響。方法 建立大鼠肝熱缺血模型。實(shí)驗(yàn)分組：術(shù)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(shù)前禁食24 h，飲水不限(L組)；正常飲食對(duì)照組(N組)和假手術(shù)組(S組)。缺血45 min，再灌注2、24 h取材。流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡及Bcl2、Bax蛋白。同時(shí)進(jìn)行肝酶學(xué)檢測(cè)、肝組織形態(tài)學(xué)觀察。結(jié)果 1.細(xì)胞凋亡及蛋白表達(dá)：(1)24 h細(xì)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(xué)指標(biāo)：各時(shí)相點(diǎn)4組間比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，S組<H組<N組< p> 【關(guān)鍵詞】  肝細(xì)胞凋亡 肝糖原 熱缺血再灌注損傷 流式細(xì)

3、胞術(shù) 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(xì)胞凋亡起重要作用。有研究1-2表明提高肝糖原貯備對(duì)肝臟WIRI過程中肝損害具有拮抗效應(yīng)，但該機(jī)制尚不明確。本實(shí)驗(yàn)主要觀察其中肝糖原對(duì)肝細(xì)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(dòng)物實(shí)驗(yàn)中心提供。AST、ALT、AKP檢測(cè)試劑盒購(gòu)自南京建成生物工程所。鼠抗鼠Bcl

11、2、Bax單克隆抗體購(gòu)自Santa Cruz公司。羊抗鼠FITCIgG由軍事醫(yī)學(xué)科學(xué)院提供。流式細(xì)胞儀FACS420，美國(guó)BD公司產(chǎn)品。    1.2  方法    1.2.1  動(dòng)物分組    Wistar雄性大鼠64只(體重250300 g)，隨機(jī)分組：高糖原組(H組)：術(shù)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(shù)前禁食24 h，飲水不限。以上各組進(jìn)行實(shí)驗(yàn)時(shí)，動(dòng)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(guān)腹，再灌注2、24 h后取材。假手術(shù)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(shí)相2、24 h隨機(jī)分為2個(gè)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(duì)熱缺血再灌注大鼠肝細(xì)胞凋亡的影響  1.2.2  動(dòng)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(jì)時(shí)。肝蒂夾閉后肝臟顏色逐漸變深、腫脹、質(zhì)地變硬，腸壁淤血。45 min后打開血管夾并關(guān)腹，再灌注2、24 h。    1.2.3  標(biāo)本采集    按缺血再灌注時(shí)相點(diǎn)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(cè)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(biāo)檢測(cè)    1.3.1  細(xì)胞凋亡及Bcl2、Bax蛋白檢測(cè)    采用流式細(xì)胞技術(shù)進(jìn)行。細(xì)胞凋亡采用溴化己啶染色法檢測(cè)。Bcl2、Bax蛋白表達(dá)量采用細(xì)胞免疫熒光標(biāo)記法檢測(cè)。    1.3.2  肝臟酶學(xué)檢測(cè)