糖基转移酶Colgalt2基因敲除对肝细胞再生过程中增殖和凋亡的作用研究  

Effect of glycosyltransferase Colgalt2 gene knockout on hepatocyte proliferation and apoptosis on course of hepatocyte regeneration

作　　者：王智强[1,2] 杨琪[1,2] 王建文[3] 刘燃[3] 郝晓花[3] 黄玉波[3] 魏红山[1,2,3] 

机构地区：[1]北京大学地坛医院教学医院 [2]北京大学医学部,北京100015 [3]首都医科大学附属北京地坛医院,北京100015

出　　处：《中国肝脏病杂志（电子版）》2015年第1期26-31,共6页Chinese Journal of Liver Diseases：Electronic Version

基　　金：国家自然科学基金(No.30872243;No.81271901);北京市自然科学基金(No.7152073)

摘　　要：目的初步探讨Colgalt2基因敲除对小鼠肝细胞再生过程中细胞增殖和凋亡的作用。方法根据Higgins和Anderson于1931年提出的肝大部分切除模型,实施Colgalt2+/+野生型对照组小鼠和Colgalt2-/-小鼠(均为C57BL/6J品系)70%肝大部分切除术(PH),分别取正常小鼠和PH后1、3、7天的小鼠再生肝脏的右外叶,于10%中性福尔马林固定,石蜡包埋,切片。采用免疫组织化学方法比较细胞增殖核抗原(PCNA)在小鼠肝部分切除术后各时间点组间的表达阳性率差异;胶原酶两步灌注法分别分离正常和PH后1、3、5天的Colgalt2+/+小鼠和Colgalt2-/-小鼠的肝细胞。流式细胞仪检测各组肝细胞凋亡率。结果 PH后1、3、7天,Colgalt2-/-小鼠肝细胞PCNA阳性表达率显著高于Colgalt2+/+小鼠肝细胞PCNA阳性表达率,且差异有显著统计学意义(P<0.01)。分离的肝细胞活细胞比率达94%,正常Colgalt2+/+小鼠和Colgalt2-/-小鼠分离的肝细胞凋亡率分别为(23.36±0.83)%、(21.04±1.16)%;PH后1天,Colgalt2+/+对照组小鼠肝细胞凋亡率和Colgalt2-/-小鼠肝细胞凋亡率分别为(8.27±0.33)%、(8.44±0.30)%,PH后1天肝细胞凋亡率均降至最低,随即肝细胞凋亡率逐渐升高;PH后3天,Colgalt2+/+小鼠和Colgalt2-/-小鼠肝细胞凋亡率分别为(15.92±0.56)%、(12.14±0.37)%,Colgalt2-/-小鼠的肝细胞凋亡率明显低于野生型对照组小鼠的肝细胞凋亡率(P<0.01);PH后5天,Colgalt2+/+小鼠和Colgalt2-/-小鼠肝细胞凋亡率分别为(21.36±0.51)%、(18.92±0.92)%,Colgalt2-/-小鼠的肝细胞凋亡率仍低于野生型对照组小鼠的肝细胞凋亡率(P<0.05),肝细胞凋亡率均接近于恢复正常水平。结论糖基转移酶Colgalt2基因敲除在肝再生过程中在一定程度上具有促进肝细胞增殖和抗肝细胞凋亡的作用,提示该基因介导的胶原Glcα1,2Galβ1-糖基化修饰对肝再生可能起着重要的调控作用。Objective To investigate the effect of Colgalt2 gene knockout on hepatocyte proliferation and apoptosis on course of hepatocyte regeneration. Methods Colgalt2＋/＋ wild type control mice and Colgalt2-/-mice models of 70% partial hepatectomy（PH） were established according to the protocols by Higgins and Anderson in 1931. Then the part of right lateral lobes of liver from normal mice and those 1, 3, 7 days after PH were harvested, fixed in 10% neutral buffered formalin, embedded in paraffin and cut into tissue sections. Immunohistochemistry was used to compare the expressive difference of proliferating cell nuclear antigen（PCNA） in the mouse liver slices at different time points after partial hepatectomy. Primary hepatocytes of normal Colgalt2＋/＋ wild type control mice, normal Colgalt2-/- mice and those treated by partial hepatectomy after 1, 3, 5 days were isolated and purified by two steps of collagenase perfusion. Flow cytometry was used to detect apoptotic rate in each group. Results The number of PCNA positive cells of Colgalt2-/- mice was much larger than that of Colgalt2＋/＋ mice on 1, 3, 7 days after PH, and the difference was statistically significant（P〈0.01）. The viability of primary hepatocytes was 94%. The apoptotic rate of normal Colgalt2＋/＋ mice was（23.36 ± 0.83）%, and that of normal Colgalt2-/- mice was（21.04 ± 1.16）%. The apoptotic rates of Colgalt2＋/＋ control mice and Colgalt2-/- mice in the first day after PH were（8.27 ± 0.33）%,（8.44 ± 0.30）%. The apoptotic rates declined to the lowest on the first day after PH and then they began to increase gradually. While, the apoptotic rates of Colgalt2＋/＋ mice and Colgalt2-/- mice in the third day after PH were（15.92 ± 0.56）%,（12.14 ± 0.37）%. The apoptotic rate of Colgalt2-/- mice was much lower than that of wild type control mice（P〈0.01）. The apoptotic rates of Colgalt2＋/＋ mice and Colgalt2-/- mice in the fifth day after PH were（21.36 ± 0.51）%,（18.92 ± 0.92）% which showed t

关 键 词：肝再生 糖基转移酶 增殖 凋亡 

分 类 号：R575[医药卫生—消化系统]