大鼠肝卵圆细胞增殖模型肝脏的GJIC功能和意义  

Function and significance of gap junction intercellular communication in rat liver for hepatic oval cell proliferation in vivo

作　　者：李学东[1] 傅华群[1] 李少华 尚西亮[1] 邢宏松[1] 胡鹏[1] 

机构地区：[1]南昌大学第二附属医院肝胆外科,江西省南昌市330006 [2]黑龙江省鸡东中医院普外科,黑龙江省鸡西市158200

出　　处：《世界华人消化杂志》2007年第13期1475-1481,共7页World Chinese Journal of Digestology

基　　金：江西省自然科学基金项目;No.0640112;江西省教育厅科技计划基金项目;No.061033;江西省科技厅科研基金项目;No.200680;江西省卫生厅中医药科研基金项目;No.2005A30;昆明制药集团股份有限公司科研基金项目~~

摘　　要：目的:研究大鼠肝卵圆细胞(HOC)增殖模型肝脏缝隙连接蛋白32,43(CX)的表达及缝隙连接细胞间通讯(GJIC)的功能,探讨HOC增殖的可能机制.方法:健康(?)Wistar大鼠,随机分成正常对照组(n=6),模型组.模型组大鼠按每天20 mg/kg剂量灌喂2-AFF,连续4 d,第5天不灌喂行2/3肝切除,术后次日按每天20 mg/kg剂量继续灌喂5 d(2-AFF/PH).模型组在术后4h,4,8,12和16d随机取6只大鼠检测.采用组织病理技术观察肝组织的形态学变化;免疫组化和细胞形态学方法计数HOC;切开标记/染料示踪技术(incision loading/dye transfer,IL/DT)技术确定GJIC;免疫组化及RT-PCR技术检测CX32蛋白及mRNA表达;免疫组化、Western blot及RT-PCR技术分析CX43蛋白及mRNA水平.结果:对照组及模型组4 h未见HOC增殖.模型组4d汇管区有HOC增殖反应,8d HOC增殖达峰值,12d HOC从汇管区向肝实质内浸润,16d HOC增殖减少较12 d减少;IL/DT检测结果显示,模型组各时点(4h,4,8,12和16d)染料扩散距离低于对照组(84.5±3.4,60.6±3.3,108.6±4.2,150.6±2.6,199.6±3.7μm vs 250.0±5.0 um,P<0.01),GJIC功能降低.模型组各时点CX32表达均低于对照组(P<0.05),在4 h下调,4d达低峰(2.85±0.39),8d后逐渐恢复;RT-PCR显示模型组CX32 mRNA在4h开始下降,4d达低峰(0.33±0.11),8 d后逐渐恢复,16 d高于对照组,但无显著差异(P>0.05).Western blot结果显示模型组CX43蛋白表达在4 h上调(P>0.05)、4-16 d明显升高;RT-PCR显示模型组4h CX43 mRNA上调(P>0.05),4 d表达明显升高,12 d达高峰(5.46±0.58),16 d低于对照组,但无显著差异(P>0.05).结论:采用Solt-Farber方法成功建立了HOC增殖动物模型;大鼠2-AAF/PH肝脏CX表达呈时空特异性变化,使肝脏GJIC功能抑制.肝脏的GJIC抑制可能启动了HOC增殖.AIM： To study the expression of connexin 32 （CX32） and connexin 43 （CX43） and function of gap junction intercellular communication （GJIC）. in rat liver during 2-acetylaminofluorene/partial hepatectomy （2AAF/PH） for hepatic oval cell （HOC） proliferation; and explore the potential mechanism of HOC proliferation in vivo.METHODS： Male Wistar rats were randomized into normal control group （n = 6） and model group. Rats in model group were used to induce HOC proliferation： 9 days of treatment with 2-AAF, 20 mg/kg per day by gavage, interrupted on day 5 to perform a 70% hepatectomy （2-AAF/PH）. At the 4^th hour, 4^th, 8^th, 12^th and 16th day, 6 rats of model group were sacrificed respectively. The morphological changes of liver tissues were observed by pathological examination and the proliferation of HOC was counted using irnmunohistochemistry and morphological recognition. GJIC was confirmed by incision loading/dye transfer （IL/DT）, and the levels of CX32 protein and mRNA were detected by immunohistochemistry and reverse transcriptionpolymerase chain reaction （RT-PCR）, respectively. The expression of CX43 protein and mRNA were determined by immunohistochemistry, Western blot and RT-PCR, respectively.RESULTS： No HOC proliferation was seen in the rat liver of control and 4-hour model group. Pathologic examination revealed that HOC ap- peared at portal area in model group on day 4, increased to the peak on day 8, intensely proliferated from the portal spaces and invaded the liver parenchyma on day 12, and decreased on day 16 as compared with day 12. In comparison with that in control group, the distance of dye transfer in model groups （4 h, 4, 8, 12, 16 d） was significantly reduced （84.5 ± 3.4, 60.6 ± 3.3, 108.6 ± 4.2, 150.6 ± 2.6, 199.6 ± 3.7 μm vs 250.0 ± 5.0 μm, P 〈 0.01）. The signal number of CX32 in the rat liver of model groups began to decrease at the 4th hour, reached to the minimum （2.85 ± 0.39） on day 4, and recovered starting from day 8, and it

关 键 词：大鼠 肝卵圆细胞 缝隙连接蛋白 缝隙连接细胞间通讯 调控 免疫组化 逆转录-聚合酶链反应 免疫印迹 切开标记/染料示踪技术 

分 类 号：R575[医药卫生—消化系统]