中华器官移植杂志
中華器官移植雜誌
중화기관이식잡지
CHINESE JOURNAL OF ORGAN TRANSPLANTATION
2014年
9期
555-560
,共6页
陈莉萍%王雅文%肖漓%樊文梅%石炳毅
陳莉萍%王雅文%肖巑%樊文梅%石炳毅
진리평%왕아문%초리%번문매%석병의
大鼠%肝移植%胆管纤维化%5-羟色胺
大鼠%肝移植%膽管纖維化%5-羥色胺
대서%간이식%담관섬유화%5-간색알
Rats%Liver transplantation%Biliary fibrosis%5-hydroxytryptamine
目的 探讨5-羟色胺受体2A亚型(5-HT2A)在大鼠肝移植术后胆管纤维化中的作用及意义.方法 建立再动脉化大鼠原位肝移植模型,分以下4组.对照Ⅰ组和对照Ⅱ组,供肝分别冷保存1和12 h后行原位肝移植术;实验组:在对照Ⅱ组基础上,于术后24 h起腹腔注射选择性5-HT2A拮抗剂Ketanserin 5 mg· kg-1·d-1,直至术后4周观察期结束;假手术组(大鼠仅行开腹和关腹操作,不行肝移植).术后1d、3d、1周、2周和4周,检测胆道酶系血清碱性磷酸酶、谷氨酰转肽酶、胆红素总量含量以评价胆道功能,采用酶联免疫吸附试验检测肝组织5-HT的含量,以增殖细胞核抗原免疫组织化学染色评价胆管上皮细胞(BEC)的增殖情况.术后4周采用Masson三色染色法观察胆管周围胶原纤维的分布;以α-平滑肌肌动蛋白(α-SMA)作为门管区成纤维细胞(PF)转化为肌纤维母细胞(MF)的标志,进行免疫组织化学染色.术后1、2和4周,采用实时荧光定量聚合酶链反应法检测肝组织内基质金属蛋白酶2(MMP2)和前胶原α1 mRNA的表达.结果 与假手术组相比,对照Ⅰ组仅术后1d胆道酶系及5-HT含量升高,术后3d少量BEC增殖,各时点肝组织MMP2和前胶原α1 mRNA的表达无显著增加;术后4周门管区未见或仅见少量胶原纤维及MF.与此相反,对照Ⅱ组和实验组术后1d胆道酶系显著升高,随后下降,术后2周时接近假手术组水平,但术后4周又显著升高,且对照Ⅱ组显著高于实验组,差异有统计学意义(P＜0.05);两组术后各时点5-HT的含量均显著高于对照Ⅰ组,峰值出现于术后1d,实验组含量有低于对照Ⅱ组的变化趋势,并在术后4周时差异有统计学意义(P＜0.05).对照Ⅱ组术后3d直至观察期结束可见BEC活跃增殖;术后2周和4周肝组织MMP2和前胶原α1 mRNA的表达显著增加;术后4周门管区可见大量胶原纤维和MF.实验组BEC增殖低于对照Ⅱ组,至术后2周和4周时差异有统计学意义(P＜0.05);术后2周和4周时肝组织MMP2和前胶原α1 mRNA的表达显著低于对照Ⅱ组,术后4周门管区胶原纤维和MF较对照Ⅱ组明显减少(P＜0.05).结论 选择性5-HT2A拮抗剂Ketanserin可延缓冷缺血时间延长引起的大鼠移植肝胆管纤维化,5-HT2A可能是潜在的肝移植后缺血性胆道疾病的干预靶点.
目的 探討5-羥色胺受體2A亞型(5-HT2A)在大鼠肝移植術後膽管纖維化中的作用及意義.方法 建立再動脈化大鼠原位肝移植模型,分以下4組.對照Ⅰ組和對照Ⅱ組,供肝分彆冷保存1和12 h後行原位肝移植術;實驗組:在對照Ⅱ組基礎上,于術後24 h起腹腔註射選擇性5-HT2A拮抗劑Ketanserin 5 mg· kg-1·d-1,直至術後4週觀察期結束;假手術組(大鼠僅行開腹和關腹操作,不行肝移植).術後1d、3d、1週、2週和4週,檢測膽道酶繫血清堿性燐痠酶、穀氨酰轉肽酶、膽紅素總量含量以評價膽道功能,採用酶聯免疫吸附試驗檢測肝組織5-HT的含量,以增殖細胞覈抗原免疫組織化學染色評價膽管上皮細胞(BEC)的增殖情況.術後4週採用Masson三色染色法觀察膽管週圍膠原纖維的分佈;以α-平滑肌肌動蛋白(α-SMA)作為門管區成纖維細胞(PF)轉化為肌纖維母細胞(MF)的標誌,進行免疫組織化學染色.術後1、2和4週,採用實時熒光定量聚閤酶鏈反應法檢測肝組織內基質金屬蛋白酶2(MMP2)和前膠原α1 mRNA的錶達.結果 與假手術組相比,對照Ⅰ組僅術後1d膽道酶繫及5-HT含量升高,術後3d少量BEC增殖,各時點肝組織MMP2和前膠原α1 mRNA的錶達無顯著增加;術後4週門管區未見或僅見少量膠原纖維及MF.與此相反,對照Ⅱ組和實驗組術後1d膽道酶繫顯著升高,隨後下降,術後2週時接近假手術組水平,但術後4週又顯著升高,且對照Ⅱ組顯著高于實驗組,差異有統計學意義(P＜0.05);兩組術後各時點5-HT的含量均顯著高于對照Ⅰ組,峰值齣現于術後1d,實驗組含量有低于對照Ⅱ組的變化趨勢,併在術後4週時差異有統計學意義(P＜0.05).對照Ⅱ組術後3d直至觀察期結束可見BEC活躍增殖;術後2週和4週肝組織MMP2和前膠原α1 mRNA的錶達顯著增加;術後4週門管區可見大量膠原纖維和MF.實驗組BEC增殖低于對照Ⅱ組,至術後2週和4週時差異有統計學意義(P＜0.05);術後2週和4週時肝組織MMP2和前膠原α1 mRNA的錶達顯著低于對照Ⅱ組,術後4週門管區膠原纖維和MF較對照Ⅱ組明顯減少(P＜0.05).結論 選擇性5-HT2A拮抗劑Ketanserin可延緩冷缺血時間延長引起的大鼠移植肝膽管纖維化,5-HT2A可能是潛在的肝移植後缺血性膽道疾病的榦預靶點.
목적 탐토5-간색알수체2A아형(5-HT2A)재대서간이식술후담관섬유화중적작용급의의.방법 건립재동맥화대서원위간이식모형,분이하4조.대조Ⅰ조화대조Ⅱ조,공간분별랭보존1화12 h후행원위간이식술;실험조:재대조Ⅱ조기출상,우술후24 h기복강주사선택성5-HT2A길항제Ketanserin 5 mg· kg-1·d-1,직지술후4주관찰기결속;가수술조(대서부행개복화관복조작,불행간이식).술후1d、3d、1주、2주화4주,검측담도매계혈청감성린산매、곡안선전태매、담홍소총량함량이평개담도공능,채용매련면역흡부시험검측간조직5-HT적함량,이증식세포핵항원면역조직화학염색평개담관상피세포(BEC)적증식정황.술후4주채용Masson삼색염색법관찰담관주위효원섬유적분포;이α-평활기기동단백(α-SMA)작위문관구성섬유세포(PF)전화위기섬유모세포(MF)적표지,진행면역조직화학염색.술후1、2화4주,채용실시형광정량취합매련반응법검측간조직내기질금속단백매2(MMP2)화전효원α1 mRNA적표체.결과 여가수술조상비,대조Ⅰ조부술후1d담도매계급5-HT함량승고,술후3d소량BEC증식,각시점간조직MMP2화전효원α1 mRNA적표체무현저증가;술후4주문관구미견혹부견소량효원섬유급MF.여차상반,대조Ⅱ조화실험조술후1d담도매계현저승고,수후하강,술후2주시접근가수술조수평,단술후4주우현저승고,차대조Ⅱ조현저고우실험조,차이유통계학의의(P＜0.05);량조술후각시점5-HT적함량균현저고우대조Ⅰ조,봉치출현우술후1d,실험조함량유저우대조Ⅱ조적변화추세,병재술후4주시차이유통계학의의(P＜0.05).대조Ⅱ조술후3d직지관찰기결속가견BEC활약증식;술후2주화4주간조직MMP2화전효원α1 mRNA적표체현저증가;술후4주문관구가견대량효원섬유화MF.실험조BEC증식저우대조Ⅱ조,지술후2주화4주시차이유통계학의의(P＜0.05);술후2주화4주시간조직MMP2화전효원α1 mRNA적표체현저저우대조Ⅱ조,술후4주문관구효원섬유화MF교대조Ⅱ조명현감소(P＜0.05).결론 선택성5-HT2A길항제Ketanserin가연완랭결혈시간연장인기적대서이식간담관섬유화,5-HT2A가능시잠재적간이식후결혈성담도질병적간예파점.
Objective To evaluate the role of 5-hydroxy trptarine 2A receptor (5-HT2A) in the pathogenesis of biliary fibrosis after liver transplantation in rats.Method Rats were randomly divided into control group Ⅰ and control group Ⅱ[(supplied livers were preserved for 1 or 12 h),ketanserin group (recipients of control group Ⅱ were intraperitoneally injected with ketanserin 24 h postoperatively at the dosage of 5 mg · kg-1 · day-1),and sham group (rats were subjected to transverse laparotomy and closure without manipulation of the liver).During 4-week observation period,serum biliary enzymes,5-HT content in the liver,the expression of fibrosis-related genes,cholangiocytes proliferation and biliary fibrosis were evaluated.Result Compared with the sham group,the serum ALP,GGT,TBil and 5-HT contents in the liver homogenate were increased on the postoperative day 1 (POD1) and then restored to the normal level.There was slight proliferation of bile duct epithelial cells on POD3 in the control group Ⅰ,with fewer collagen fibers and α-sooth muscle actin (α-SMA)-positive myofibroblasts in the portal area.The expression of matrix metalloproteinase-2 (MMP-2) and procollagen α1-mRNA in graft livers was not significantly increased in the control group Ⅰ.To the contrast,the control group Ⅱ demonstrated high levels of serotonin in the liver homogenate and enhanced serum biliary enzymes.Active cholangiocytes proliferation was triggered on POD3 and remained higher than in the control group Ⅰ and the sham group.The control group Ⅱ showed a large number of α-SMA-positive myofibroblasts and collegan fibers at the postoperative week 4.In parallel,the major profibrogenic transcripts MMP2 and procollagen α1 were significantly increased at 2nd,and 4th week postoperation in the control group Ⅱ.Importantly,we also found that ketanserin relieved the signs of biliary fibrosis at 4th week postoperation in 5-HT2A group by the demonstration of reduced collagen fibers and a-SMA-positive myofibroblast in the portal area,as well as the decrease in the fibrosis-related gene expression.In addition to the lower cholangiocytes proliferation,serum levels of biliary enzymes including GGT,ALP and TBil in 5-HT2A group were significantly decreased at 4th week postoperation as compared with the control group Ⅱ.Conclusion Selective 5-HT2A receptor antagonist,Ketanserin retards biliary fibrosis progression posttransplantation,suggesting that 5-HT2A receptor is a potential therapeutic target for ischemia-related biliary fibrosis after DCD liver transplantation.
