【作者】 吴田田；

【导师】 杨广顺；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 随着器官移植技术的发展、移植手术成功率的不断提高.移植物、移植受体术后长期生存及生存质量成为学界研究的焦点。抗排异是术后治疗的主要组成部分,其不良反应影响移植物、移植受体的长期生存,降低患者生活质量。近年来,为减少免疫抑制剂导致的不良反应,新型免疫抑制药物、新的抗排异方案以及给药剂量的合理调节备受关注。在诸多抗排异策略中,钙神经蛋白抑制剂（calcineurin inhibitor,CNI）曾处于基础地位,但可能导致肾功能不全,神经毒性等严重不良反应。1999年,美国FDA批准雷帕霉素作为肾移植术后抗排异用药。雷帕霉素是mTOR（mammaliantagert of rapamycin）特异性抑制剂,与细胞内受体FKBP12结合,形成FKBP—rapamycin复合物,该复合物与mTOR分子结合,抑制mTOR活性,阻断其下游信号p70S6k及4EBP1的活化,影响细胞G₁期至S期关键蛋白mRNA的转录,最终导致细胞G₁期停滞,抑制细胞增殖。进入临床后,大量资料显示雷帕霉素无明显肾毒性,这使得雷帕霉素常常与CNI类药物联合应用,甚至替代CNI成为免疫抑制方案的基础用药。最近的研究发现雷帕霉素具有抗肿瘤活性,降低移植术后新发恶性肿瘤患病风险;雷帕霉素还可以防治冠脉成型术后的再狭窄;在动物模型中,雷帕霉素明显减少肝硬化大鼠肝细胞外基质的沉积,改善肝功能,提高生存率;还有一些研究显示,雷帕霉素对蛋白尿性肾小球肾病,多囊肾病,顽固性葡萄膜炎等疾病有一定的治疗作用。然而,随着雷帕霉素在临床越来越广泛的应用,其副作用也逐渐引起人们的注意。除了可以引起血液学变化,如贫血,低白细胞血症,低血小板血症,高胆固醇血症;关节痛;肢体水肿;切口愈合不良等副作用外,研究发现,雷帕霉素能导致肺毒性等严重并发症。最近有资料显示雷帕霉素抑制边缘供肾内皮细胞的自身修复;抑制细胞增殖关键蛋白p70S6激酶的活化,影响受损肾小管上皮细胞的增生修复。这提示雷帕霉素抗增殖活性可能对组织自身修复造成影响。肝脏外科领域中,手术操作、肝脏移植、脏器保存、介入治疗等都可能造成肝组织损伤。作为代偿机制,肝细胞、胆管上皮细胞出现增生反应。此时应用雷帕霉素是否会抑制代偿性增生、影响肝组织自身修复,目前还不明确。本研究将探讨雷帕霉素对胆管缺血后代偿性增生的影响。第一部分雷帕霉素对胆管缺血后代偿性增生的影响目的观察离断大鼠胆管动脉血供后,肝内胆管代偿性增生反应,探讨雷帕霉素对于该反应是否存在抑制作用。方法雄性SD大鼠随机分配到:（1）缺血组（n=32）;（2）缺血+雷帕霉素组（n=32）;（3）假手术组（对照组,n=28）;（4）假手术+雷帕霉素处理组（n=28）。缺血组完全离断胆管动脉血供,假手术组仅给予开关腹手术,雷帕霉素处理组自手术当日始每日给予雷帕霉素2.0mg/kg灌胃,非雷帕霉素处理组予生理盐水2ml/kg灌胃。各实验组于术后第1、3、7天分别处死6只实验动物,术后14天处死全部动物。取肝脏组织行H-E及Ki-67免疫组化染色。高倍镜视野下,每只实验动物病理切片观察15～20个汇管区。H-E切片分别计数每个汇管区内小叶间胆管及汇管区周围小胆管数目,分析单个汇管区内小叶间胆管平均数目及单个汇管区周围小胆管平均数目。Ki-67染色切片分别计数每个汇管区内Ki-67阳性及阴性胆管细胞,计算阳性—阴性胆管细胞比值,分析单个汇管区内Ki-67阳性—阴性胆管细胞平均比值。统计学处理采用SPSS 13.0软件,结果以X±S_X表示,多个独立样本比较采用Kruskal-Wallis H检验,多个独立样本间的两两比较采用秩转换处理后行ONEWAY ANOVA检验,P＜0.05时认为存在统计学差异。结果H-E染色切片显示:对照组及假手术+雷帕霉素组术后未见明显胆管增生,缺血组胆管增生明显,既包括汇管区内小叶间胆管增生,也包括汇管区周围小胆管增生。汇管区内小叶间胆管计数结果显示:与对照组比较,缺血组术后汇管区小叶间胆管数目明显增加,术后2周内与时间变化呈现正相关趋势,雷帕霉素处理胆管缺血大鼠后,小叶间胆管数目增加幅度下降,术后7天、14天与缺血组间表现出显著差异（P分别为0.034及0.026）。汇管区周围小胆管计数结果显示:与对照组比较,缺血组术后汇管区周围小胆管数目增加;给予雷帕霉素后,胆管缺血大鼠汇管区周围小胆管数目增加幅度下降,术后1、7天与缺血组差异明显（P分别为0.042及0.009）。Ki-67染色结果显示:对照组及假手术+雷帕霉素组术后汇管区内未见明显Ki-67阳性染色胆管细胞,缺血组术后汇管区内Ki-67阳性胆管细胞明显增多,但肝细胞染色无明显增强。术后3天缺血组Ki-67阳性—阴性胆管细胞数目比值达到峰值,雷帕霉素处理后该峰值水平下降37.74%（P=0.002）。结论雷帕霉素抑制缺血后肝内胆管上皮细胞Ki-67抗原表达,明显抑制汇管区内小叶间胆管的代偿性增生反应;对于汇管区周围小胆管的增生,雷帕霉素也具有一定抑制作用。第二部分雷帕霉素对肝功能及生存率的影响目的探讨雷帕霉素对于实验大鼠胆管缺血后肝功能、体重及生存率变化的影响。方法雄性SD大鼠随机分配到缺血组、缺血+雷帕霉素组、对照组及假手术+雷帕霉素组。缺血组完全离断胆管动脉血供,假手术组仅给予开关腹手术,雷帕霉素处理组自手术当日始每日予雷帕霉素2.0mg/kg灌胃,非雷帕霉素处理组予生理盐水2ml/kg灌胃。术前、术后7天及14天测量动物体重,计算术后7天、14天相对术前体重变化。各实验组于术后第1、3、7天分别处死6只实验动物,术后14天处死全部动物,下腔静脉抽血2～3ml,检测血清TBIL、ALP、GGT及ALT。统计学处理采用SPSS13.0软件,结果以X±S_X表示,多个独立样本比较采用Kruskal-Wallis H检验,多个独立样本间的两两比较采用秩转换处理后行ONE WAYANOVA检验,P＜0.05时认为存在统计学差异。Kaplan—Meier分析生存率,组间生存差异采用log-rank检验,P＜0.05时认为存在统计学差异。结果与对照组比较,缺血组术后血清TBIL升高明显。雷帕霉素处理胆管缺血大鼠,术后7、14天血清TBIL水平显著高于缺血组（P=0.005及0.003）。术后1-7天,缺血组ALP明显升高,术后14天升高趋势缓和。缺血+雷帕霉素组术后血清ALP持续升高,至术后14天仍保持上升趋势,此时ALP水平明显高于缺血组（P=0.012）。根据体重测量结果,缺血组术后体重下降明显;给予雷帕霉素后,术后7、14天体重下降幅度增加（P＜0.01）。Kaplan—Meier生存分析结果显示:缺血组生存率较对照组无明显下降,但同时给予雷帕霉素后,生存率下降明显（P＜0.05）。结论雷帕霉素加重胆管缺血后肝内胆汁淤积,影响肝功能恢复,并对实验动物术后生存状态及生存率造成影响。第三部分雷帕霉素对VEGF mRNA、p70S6激酶及磷酸化p70S6激酶的影响目的探讨雷帕霉素对实验大鼠胆管缺血后肝内VEGF mRNA、p70S6激酶及磷酸化p70S6激酶含量的影响。方法雄性SD大鼠随机分配到缺血组、缺血+雷帕霉素组、对照组（假手术组）以及假手术+雷帕霉素组。缺血组完全离断胆管动脉血供,假手术组仅给予开关腹手术,雷帕霉素处理组自手术当日始每日予雷帕霉素2.0mg/kg灌胃,非雷帕霉素处理组予生理盐水2ml/kg灌胃。各实验组于术后第1、3、7天分别处死6只实验动物,术后14天处死全部动物,取肝脏标本于-80℃或10%中性甲醛中保存。免疫组化染色观察肝内VEGF表达情况,实时定量PCR检测VEGF mRNA水平,Western-blot检测p70S6k及其磷酸化物的含量。统计学处理采用SPSS13.0软件,结果以X±S_X表示,多个独立样本比较采用Kruskal-Wallis H检验,多个独立样本间的两两比较采用秩转换处理后行ONE WAYANOVA检验,P＜0.05时认为存在统计学差异。结果免疫组化染色结果显示:对照组及假手术+雷帕霉素组未见明显VEGF阳性染色;缺血组及缺血+雷帕霉素组汇管区内可见VEGF阳性染色,主要定位于血管内皮细胞。实时定量PCR结果显示:缺血组VEGF mRNA升高明显,术后3天达到峰值,为对照组7.57倍,术后1～14天,缺血组VEGF mRNA含量均明显高于对照组（P＜0.001）。用雷帕霉素处理胆管缺血大鼠,VEGF mRNA含量变化趋势虽与缺血组相似,但峰值水平仅为缺血组54%（P=0.005）;另外,术后1、7天缺血+雷帕霉素组VEGF mRNA含量明显低于缺血组（P＜0.05）。Western-blot结果显示,缺血组p70S6k表达上调,其峰值水平为对照组的4.43倍（P＜0.001）;同时,磷酸化p70S6k表达上调,峰值水平为对照组9.64倍（P＜0.01）。用雷帕霉素处理胆管缺血大鼠,术后p70S6k含量虽有增加,但是峰值水平仅为缺血组峰值50%（P=0.004）;术后1、7、14天,缺血+雷帕霉素组p70S6k含量明显低于缺血组（P＜0.005）。用雷帕霉素处理胆管缺血大鼠,术后磷酸化p70S6k含量无增高趋势;术后3、7天,缺血+雷帕霉素组磷酸化p70S6k含量明显低于缺血组（P＜0.005）。结论雷帕霉素抑制VEGF mRNA表达,下调p70S6激酶及其磷酸化物含量。这在一定程度上解释了雷帕霉素对胆管缺血后代偿性增生的抑制作用。更多还原

【Abstract】 As technical improvement in organ transplantation field,more and more researches concentrated on postoperative long-term survival and better quality of life.Side-effects of immunosuppressant,which is the most important element in post-operative therapy,may harm postoperative survival and life quality.In recent years,numerous researches have been carried out to explore new-type immunosuppressant,new anti-rejection strategies and optimal dosage adjusting according to blood concentration of drug.Calcineurin inhibitor （CNIs）,fundamental agent in a varity of anti-rejection strategies,increased incidence of renal dysfunction and neural disorder postoperatively.In 1999,rapamycin was permitted into immunosuppressant therapy after renal transplantation.As a mTOR specific inhibitor, rapamycin binds to its intracellular receptor FKBP12 to form FKBP—rapamycin compounds,which conjugate to FRB domain on mTOR moleculi,inactivate mTOR,block downstream signal activation of p70S6k and 4EBP1,hamper transcription of key mRNA answered for transformation from G₁ to S phase,and finally arrest cells in G₁ phase and inhibit cell proliferation.Since its initiation of clinical use,rapamycin caused less burden to renal function in contrast to CNIs.In combination with CNIs,or even in replacement of CNIs,rapamycin gradually occupied the fundamental place in postoperative anti-rejection. Recently,it has been reported that rapamycin had anti-tumor activity and lowered malignancy incidence after transplantation.What’s more,rapamycin prevented coronary arteries from re-stenosis after coronary arterioplasty.In a animal model,rapamycin reduced extracellular matrix deposit,improved liver function and increased survival.Some clinical data indicated that rapamycin contributed to treatment of proteinuric glomerulopathy,polycystic renal pathy and refractory uveitis.However,as increasing clinical use of rapamycin,its side-effects attracted more and more concern.Besides arthralgia,extremity edema,insufficient wound healing and hematologic changes,such as anemia,hypercholestemia,thrombocytopenia and leukocytopenia,rapamycin caused pulmonary toxity and angioedema according to updated data.Further more,some papers held that rapamycin harmed self-repair of marginal renal donor,inhibit activation of p70S6k and put obstacle to repair of injured renal tubuli.It seemed that anti-proliferative nature of rapamycin could cause negative effect on tissue repair.In field of liver surgery,operation,transplantation,organ reservation and intervention may cause injury to liver tissue.As a compensatory mechanism,hepatocytes and cholangiocytes proliferate.Whether rapamycin inhibits proliferation,hampers self-repair of liver tissue remains unclear.In the present,paper,effect of rapamycin on adaptive bile duct proliferation in response to ischemia will be explored.PartⅠEffect of Rapamycin on Adaptive Bile Duct Proliferation in Response to IschemiaAimObserve intra-hepatic adaptive bile duct proliferation after complete deprivation of bile duct arterial supply.Explore the effect of rapamycin on the proliferation of bile ducts.MethodMale SD rates were randomly assigned into 4 groups:ischemia（n=32）, ischemia+rapamycin（n=32）,sham（n=28） and sham+rapamycin（n=28）.In ischemia groups,complete deprivation of bile duct arterial supply was performed while in sham groups,open-close operation was carried out.Daily intake of rapamycin（2mg/kg） was given in rapamycin groups and the same volume of saline was given in non-rapamycin groups.On day 1,3,7 and 14,fresh live tissue were obtained,followed by H-E and Ki-67 immunohistochemical staining.In high magnification fields,15～20 portal areas were examined per animal.In H-E sections,interlobular bile duct within portal areas and ductuli at periphery of portal areas were counted separately and average number of bile ducts per portal area were calculated.In Ki-67 sections,Ki-67 positive and negative bile duct cells were counted and ratio of Ki-67 positive to negative bile duct cells per portal area were calculated.Statistic analysis was performed by SPSS 13.0 software,results showed as X±S_X. Kruskal-Wallis H test was used for comparison of multiple independent samples and the following pair-wise comparisons were performed by rank transformation and ONE WAY ANOVA.Statistic difference was considered when P＜0.05.ResultsFor H-E sections,there were no obvious bile duct proliferation in sham and sham+rapa group,while bile duct proliferation were observed obviously in ischemia group, including proliferation of interlobular bile duct within portal areas and ductuli at periphery of portal areas.Compared with sham,a sharp increase of interlobular bile duct number was found within 2 weeks postoperatively.When rapamycin was administrated,the increase shrunk and obvious differences appeared on day 7 and 14 in contrast to ischemia group （P=-0.034 and 0.026,respectively）.Compared with sham,number of peri-portal ductuli increased in ischemia group.When rapamycin was given,the increase also shrunk and resulted statistic differences on day1 and 7（P=0.042 and 0.009,respectively）.Ki-67 stained sections showed there was no obvious positive staining within portal area in sham group,while a large number of Ki-67 positive bile duct cells were found in ischemia group. On postoperative day 3,ratio of Ki-67 positive to negative bile duct cells reached peak level,which descended by 37.74%（P=0.002） due to rapamycin administration.ConclusionRapamycin inhibits expression of Ki-67 antigen in bile duct epithelium,inhibits interlobular bile duct proliferation within portal areas and as for peri-portal ductuli, rapamycin may also put obstacle to their proliferation.PartⅡEffect of Rapamycin on Liver Function and SurvivalAimExplore the effect of rapamycin on liver function,body weight and survival of experimental rats.MethodMale SD rates were randomly assigned into 4 groups as previously described.In ischemia groups,complete deprivation of bile duct arterial supply was performed while in sham groups,open-close operation was carried out.Daily intake of rapamycin（2mg/kg） was given in rapamycin groups and the same volume of saline was given in non-rapamycin groups.Body weight was measured before operation,postoperative day 7 and postoperative day 14.On day 1,3,7 and 14,blood sample（2～3ml） were obtained, followed by liver function examination including TBIL,ALP,GGT and ALT.Statistic analysis was performed by SPSS 13.0 software,results showed as X±S_X. Kruskal-Wallis H test was used for comparion of multiple independent samples and the following pairwise comparisons were performed by rank transformation and ONE WAY ANOVA.Kaplan—Meier survival analysis was performed followed by log-rank test. Statistic difference was considered when P＜0.05.ResultsSerum TBIL in ischemia group ascended sharply postoperatively.When rapamycin administrated,TBIL of bile duct ischemic rats reached higher levels on day 7（P=0.005） and day 14（P=0.003） compared with ischemia group.Serum ALP of ischemia group increased from day 1 to day 7 postoperatively and on day 14,increasing tendency ceased. In ischemia+rapamycin group,serum ALP ascended continuously,without any remission even on day 14,when ALP was higher than ischemia group（P=0.012）.Based on measurement of body weight,in ischemia group,experimental rats experienced weight loss after operation.While in ischemia+rapamycin group,animals suffered more weight loss than in ischemia group（P＜0.01）.Kaplan—Meier survival analysis showed compared with sham,there was no statistical decrease of cumulative survival in ischemia group,while decrease in ischemia+rapamycin group was significant（P＜0.05）.ConclusionAfter ischemia injury,liver experienced cholestasis.Rapamycin exaggerated intra-hepatic cholestasis,deteriorated liver function and affected negatively on postoperative nutrition and survivalPartⅢEffect of Rapamycin on VEGF mRNA,p70S6k and phosphorylated p70S6kAimExplore the effect of rapamycin on expression of VEGF mRNA and content of p70S6k and phosphorylated p70S6k in liver tissue of experimental rats.Method Male SD rates were randomly assigned into 4 groups as previously described.In ischemia groups,complete deprivation of bile duct arterial supply was performed,while in sham groups,open-close operation was carried out.Daily intake of rapamycin（2mg/kg） was given in rapamycin groups and the same volume of saline was given in non-rapamycin groups.On day 1,3,7 and 14,fresh liver tissue were obtained,followed by VEGF immunohistochemical staining,real-time PCR（dye method） and Western-blot to detect content of VEGF mRNA,p70S6k and phosphorylated p70S6k.Statistic analysis was performed by SPSS 13.0 software,results showed as X±S_X. Kruskal-Wallis H test was used for comparion of multiple independent samples and the following pairwise comparisons were performed by rank transformation and ONE WAY ANOVA.Statistic difference was considered when P＜0.05.ResultsIn ischemia group,obvious VEGF positive staining was found within potal areas, which mainly located at bile duct epithelium and vessel endothelium.While in sham or sham+rapamycin groups,less obvious positive staining was found.Right after surgey, VEGF mRNA increased sharply in ischemia group and reached peak level on day 3 postoperatively,which was 7.57 folds to sham（P＜0.001）.On day1,3,7,and 14,content of VEGF mRNA in ischemia group was signifantly higher than in sham（P＜0.001）.During rapamycin administration,VEGF mRNA of ischemia+rapamycin rats fluctuated in a similar manner to ischemia group,with a peak level on day 3,which was only 54%of that in ischemia group（P=0.005）.On day1 and day 7,VEGF mRNA was significantly lower in ischemia+rapamycin group than in ischemia group（P＜0.05）.According to Western-blot, p70S6k was up-regulated in ischemia group,with the peak level 4.43 folds to sham （P＜0.001）.Simultaneously phosphorylated p70S6k was up-regulated,with a peak level 9.64 folds to sham（P＜0.01）.Giving rapamycin to bile duct ischemia rats,though there was a increase of p70S6k content,the peak was only 50%of that in ischemia group （P=0.004）.On day1,7 and 14,p70S6k was lower in ischemia+rapamycin group than in ischemia group（P＜0.005）.In addition,there was no obvious increase of p70S6k when bile duct ischemia rats were administrated with rapamycin.On day 3 and 7,obvious difference was found between ischemia+rapamycin and ischemia group（P＜0.005）.Conclusion Rapamycin inhibits VEGF expression at transcriptional level,down-regulated p70S6k and its phophorylated formation.These results provided explanation to negative effect of rapamycin on adaptive bile duct proliferation in response to ischemia.更多还原