【作者】 刘其雨；

【导师】 李立；

【作者基本信息】 昆明医科大学 ， 外科学， 2011， 博士

【摘要】 目的：通过不同灌注方法的比较,探讨原位肝移植中经下腔静脉逆行灌注对大鼠移植肝缺血再灌注损伤的影响。方法：成功建立54例大鼠肝移植模型,并随机分为三组,每组18例。第一组门静脉组,即经门静脉顺行灌注；第二组肝动脉+门静脉组,即同时开放肝动脉及门静脉顺行灌注；第三组下腔静脉组,即先吻合下腔静脉后开放逆行灌注,然后吻合门静脉及肝动脉。分别检测肝移植术后1h、6h及24h的血清谷草转氨酶、血清谷丙转氨酶、移植肝组织病理变化及肝细胞凋亡指数。结果：第三组逆行灌注组的血清转氨酶、血清谷丙转氨酶水平明显低于第一、二两组的顺行灌注组,其中第二组肝动脉+门静脉组酶学水平最高；移植肝的病理损伤情况第二组最重,第一组次之,第三组最轻；肝细胞凋亡指数第二组>第一组>第三组。结论：移植肝的缺血再灌注损伤与灌注方法有关；与传统的顺行灌注相比,经下腔静脉逆灌注可以减轻移植肝的缺血再灌注损伤。目的：通过观察不同组别中血清谷丙转氨酶、血清胆红素及肝组织排斥反应的程度,探讨CYP3AmRNA的表达在肝移植大鼠的他克莫司血药浓度中的意义,进而评估其在他克莫司免疫抑制中的作用。方法：建立SD-Wistar大鼠肝移植急性排斥反应模型72例,同时随机分为空白对照组、实验组、肝酶诱导组及肝酶抑制组四组,分别于肝移植术后1d、4d、7d三个时间点分批处死取材,抽血检测血清谷丙转氨酶、胆红素及他克莫司的血药浓度,光镜下观察肝组织病理学变化,并检测肝组织中CYP3AmRNA的表达情况。结果：空白对照组中18对大鼠成功建立16对急性排斥反应模型,成功率为88.9%；血清谷丙转氨酶及胆红素,空白对照组依次高于肝酶诱导组、实验组及肝酶抑制组,与光镜下肝组织的病变程度一致；他克莫司的血药浓度,肝酶抑制组最高,依次为实验组、肝酶诱导组,空白对照组最低；而肝组织中CYP3AmRNA的表达量,肝酶诱导组最高,其次为空白对照组和实验组,肝酶抑制组最低。结论：使用SD-Wistar大鼠的肝移植,可以成功稳定的建立大鼠肝移植的急性排斥反应模型；肝组织中CYP3AmRNA的表达量会影响他克莫司的血药浓度,进而影响其免疫抑制的作用；他克莫司既是CYP3A酶的底物,可能又是CYP3A酶的抑制剂。目的：1.总结肝移植术后免疫抑制剂他克莫司用量及其血药浓度的个体化差异的特点；2.研究肝移植人群中CYP3A5和ABCBl基因多态性分布的特点；3.探讨肝移植术后他克莫司用量及其血药浓度的个体化差异与CYP3A5和ABCBl基因多态性的关系。方法：1.统计我院67例肝移植病人的病历资料,监测肝移植患者术后不同时间的他克莫司的用量(D)、血药浓度(C)及校正后的C/D比值；2.利用DNA直接测序检测所有肝移植受体CYP3A5和ABCBl的基因型；3.67例肝移植病人,于移植后按照目标血药浓度一天分两次给予口服他克莫司,并根据血药浓度不断调整剂量,以达到目标血药浓度(7-10ng/m1),分为术后1周、2周及1月三组。结果：1.肝移植术后他克莫司的口服需药量在个体间存在很大差异,校正后的C/D值也存在很大差异；2.67例肝移植患者中,CYP3A5*1/*1基因型有15例、CYP3A5*1/*3基因型有23例、CYP3A5*3/*3基因型有29例,发生频率分别为22.4%、34.3%、43.3%。相比CYP3A5*1/*1基因型和CYP3A5*1/*3基因型两组,CYP3A5*3/*3组他克莫司的需药量最低,而校正后的C/D比值最高。3.67例肝移植患者中,ABCBl不同位点的基因多态性分布不同,其中仅ABCBl3435C>T基因多态性与他克莫司用量有关。ABCBl3435>T C/C基因型22例、C/T基因型40例、T/T基因型5例,发生频率分别为32.8%、59.7%、7.5%。结论：1.肝移植患者术后免疫抑制剂他克莫司的治疗用量存在很大的个体化差异；2.CYP3A5和ABCBl的基因多态性可能是肝移植患者术后他克莫司药代动力学显著个体差异的重要因素；3.CYP3A5非表达型(*3/*3)较表达型(*1/*1、*1/*3)基因型的患者需要更低剂量的他克莫司便可达到目标血药浓度水平,ABCBl3435C>T低表达组(C/T、T/T)较高表达组(C/C)需要更低剂量的他克莫司便可达到目标血药浓度水平；4.检测CYP3A5和ABCBl的基因多态性可以优化肝移植患者他克莫司个体化治疗的方案。更多还原

【Abstract】 Part IThe experimental study of liver graft injury by ischemia and reperfusion secondary to retrograde reperfusionAim:The aim of this study was to evaluate the influence of retrograde reperfusion via the vena cava on ischemic/reperfusion injury (I/R-injury) in a rat liver transplantation model, under comparing different kinds of reperfusion.Methods:Fifty four orthoptic rat liver transplantations were performed on male SD rats. Three groups (n=18) were formed. Group I:Antegrade reperfusion via the portal vein. Group II:Antegrade reperfusion, simultaneously, via the portal vein and the hepatic artery. Group III:Retrograde reperfusion via the vena cava. Serum parameters were determined one,6and24h after operation. Furthermore, the liver was taken for histological assessment. The early cell apoptosis index was examined also.Results:Rats of group III showed significantly lower aspartate amino transferase and alanine aminotransferase serum levels compared with group I and group II rats. Aspartate amino transferase and alanine aminotransferase serum level were significantly lower in group I than in group II. In histology, group III livers showed significantly less leukocytic infiltrate than group I and group II livers, however, group II livers showed the most serious. The apoptosis index in the group III livers were lower than in the group I and group II livers.Conclusions:Our data suggested that the expression of I/R-injury correlates with the type of reperfusion. Furthermore, this study was able to demonstrate that in a rat model, the retrograde reperfusion leads to a lower expression of I/R-injury than the antegrade reperfusion. Objectives:According to the serum alanine transarninase, serum total bilirubin and the characteristics of the acute rejection in hepatic tissue, to investigate the significance of CYP3A mRNA expression in rat’s blood concentration of tacrolimus following liver transplantation and immunosuppressive effect of tacrolimus.Methods:With Sprague-Dawley rats as donors and male Wistar rats as recipients, seventy two acute rejection models of orthotopic liver transplantation in the rat were established. Recipients were divided into4groups:blank control group, experimental group, enzyme inhibition group and enzyme induction group. Every six recipients were sacrificed respectively at day1,4,7after OLT to detect the changes of serum alanine transarninase, serum total bilirubin, tacrolimus whole-blood trough concentrations and the grafts histology. CYP3A mRNA levels in rat hepatic tissue were determined by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).Results:The success rate of acute rejection model of orthotopic liver transplantation in blank control group was88.9%(16/18). Rats of blank control group showed the highest levels of serum alanine transarninase, serum total bilirubin and the histological score of the acute rejection in hepatic tissue compared with other three groups, however, rats of enzyme inhibition group showed the lowest levels, enzyme induction group was higher than experimental group. The blood drug level of tacrolimus in rats of enzyme inhibition group was the highest compared with other three groups, however, blank control group showed the lowest, enzyme induction group was lower than experimental group. CYP3A mRNA levels in hepatic tissue of enzyme induction group showed the highest compared with other three groups, however, enzyme inhibition group showed the lowest, blank control group was higher than experimental group.Conclusions:Acute rejection experimental model of liver transplantation in rat could be stably established using SD rat as donor and Wistar rat as recipient. The expression of CYP3A mRNA levels in hepatic tissue may influence the tacrolimus whole-blood trough concentration and immunosuppressive effect of tacrolimus furthermore. Objectives:1. To summarize the clinical data in considerable interindividual differences of tacrolimus dosage and trough blood concentration during the early period after liver transplantation in patients.2. To evaluate the distribution characteristics of CYP3A5and ABCB1gene polymorphisms in Chinese liver transplantation recipients.3. To investigate the possible association of the ABCBl gene C3435T polymorphism and the CYP3A5gene A6986G polymorphism with tacrolimus trough concentration and dose requirements during the early period after liver transplantation in patients.Methods:1. The clinical data of sixty seven liver transplantation recipients were analyzed and tacrolimus whole-blood trough concentrations were measured by enzyme-linked immunospecific assay. Dose requirements and dose-adjusted trough concentrations (concentration/dose [C/D] ratios) were analyzed.2. CYP3A5and ABCBl genotyping were performed by direct DNA sequencing using the polymerase chain reaction restriction sites polymorphism-based procedure.3. Tacrolimus was administered twice daily at specified times post-transplant period according to the trough-targeting strategy. Sixty recipients with stable graft function were studied beyond1-month post transplantation. Whole blood samples were collected lw,2w and lm after both the morning and evening doses during hospitalization.Results1. Tacrolimus dose requirements and dose-adjusted trough concentrations (concentration/dose [C/D] ratios) after liver transplantation had great variation between individuals.2. The CYP3A5*1/*1was observed in15subjects (22.4%),23(34.3%) carried*l/*3, and29(43.3%) carried*3/*3. CYP3A5*3/*3variant was associated with significant lower tacrolimus dose at lw,2w and lm compared with patients who did not (CYP3A5*3/*3) after transplantation. The tacrolimus C/D ratios were obviously higher in recipients carrying CYP3A5*3/*3.3. A synonymous single nucleotide polymorphism (SNP) of ABCBl in various exon was different. Furthermore, ABCBl3435C>T polymorphisms was significantly correlated with tacrolimus dose-adjusted pre-dose concentrations at various time points. The ABCB13435C/C was observed in22subjects (32.8%), whereas40(59.7%) carried3435C/T and5(7.5%) carried3435T/T.Conclusions1. Tacrolimus dose requirement is characterized by a large interindividual variability requiring the use of therapeutic drug monitoring in daily clinical practice.2. The present study shows that genetic polymorphisms in CYP3A5and ABCB1may be responsible, in part, for the large interindividual variability of tacrolimus pharmacokinetics during the early period after liver transplantation in patients.3. Patients in CYP3A5non-expressors require a low dose of tacrolimus to reach target levels compared with expressors. Patients in ABCB1low-expressors require a low dose of tacrolimus to reach target levels compared with high-expressors.4. In the future, genotyping patients for CYP3A5and ABCB1polymorphisms may help to optimize the tacrolimus individualized immunosuppressive therapy in liver transplantation.更多还原