重组生长激素对肝移植术后早期胆汁淤积和肝脏功能影响的实验研究
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摘要
第一部分:大鼠原位肝移植(OLT)模型的建立
目的
探讨大鼠原位肝移植(OLT)模型的建立方法,为肝移植的相关研究提供稳定的模型基础。
方法
通过改良的Kamada“二袖套法”,对Wistar大鼠实施了同种原位肝移植手术,并观察大鼠术后存活率和并发症的发生情况。
结果
总共实行大鼠原位肝脏移植近220例,在40例定型手术中,供体手术时间(22.3±7)min,全肝移植组供肝修整时间(15.6±4)min,受体手术时间(50.6±11.2)min,无肝期为(20.6±2.5)min,手术成功率为100%,一周存活率为92.5%。成功地建立了稳定的大鼠肝移植模型。
结论
大鼠原位肝移植模型的建立,需要一定的显微外科基础和熟悉大鼠的腹部解剖结构的前提下,经过一段时间的专门训练,方能获得成功。其中娴熟的显微外科技术和耐心细致地手术操作是大鼠原位肝移植成功的关键。
第二部分重组生长激素对大鼠肝移植术后早期胆汁淤积和肝脏功能的影响
目的
探讨重组生长激素(rhGH)对肝移植术后早期胆汁淤积和肝功能恢复的影响。
材料与方法
采用Kamada的双袖套法并加以改进,建立Wistar大鼠原位肝移植模型。按随机原则将大鼠分为三组:假手术组(SO组)、肝移植组(OLT组)、肝移植术后生长激素干预组(OLT+rhGH组)。每组按术后1d、3d、7d三个时相点再分成三个亚组,每一时相点6只大鼠,于预定时相点处死分别留取血清及肝脏组织标本,测定血清ALP、GGT、ALT、TBIL水平评估肝功能;酶联免疫法检测血清TNFα、IL-1β水平;放射免疫法测定血清生长激素(GH)水平;放射配体结合分析法分析移植肝生长激素受体(GHR)和胰岛素样生长因子-1受体(IGF-1R)表达;高效液相色谱法(HPLC)检测肝组织ATP含量;用化学比色法测量肝脏中Na~+/K~+ATP酶等的浓度变化;免疫荧光染色半定量分析Mrp2的定位;RT-PCR法检测肝组织Mrp2mRNA表达;并将Mrp2mRNA与血清TBIL、TNFα作相关分析;常规病理结合电镜分别观察肝脏组织学及超微结构的改变。
结果
1.OLT+rhGH组血清ALT、ALP、TBIL、GGT、TNFα低于OLT组,差异均具有显著性意义(P＜0.05)。OLT+rhGH组血清IL-1β与OLT组比较无差别。
2.在肝移植术后1d组和3d组,ATP含量和Na~+/K~+ATPase活性均有明显下降,至术后7d组各指标和SO组比较无差别。GH处理后各指标水平均较OLT组明显上升,差异具有显著性意义(P＜0.05)。
3.OLT+rhGH组GH水平在术后1天,和SO、OLT组差别无统计学意义,而在术后3、7d组明显高于OLT组同时相点水平(P＜0.05),和SO组比较差别无统计学意义。大鼠肝移植术后GH-IGF-1轴发生改变,术后7d内GHR和IGF-1R表达呈下降趋势。rhGH治疗组术后7d GHR数恢复明显,术后3、7d IGF-1R数恢复明显(P＜0.05)。
4.Mrp2转运蛋白特异性定位于肝细胞膜的毛细胆管面,荧光染色呈线条形勾勒出毛细胆管轮廓,给予rhGH的肝移植大鼠肝脏Mrp2表达水平明显增高,恢复加快。Mrp2的RT-PCR结果表明:其表达于术后3d达高峰。OLT术后Mrp2表达亦明显下调,给予rhGH的肝移植大鼠肝脏Mrp2RNA表达明显上调,水平明显增高,恢复加快。肝组织Mrp2基因的表达与血清TBIL、TNFα存在密切的负相关(P＜0.05)。
5.肝组织常规HE染色和电镜检查均提示:重组生长激素干预的肝移植组的肝细胞损伤改变较未干预组轻。
结论
1.大鼠肝移植术后早期存在胆汁淤积。肝细胞功能恢复早于其形态学恢复。肝移植术后应用外源性GH对大鼠肝脏热缺血再灌注损伤具有保护作用可促进热缺血损伤移植肝功能恢复。
2.肝移植术后肝组织的能量代谢有显著改变。在术后肝组织ATP含量和肝细胞胞质膜Na~+/k~+ATP酶活性均显著降低,这表明此时胆汁淤积的发生有泵功能显著不足的因素参与作用。肝组织的泵功能不足是导致大鼠肝移植术后并发胆汁淤积的因素之一。
3.大鼠原位肝移植后产生一过性GH抵抗,存在GH相对不足;热缺血损伤移植肝GH-IGF-1轴的改变与热缺血损伤移植肝恢复延迟有关;肝移植术后应用外源性GH可促进移植肝GH-IGF-1轴的恢复,并促进肝功能的恢复。
4.Mrp2表达的减少以及定位的异常是鼠肝移植术后胆汁淤积发生的主要分子机制之一,rhGH促进胆红素的转运的作用机制可能是通过直接上调Mrp2表达来实现;也可能通过限制TNFα的过度释放间接上调Mrp2来促进胆红素的转运。
PARTⅠESTABLISHMENT OF THE RAT LIVER TRANSPLANTATION MODEL
Objective
To study the operation technique of rat's orthotopic liver transplantation(OLT) and to provide a reliable model for the study of OLT.
Methods
The models of the Wistar rat were established with modified Kamada's two cuff technique.We monitored the survival rate complications of operated rat.
Results
About 220 cases of rat orthotopic and liver transplantation operations were performed in total.In 40 cases of formal operations,the operation time of donor was(22.3±7)min,cuff preparation time of the whole graft group was(15.6±4)min,the operation time of recipient was(50.6±11.2)min,anhepatic phase was(20.6±2.5)min.The successful rate and 1 week survival rate were 100%and 92.5%respectively.We successfully establish a stable rat liver transplantation model.
Conclusions
If you want to establish a stable rat liver transplantation model,you must master enough microsurgery skills and an intimate knowledge of abdomen anatomy of rat. Moreover,you have to repeat training in OLT for a phase.The sophisticated microsurgical technique and the delicate surgical manipulation is the key to successfully perform rat orthotopic liver transplantation.
PARTⅡEFFECT AND MECHANISM OF rhGH ON CHOLESTASIS AND LIVER FUNCTION AT EARLY STAGE AFTER TRANSPLANTATION
Objective
Our experiments were designed to study the influence and regulatory mechanisms of rhGH on cholestasis and liver function after liver transplantation in rats.
Materials and Methods
Orthotopic liver transplantation(OLT) was performed in male Wistar rats using the cuff technique as described by Kamada with modifications.Rats were divided into three groups randomly:SO group(n=18,sham operation group),OLT group(n=18,liver transplantation control group),OLT+rhGH group(n=18,rhGH administrated group in liver transplantation),Each group was further divided into 3 subgroups respectively according to different stage after operation.Three time points were predetermined as 1d,3d,7d postoperation.Six animals were used per time-point.Rats were sacrificed at each time-point and the liver specimens and blood samples were collected.The liver function was evaluated by serum ALP,GGT,ALT,TBIL levels.Levels of TNFα,IL-1βwere measured by ELISA.Serum GH by radioimmunoassay.The GH receptor and IGF-1 receptor binding in grafts were analyzed by radioligend binding assay(RBA).Levels of ATP in hepatic tissues were examined by high performance liquid chromatography(HPLC).The change of Na~+/K~+ATPase was measured by chromatometry.Finally,Mrp2 of liver tissue was determined by immunofluorescent microscopy and RT-PCR.Meanwhile,expression of Mrp2mRNA correlatively analyzed with TNFαand TBIL in the serum.Morphometry was observed under light microscope and TEM.
Results
1.Biochemical data showed that the levels of serum ALP,TBIL,GGT,ALT and TNFαwere significantly lower in OLT+rhGH group than that in OLT group(P＜0.05), while the level of IL-1βbetween the two groups has no significant difference.
2.At 1,3 days after OLT,the levels of ATP and the activity of Na~+/K~+ ATPase were significantly lower in OLT group than that in SO group(P＜0.05).Treatment with GH,the levels of ATP and the activity of Na~+/K~+ ATPase significantly increased.
3.At 1d after operation,the level of serum GH in group OLT was not different compared with that in group OLT+rhGH and SO.while At 3,7 days after OLT,the level of serum GH was significantly lower in OLT group than that in SO and OLT+rhGH group(P＜0.05).In OLT group,the liver grafts showed a significant decrease in GH receptor and IGF-1 receptor number on postoperative day 7(P＜0.05).After the rhGH treatment, GH receptor markedly recovered on postoperative day 7 while IGF-1R did on postoperative day 3 and 7(P＜0.05).
4.At 1d after operation,the expression of Mrp2 in OLT group was so weak that could hardly be detected under fluorescence microscope,while obvious fluorescence could be detected from the seventh day postoperatively,showing significant discrepancies when compared with SO group.As duration elongated,both the intensity and range of specific fluorescence grew,moderate level of fluorescence was maintained.Compared with OLT rats at the same timepoints,the expression of Mrp2 in rhGH-treated rats was much stronger.Expression of Mrp2mRNA correlated negatively with TBIL and TNFα..
5.The histological changes were more severe in OLT group than that in OLT+rhGH group.
Conclusions
1.Cholestasis was evident after liver transplantation at early phase.Restoration of the function was faster than restoration of morphology.Exogenous GH treatment can protect ischemic/reperfusion injury in rat liver,and facilitate recovery of function of liver graft.
2.The level of ATP and Na~+/K~+ ATPase in early time of cryopreservationreperfusion in donor liver are related to the graft function.The changes of Na~+/K~+ ATPase in graft indicated that liver transplantation might lead to energy metabolism disturbance of the membrane of liver cell,which might be responsible for the metabolism disorder of cholestasis.
3.Changes of GH-IGF-1 axis appeared,expression of GHR and IGF-1R trended to descend within the first 7 days postoperatively.Injury from warm ischemia caused delayed recovery of GH-IGF-1 axis,and transient GH resistance was present.Both GHR and IGF-1R decreased and resulted in a relative deficiency of GH.The delayed recovery of function of liver graft was implicated to the changes of GH-IGF-1 axis.After OLT, exogenous GH treatment facilitated recovery of GH-IGF-1 axis,and recovery of function of liver graft was promoted by postoperative rhGH treatment.
4.The level of Mrp2 in the liver tissue was upregular by rhGH and was significantly.Liver transplantation might lead to down-regulation and abnormal localization of Mrp2 in liver cell,which might be responsible for the cholestasis after rat liver transplantation,rhGH can protect the liver from the injury of intrahepatic cholestasis.The promotion of bilirubin transport by rhGH can upregulate the expression of Mrp2 directly.On the other hand,The application of rhGH might limit exaggerated hepatic production of TNFαto upregulate the expression of Mrp2.
目的
探讨大鼠原位肝移植(OLT)模型的建立方法,为肝移植的相关研究提供稳定的模型基础。
方法
通过改良的Kamada“二袖套法”,对Wistar大鼠实施了同种原位肝移植手术,并观察大鼠术后存活率和并发症的发生情况。
结果
总共实行大鼠原位肝脏移植近220例,在40例定型手术中,供体手术时间(22.3±7)min,全肝移植组供肝修整时间(15.6±4)min,受体手术时间(50.6±11.2)min,无肝期为(20.6±2.5)min,手术成功率为100%,一周存活率为92.5%。成功地建立了稳定的大鼠肝移植模型。
结论
大鼠原位肝移植模型的建立,需要一定的显微外科基础和熟悉大鼠的腹部解剖结构的前提下,经过一段时间的专门训练,方能获得成功。其中娴熟的显微外科技术和耐心细致地手术操作是大鼠原位肝移植成功的关键。
第二部分重组生长激素对大鼠肝移植术后早期胆汁淤积和肝脏功能的影响
目的
探讨重组生长激素(rhGH)对肝移植术后早期胆汁淤积和肝功能恢复的影响。
材料与方法
采用Kamada的双袖套法并加以改进,建立Wistar大鼠原位肝移植模型。按随机原则将大鼠分为三组:假手术组(SO组)、肝移植组(OLT组)、肝移植术后生长激素干预组(OLT+rhGH组)。每组按术后1d、3d、7d三个时相点再分成三个亚组,每一时相点6只大鼠,于预定时相点处死分别留取血清及肝脏组织标本,测定血清ALP、GGT、ALT、TBIL水平评估肝功能;酶联免疫法检测血清TNFα、IL-1β水平;放射免疫法测定血清生长激素(GH)水平;放射配体结合分析法分析移植肝生长激素受体(GHR)和胰岛素样生长因子-1受体(IGF-1R)表达;高效液相色谱法(HPLC)检测肝组织ATP含量;用化学比色法测量肝脏中Na~+/K~+ATP酶等的浓度变化;免疫荧光染色半定量分析Mrp2的定位;RT-PCR法检测肝组织Mrp2mRNA表达;并将Mrp2mRNA与血清TBIL、TNFα作相关分析;常规病理结合电镜分别观察肝脏组织学及超微结构的改变。
结果
1.OLT+rhGH组血清ALT、ALP、TBIL、GGT、TNFα低于OLT组,差异均具有显著性意义(P＜0.05)。OLT+rhGH组血清IL-1β与OLT组比较无差别。
2.在肝移植术后1d组和3d组,ATP含量和Na~+/K~+ATPase活性均有明显下降,至术后7d组各指标和SO组比较无差别。GH处理后各指标水平均较OLT组明显上升,差异具有显著性意义(P＜0.05)。
3.OLT+rhGH组GH水平在术后1天,和SO、OLT组差别无统计学意义,而在术后3、7d组明显高于OLT组同时相点水平(P＜0.05),和SO组比较差别无统计学意义。大鼠肝移植术后GH-IGF-1轴发生改变,术后7d内GHR和IGF-1R表达呈下降趋势。rhGH治疗组术后7d GHR数恢复明显,术后3、7d IGF-1R数恢复明显(P＜0.05)。
4.Mrp2转运蛋白特异性定位于肝细胞膜的毛细胆管面,荧光染色呈线条形勾勒出毛细胆管轮廓,给予rhGH的肝移植大鼠肝脏Mrp2表达水平明显增高,恢复加快。Mrp2的RT-PCR结果表明:其表达于术后3d达高峰。OLT术后Mrp2表达亦明显下调,给予rhGH的肝移植大鼠肝脏Mrp2RNA表达明显上调,水平明显增高,恢复加快。肝组织Mrp2基因的表达与血清TBIL、TNFα存在密切的负相关(P＜0.05)。
5.肝组织常规HE染色和电镜检查均提示:重组生长激素干预的肝移植组的肝细胞损伤改变较未干预组轻。
结论
1.大鼠肝移植术后早期存在胆汁淤积。肝细胞功能恢复早于其形态学恢复。肝移植术后应用外源性GH对大鼠肝脏热缺血再灌注损伤具有保护作用可促进热缺血损伤移植肝功能恢复。
2.肝移植术后肝组织的能量代谢有显著改变。在术后肝组织ATP含量和肝细胞胞质膜Na~+/k~+ATP酶活性均显著降低,这表明此时胆汁淤积的发生有泵功能显著不足的因素参与作用。肝组织的泵功能不足是导致大鼠肝移植术后并发胆汁淤积的因素之一。
3.大鼠原位肝移植后产生一过性GH抵抗,存在GH相对不足;热缺血损伤移植肝GH-IGF-1轴的改变与热缺血损伤移植肝恢复延迟有关;肝移植术后应用外源性GH可促进移植肝GH-IGF-1轴的恢复,并促进肝功能的恢复。
4.Mrp2表达的减少以及定位的异常是鼠肝移植术后胆汁淤积发生的主要分子机制之一,rhGH促进胆红素的转运的作用机制可能是通过直接上调Mrp2表达来实现;也可能通过限制TNFα的过度释放间接上调Mrp2来促进胆红素的转运。
PARTⅠESTABLISHMENT OF THE RAT LIVER TRANSPLANTATION MODEL
Objective
To study the operation technique of rat's orthotopic liver transplantation(OLT) and to provide a reliable model for the study of OLT.
Methods
The models of the Wistar rat were established with modified Kamada's two cuff technique.We monitored the survival rate complications of operated rat.
Results
About 220 cases of rat orthotopic and liver transplantation operations were performed in total.In 40 cases of formal operations,the operation time of donor was(22.3±7)min,cuff preparation time of the whole graft group was(15.6±4)min,the operation time of recipient was(50.6±11.2)min,anhepatic phase was(20.6±2.5)min.The successful rate and 1 week survival rate were 100%and 92.5%respectively.We successfully establish a stable rat liver transplantation model.
Conclusions
If you want to establish a stable rat liver transplantation model,you must master enough microsurgery skills and an intimate knowledge of abdomen anatomy of rat. Moreover,you have to repeat training in OLT for a phase.The sophisticated microsurgical technique and the delicate surgical manipulation is the key to successfully perform rat orthotopic liver transplantation.
PARTⅡEFFECT AND MECHANISM OF rhGH ON CHOLESTASIS AND LIVER FUNCTION AT EARLY STAGE AFTER TRANSPLANTATION
Objective
Our experiments were designed to study the influence and regulatory mechanisms of rhGH on cholestasis and liver function after liver transplantation in rats.
Materials and Methods
Orthotopic liver transplantation(OLT) was performed in male Wistar rats using the cuff technique as described by Kamada with modifications.Rats were divided into three groups randomly:SO group(n=18,sham operation group),OLT group(n=18,liver transplantation control group),OLT+rhGH group(n=18,rhGH administrated group in liver transplantation),Each group was further divided into 3 subgroups respectively according to different stage after operation.Three time points were predetermined as 1d,3d,7d postoperation.Six animals were used per time-point.Rats were sacrificed at each time-point and the liver specimens and blood samples were collected.The liver function was evaluated by serum ALP,GGT,ALT,TBIL levels.Levels of TNFα,IL-1βwere measured by ELISA.Serum GH by radioimmunoassay.The GH receptor and IGF-1 receptor binding in grafts were analyzed by radioligend binding assay(RBA).Levels of ATP in hepatic tissues were examined by high performance liquid chromatography(HPLC).The change of Na~+/K~+ATPase was measured by chromatometry.Finally,Mrp2 of liver tissue was determined by immunofluorescent microscopy and RT-PCR.Meanwhile,expression of Mrp2mRNA correlatively analyzed with TNFαand TBIL in the serum.Morphometry was observed under light microscope and TEM.
Results
1.Biochemical data showed that the levels of serum ALP,TBIL,GGT,ALT and TNFαwere significantly lower in OLT+rhGH group than that in OLT group(P＜0.05), while the level of IL-1βbetween the two groups has no significant difference.
2.At 1,3 days after OLT,the levels of ATP and the activity of Na~+/K~+ ATPase were significantly lower in OLT group than that in SO group(P＜0.05).Treatment with GH,the levels of ATP and the activity of Na~+/K~+ ATPase significantly increased.
3.At 1d after operation,the level of serum GH in group OLT was not different compared with that in group OLT+rhGH and SO.while At 3,7 days after OLT,the level of serum GH was significantly lower in OLT group than that in SO and OLT+rhGH group(P＜0.05).In OLT group,the liver grafts showed a significant decrease in GH receptor and IGF-1 receptor number on postoperative day 7(P＜0.05).After the rhGH treatment, GH receptor markedly recovered on postoperative day 7 while IGF-1R did on postoperative day 3 and 7(P＜0.05).
4.At 1d after operation,the expression of Mrp2 in OLT group was so weak that could hardly be detected under fluorescence microscope,while obvious fluorescence could be detected from the seventh day postoperatively,showing significant discrepancies when compared with SO group.As duration elongated,both the intensity and range of specific fluorescence grew,moderate level of fluorescence was maintained.Compared with OLT rats at the same timepoints,the expression of Mrp2 in rhGH-treated rats was much stronger.Expression of Mrp2mRNA correlated negatively with TBIL and TNFα..
5.The histological changes were more severe in OLT group than that in OLT+rhGH group.
Conclusions
1.Cholestasis was evident after liver transplantation at early phase.Restoration of the function was faster than restoration of morphology.Exogenous GH treatment can protect ischemic/reperfusion injury in rat liver,and facilitate recovery of function of liver graft.
2.The level of ATP and Na~+/K~+ ATPase in early time of cryopreservationreperfusion in donor liver are related to the graft function.The changes of Na~+/K~+ ATPase in graft indicated that liver transplantation might lead to energy metabolism disturbance of the membrane of liver cell,which might be responsible for the metabolism disorder of cholestasis.
3.Changes of GH-IGF-1 axis appeared,expression of GHR and IGF-1R trended to descend within the first 7 days postoperatively.Injury from warm ischemia caused delayed recovery of GH-IGF-1 axis,and transient GH resistance was present.Both GHR and IGF-1R decreased and resulted in a relative deficiency of GH.The delayed recovery of function of liver graft was implicated to the changes of GH-IGF-1 axis.After OLT, exogenous GH treatment facilitated recovery of GH-IGF-1 axis,and recovery of function of liver graft was promoted by postoperative rhGH treatment.
4.The level of Mrp2 in the liver tissue was upregular by rhGH and was significantly.Liver transplantation might lead to down-regulation and abnormal localization of Mrp2 in liver cell,which might be responsible for the cholestasis after rat liver transplantation,rhGH can protect the liver from the injury of intrahepatic cholestasis.The promotion of bilirubin transport by rhGH can upregulate the expression of Mrp2 directly.On the other hand,The application of rhGH might limit exaggerated hepatic production of TNFαto upregulate the expression of Mrp2.
引文
1.Otero A ,Gomez-Gutierrez M ,Suarez F ,et al .Liver transplantation from Maastricht category non-heart-beating donors: a source to increase the donor pool? Transplant Proc,2004,36(3):747-50
2.Non-heart-beating organ transplantation: medical and ethical issues in procurement(1997).Institute of Medicine, National Academy Press.Available online at http://books.nap.edu/books/0309064244 / html/index.html
3.Reich DJ,Munoz SJ,Rothstein KD,et al. Controlled non-heart-beating donor liver transplantation:a successful single center experience, with topic update. Transplantation,2000,70(4): 1159-66
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