【作者】 刘静；

【导师】 李立； 冉江华； 李晓延；

【作者基本信息】 昆明医学院 ， 外科学， 2010， 博士

【摘要】 目的:探讨改良后的大鼠减体积肝移植模型的效果;为进一步进行大鼠减体积肝移植术后肝脏再生及免疫的研究提供重要的技术平台和前提条件。方法:1.实验大鼠均为健康的SD大鼠,体重260-280 g,供体为雌性,受体为雄性,供、受体体重相差10 g左右,一般为供体体重比受体体重轻。2.供体采用单人裸眼操作,在取肝的过程中即进行减体积操作;修肝时将套管柄置于门静脉和肝下下腔静脉的正前方,将幽门静脉结扎点外翻于套管外并置于套管柄的左侧,即肝脏的左侧;将右肾上腺静脉结扎点外翻于套管外并置于套管柄的右侧,即肝脏的右侧;供肝套管完成后用灌注液对门静脉和肝下下腔静脉进行冲洗;然后以左膈静脉为标识点进行7-0无损伤血管缝线吊线。3.受体切肝前采用单人裸眼操作,从新肝移植开始采用双人裸眼配合操作;供体完成胆道支撑架安置以后,受体即开始手术;肝上下腔静脉吻合时,左右固定位点采用“8”字形外翻缝合,后壁和前壁分别采用连续吻合,门静脉和肝下下腔静脉采用改良的双袖套法,胆道支撑管法建立大鼠减体积的稳定模型。4.改良的的大鼠减体积肝移植采用文献报道的方法进行,供肝在修肝盆中进行相应的肝叶切除。5.改良前和改良后两组术中和术后均观察大鼠的症状和体征、各种并发症的发生情况、术后生存情况、肝功能的变化等等。结果:1.改良后的减体积肝移植模型供体手术时间为32±2 min,修肝时间为6±2 min,受体手术时间为40±3 min,无肝期时间为14±3 min,供肝的冷保存时间为51±3min;手术的成功率为92%,术后3 d的存活率为85%,术后2周存活率为83%;术后并发症较改良前明显减少,差异有显著性（P<0.05）。2.术后1 d、3 d和7 d的肝功能ALT变化为改良后较改良前低,改良后血清ALT与同期的改良前组比较差异有显著性（P<0.05）;术后第14 d和21 d,改良后血清ALT与同期的改良前组比较差异无显著性（P>0.05）。3.术后1 d、3 d和7 d的肝功能TBIL变化为改良后较改良前低,改良后血清TBIL与同期的改良前组比较差异有显著性（P<0.05）;术后第14 d和21 d,改良后血清TBIL与同期的改良前组比较差异无显著性（P>0.05）。4.术后1 d、3 d和7 d的胆碱酯酶变化为改良后较改良前升高,改良后血清胆碱酯酶与同期的改良前组比较差异有显著性（P<0.05）;术后第14 d和21 d,改良后血清胆碱酯酶与同期的改良前组比较差异无显著性（P>0.05）。5.术后1 d和3 d的血氨变化为改良后较改良前降低,改良后血清血氨与同期的改良前组比较差异有显著性（P<0.05）;术后第7 d、14 d和21 d,改良后血清血氨与同期的改良前组比较差异无显著性（P>0.05）。结论:1.改良大鼠减体积模型采用供体灌注完成后,在切取供肝的过程中进行相应肝叶的切除;可获得高质量、满意的减体积供肝;供受体手术配合,尽量的缩短供肝的冷保存时间;改进的血管吻合技术缩短无肝期、减少术后出血;对诸多细节操作的改进等;这些可以尽量的减少大鼠减体积肝移植术中和术后的并发症,提高大鼠减体积肝移植模型的成功率和长期生存率。是一种值得推广的大鼠减体积肝移植模型。2.成功的改良大鼠减体积肝移植模型为我们下一步进行大鼠减体积肝移植术后肝脏的再生和免疫的研究提供了研究的基础和前提。目的:探讨在成功建立大鼠减体积肝移植模型的基础上;利用蛋白质组学的技术研究大鼠减体积肝移植术后肝脏再生的蛋白质组学的变化,找到与肝脏再生有关的差异蛋白质。方法:1.实验大鼠供体均为健康的Lewis雌性大鼠,体重200-230 g左右;受体为均为健康的Wistar雄性大鼠,体重220-250 g左右。2.采用改良法建立Lewis-Wistar的大鼠减体积肝移植模型,供肝与受体肝之比大约50%左右。术后使用FK506抗免疫排斥药物,按照0.1 mg/kg·d服用。在肝移植术后1 d、3 d和7 d获取移植后的肝脏组织标本,供体和受体的正常肝脏组织标本在进行移植手术时获取,将标本放置在-70℃冰箱保存。3.将获取的标本进行大鼠减体积肝移植术后的蛋白质组学的检测分析。对移植后的肝脏组织与供体和受体正常肝脏组织进行比较蛋白质组学的研究,即在双向电泳的基础上进行MS-MS串联质谱分析,从而鉴定表达差异在10倍以上的蛋白质点,分析差异表达的蛋白质的功能,以及与大鼠减体积肝移植术后肝脏再生的相关性等。结果:1.得到了分辨率较高、重复性较好的双向凝胶电泳图谱,以差异10倍以上为标准,共找到了72个差异蛋白质点。2.对72个差异表达的蛋白质点进行MS-MS串联质谱鉴定和肽谱指纹图分析,72个点全部鉴定出,共鉴定到40种蛋白。3.这些蛋白主要参与细胞信号转导、应激反应、氧化还原、碳水化合物代谢、能量代谢、氨基酸代谢和细胞骨架等生理过程。有些蛋白直接或者间接参与肝移植术后肝脏再生的过程。结论:1.成功建立Lewis-Wistar大鼠减体积肝移植模型。2.采用比较蛋白质组学的方法成功鉴定了72个差异蛋白质点40种蛋白。3.找到与大鼠减体积肝移植术后肝脏组织细胞再生有关的差异蛋白,这些蛋白质对大鼠减体积肝移植术后肝脏组织细胞的再生作用可能主要通过两个方面完成:一是直接促进肝脏组织细胞的再生:一是间接的促进肝脏组织细胞的再生。4.为下一步深入研究大鼠减体积肝移植术后肝脏再生的机理等相关问题提供了一定的基础研究成果。目的:在成功建立大鼠减体积肝移植模型的基础上,研究减体积肝移植术后肝脏再生的一般情况,受体骨髓干细胞跨细胞分化成肝细胞;在肝脏再生基本完全后（大约在减体积肝移植术后第9 d）,撤除抗免疫排斥药物以后观察各组排斥反应发生的情况,最终得出:减体积肝移植术后,肝脏再生诱导免疫低反应并探讨可能的机理。方法:1.实验大鼠供体均为健康的Lewis雌性大鼠,体重200-230 g左右,受体均为健康的Wistar雄性大鼠,体重240-380 g左右。2.采用改良法建立Lewis-Wistar的大鼠减体积肝移植模型,详细见第一部分模型的建立。3.实验分组（1）实验一组（A组）:全肝移植组。供体和受体体重相差在10 g以内。（2）实验二组（B组）:50%肝移植组。供体与受体肝脏质量比大约为50%。（3）实验三组（C组）:30%肝移植组。供体与受体肝脏质量比大约为30%。4.实验分为两个小部分（1）第一小部分:观察不同体积的大鼠肝移植术后肝脏的再生情况。术后使用FK506抗免疫排斥药物,按照0.1 mg/kg·d服用。在肝移植术后1 d、2 d和7 d获取血和移植后的肝脏组织标本,留待作相关指标的检测。（2）第二小部分:观察肝脏再生诱导免疫低反应。不同减体积肝移植术后,待肝脏再生基本完全后,同时撤除FK506,观察其三组的排斥反应的发生情况。在撤除FK506后的0 d、3 d、7 d和11 d取血和肝脏组织标本,留待作相关指标的检测。5.移植大鼠生存状态观察和移植排斥反应的判断:大鼠术后3 d内死亡,则弃之不用,并给与补充。观察每组的生存时间,根据国际公认的Banff评分系统判断排斥反应程度。6.血清生化检测ALT、AST、TBIL;血清细胞因子检测IL-2、IFN-γ、IL-4、IL-10、TGF-β₁的水平。7.肝脏组织的ELISA检测:观察肝脏再生的IL-6和TNF-α水平;观察排斥反应TGF-β₁水平。8.肝脏组织免疫组化检测:观察肝脏再生的IL-6、TNF-α、PCNA和Ki-67。观察肝脏排斥反应的ICAM-1、NFκB/p65和TGF-β₁。9.应用原位杂交技术检测肝脏组织的Y染色体,以证实受体骨髓干细胞跨细胞分化成肝细胞。10.肝脏组织的HE染色、电镜和凋亡检测。观察肝脏织形态结构和排斥反应。结果:1.大鼠减体积肝移植术后肝脏再生先增快（在减体积肝移植术后第2 d最明显）,后减慢,在术后第7 d,肝脏基本达到移植前受体的肝脏体积和质量。2.肝脏再生的过程中,C组中Y染色体的阳性率最高,B组次之,而A组的Y染色体的阳性率最低。3.大鼠减体积肝移植肝脏再生过程中,肝脏组织中的IL-6和TNF-α水平表现为先升高,后减低,即在术后第2 d,肝脏组织中的IL-6和TNF-α水平最高,而后降低。4.观察排斥反应时,血清中的IL-2和IFN-γ水平,A组最高,B组次之,C组最低;相反,IL-4和IL-10的水平,C组最高,B组次之,而A组最低。5.血清中的TGF-β₁表现为先升高,后降低。升高时,A组升高最快、最高,B组次之,C组升高得最慢、最低;降低时,A组降低的速度最快、降低最明显,B组次之,而A组下降最慢、减低的程度最小。肝脏组织中的TGF-β₁则表现为持续升高,以A组最为明显,B组次之,C组最低。6.肝脏组织的免疫组化ICAM-1、NFκB/p65、TGF-β₁,肝细胞凋亡以及HE染色等进一步证实了A组的组织局部的炎症反应最重,B组次之,而C组最轻。结论:1.成功建立Lewis-Wistar大鼠减体积肝移植模型。2.大鼠减体积肝移植术后肝脏再生过程中,有骨髓干细胞跨细胞分化成肝细胞。3.在大鼠减体积肝移植肝脏再生基本完全的基础上,撤除FK506后,C组发生的排斥反应程度最低,B组次之,而A组的排斥反应程度最重。4.大鼠减体积肝移植术后肝脏再生诱导免疫低反应的原理可能与受体骨髓干细胞跨细胞分化成肝细胞以及机体的多因素共同参与了肝脏的再生过程有关系。更多还原

【Abstract】 Objective:It was the foundation and prerequisite for the next research of liver regeneration and it’s immune hyporesponsiveness that The improved model of reduced-size liver transplantation in the rat was investigated.Methods:1.The donors were female and the receptors were male,which were healthy SD rats and weight range was 260-280 g,weight of the receptor was more than that of the donor,about 10g.2.Operation of donor was performed by only one person with the naked eye,during which reduced-size donor liver was performed.The handle of self-made cuff was placed in the good front of portal vein and inferior vena cava respectively,which the tied ligature of pyloric veins was turned inside out of the self-made cuff, furthermore,the tied ligature was placed in the left of the self-made cuff;the same to inferior vena cava except that the tied ligature of right renal vein was placed in the right of the self-made cuff.Then the portal vein and inferior vena cava were received washing with self-made perfusate respectively.3.Operation of the receptor was performed by two person with the naked eye,with improved dual-cuff technique of Kamada and stay pipe of biliary tract.The fixed points of left and right were connected by anastomosis of "8" type turning inside out while inosculating inferior vena cava.4.The pre-improved model of reduced-size liver transplantation in rat was performed by reported in the literature,in which lobes of rat liver were removed in liver repair basin.5.The symptoms and signs> all complications、survival conditions、liver function change of rats were observed after liver transplantation in the improved and pre-improved model groups.Results:1.The average operation time of gaining the donor and preparating the donor liver were 32±2 minutes and 6±2 minutes respectively.The average operation time of gaining the receptor and the anhepatic were 40±3 minutes and 14±3 minutes respectively.And cold preservation time of liver donor was 51±3 minutes.The general successful rate was 92%,three-day survival rate was 85%and two-week survival rate was 83%.The postoperative complications was reduced clearly.the contrast was significant difference（P<0.05）.2.ALT values of ldpo（days post operation）,3dpo and 7dpo in improved group were reduced progressively in pre-improved group in comparation with that of pre-improved group,which was significantly difference（P<0.05） in two groups at the same time.But ALT values of 14dpo and 21dpo in improved group were non-significant difference（P>0.05） in comparation with that of pre-improved group.3.Compared with pre-improved group,TBIL values in improved group were reduced progressively advanced at 1dpo,3dpo and 7dpo,which were significantly difference （P<0.05） in two groups at the same time.But the level of 14dpo and 21dpo in two groups were non-significant difference（P>0.05）.4.Compared with pre-improved group,XCHE values in improved group was progressive advanced at 1dpo,3dpo and 7dpo,which were significantly difference （P<0.05） in two groups at the same time.But the level of 14dpo and 21dpo in two groups were non-significant difference（P>0.05）.5.Compared with pre-improved group,AMON values in improved group was reduced progressively at ldpo and 3dpo,which were significant difference（P<0.05） in two groups at the same time.But the level of it in two groups at 7dpo,14dpo and 21dpo were non-significant difference（P>0.05） at the same time.Conclusions:1.It was better method of obtaining high quality and satisfied reduced-size liver in the rat that lobes of rat liver in operating on donor liver were removed after liver donor perfusion.The operation of donor and receptor was intercoordination,which may shorten cold preservation time of liver donor;The improved vascular anastomosis may shorten anhepatic time and reduce bleeding complications;It was worthy of being accepted model of reduced-size liver transplantation in the rat that the improved model may reduce in-operation and post-operation complications after liver transplantation,and raise succeed rate of rat liver transplantation and survival rate of receptor.2.It was maybe the foundation and prerequisite for hepatic rebirth and immune experimental research after liver transplantation that improved model of reduced-size liver transplantation in the rat was established successfully. Objective:To search for the differential proteins related to liver regeneration after transplantation,it was studied on the base of the success in the reduce-size liver transplantation in rat applying of technology of proteomics.Methods:1.Experimental donors were healthy and female Lewis rats,weight range was 200-230 g.The receptor were healthy and male Wistar rats,weight range was 220-250 g.2.Models of reduced-size liver transplantation in rats were established successfully, which weight ratio of the donor and the receptor liver was about 50%.The postoperative rats accepted immunosuppressive treatment of KF506,with 10mg/kg·d.The postoperative liver specimens were harvested by 1 d,3 d and 7 d after reduce-size liver transplantation.And the normal liver specimens of the donors and the receptors were harvested during reduce-size liver transplantation,which were kept in a -70℃refrigerator for the proteomics test.3.The harvested liver tissue specimens were accepted proteomic test and analysis.The first step,comparative proteome analysis with postoperative liver tissue and normal donor,receptor liver tissue.Namely,they were accepted MS-MS spectrometry after two-dimensional electrophoresis（2-DE） for identifying proteins stains with differentiation exceeding 10 times.At last,the function of differential proteins related to liver regeneration was analyzed after reduce-size liver transplantation in rats.Results:1.The results showed the high-resolution and reproduciblity 2-DE patterns about postoperative liver tissue and normal donors and receptors,and found 72 differential protein stains by taking 10 times measure.2.Seventy-two differentially expressed protein spots were all identified by MS-MS spectrometry and PMF.At last,Forty proteins were identified successfully.3.The identified proteins mostly participated in physiology pathway:signal transduct -ion,stress defence,redox homeostasis,carbohydrate metabolism,energy, aminoacid metabolism and cell cytoskeleton,and so on.Some of which participated in liver regeneration after reduce-size liver transplantation by means of direct and indirect physiology pathway.Conclusion:1.Reduce-size liver transplantation models in rats were established successfully,with Lewis to Wistar.2.Sevety-two differential protein spots and forty proteins were identified successfully. 3.The differential proteins related to liver regeneration were found successfully after reduce-size liver transplantation,which maybe participate in liver regeneration after reduce-size liver transplantation through two aspects as follows:on one hand,to promote liver regeneration by direct physiology pathway;on other hand,to promote liver regeneration by indirect physiology pathway.4.provided fundamental research results for next research related to liver regeneration mechanism after reduce-size liver transplantation.Objective:Liver regeneration and how many the recipients’ bone marrow stem cells differentiated to hepatocytes,and it’s possible mechanism of immune hyporesponsiveness induced by liver regeneration after removing immunosuppressive drugs were studied on the basis of successful murine model of reduced-size liver transplantation.Methods:1.Experimental donors were healthy and female Lewis rats,whose weights varied from 200 to 230 g.Recipients were healthy and male Wistar rats,whose weights varied from 240 to 380 g.2.Models were operated via method of improved Lewis-Wistar reduced-size liver transplantation;details can be seen in the first part.3.Experimental groups:（1） Group A:whole liver transplantation group.The donors’ weight was less than that of receptors.（2） Group B:50%liver transplantation group.The ratio of donors’ to recipients’ liver weights was about 50%.（3） Group C:30%liver transplantation group.The ratio of donors’ to recipients’ liver weights was about 30%.4.The entire experiment can be divided into two parts.（1） To observe situations of liver regeneration among groups that accepted different volume liver transplantations.After operations,used FK506 as immunosuppressive drug,whith 0.1 mg/kg·d.On the first day and 3rd day and 7th day after transplantation,sampled serum and liver tissue for relevant tests.（2） To observe low immunological reject reaction induced by liver regeneration. When liver regeneration followed partial liver transplantation finished, removed usage of FK506,then observed rejection among 3 groups.On the 0 dpo,3 dpo,7 dpo and 11 dpo after removal FK506,sampled serum and liver tissue for relevant tests.5.Survival situation and rejection reaction of rats were observed and judged:If rat died within 3 days after operation,the rat would be excluded,another would be compensated.The degree of rejection was judged by Banff score system.6.The level of ALT,AST,TBIL in serum and level of serum cytokines IL-2,IFN-γ, IL-4,IL-10 and TGF-β₁ were tested.7.Levels of IL-6 and TNF-αin liver tissues were detested by ELISA,which related to regeneration after liver transplantation,and the level of TGF-β₁ related to rejection was detested.8.To detect variation of IL-6,TNF-α,PCNA and Ki-67 related regeneration after liver transplantation,liver tissues immunochemical methods were performed.And then, expression variation of ICAM-1,NFκB/p65 and TGF-β₁ related to rejection after operation was detested in same methods.9.Y chromosome in liver tissues was detested by hybridization in situ,In ordor to prove bone marrow stem cells of recipients differentiated into hepatocytes.10.To observe morphous and rejection reaction of liver tissues.Liver tissues were treated by hematoxylin and eosin stain,and seen under electron microscopy and detested by Tunnel methods to showe celluar apoptosis,Results:1.In the early stage of regeneration followed reduced-size liver transplantation, regeneration was accelerated（on the second day of operation,it got to climax）,then decelerated.On the 7th day of operation,liver’ weight achieved to as big as that of pre-transplantation.2.In the process of regeneration,Y chromosome positive rate was the highest in the group C,lower in group B,lowest in group A.3.In the process of regeneration,levels of IL-6 and TNF-a in liver tissues were ascend -ing in early stage（the summit appeared in the second day）,then they were descending.4.When observing rejection,levels of IL-2 and IFN-γin serum were the highest in group A,lower in group B,the lowest in group C.On the opposite,the levels of IL-4 and IL-10 in serum were the highest in group C,lower in group B,the lowest in group A.5.The serum level of TGF-Pi were ascending at the beginning,then carried on descending.When it was ascending,group A appeared fastest and highest,then the group B,but group C was last one.On the other hand,in the process of TGF-β₁ descending,it can be seen to decrease fastest and most in group A,then group B, group C followed.Meanwhile,TGF-β₁ level in liver tissues appeared increasing persistently,same mode as IL-2 and IFN-γin serum,the most apparent is group A, group B is the second,at last,group C.6.By immunochemical examination of ICAM-1、NFκB/p65、TGF-β₁,hepatocyte apoptosis and hematoxylin and eosin stain analysis,the most severely imflammation reaction was observed in group A,better in group B,then group C.Conclusions:1.Lewis-Wistar improved reduced-size liver transplantation models was builted successfully.2.In the process of liver regeneration after improved reduced-size liver transplantation, bone marrow stem cells differentiated into hepatocytes.3.On the basis of complete liver regeneration after improved reduced-size liver transplantation,most severel rejection was observed in group A,better in group B, and best in group C after removing FK506.4.The mechanism of low immunological reaction induced by improved reduced-size liver transplantation maybe relate to the process of bone marrow stem cells differentiated into hepatocytes,and multiple factors maybe contribute to this process.更多还原