一种小鼠胰岛经门静脉肝内移植方法及疗效评价

目的 介绍一种糖尿病小鼠经门静脉到肝内胰岛移植的方法并进行移植前后的血糖、口服糖耐量、免疫组化等评价。方法 采用Balb/c小鼠,将分离纯化的小鼠胰岛培养6 h后,用自制的移植工具进行糖尿病模型小鼠的肝门静脉插管和输注胰岛,移植胰岛量为（320±30）IEQ,对移植前后的小鼠血糖进行测定,在移植后第10天进行糖耐量试验,并取受体小鼠肝脏,进行HE染色和免疫组化分析,观察胰岛细胞团在肝内的存活情况。结果受体小鼠胰岛移植后血糖均能长期维持正常,糖耐量试验结果显示与正常小鼠无统计学差异（P =0.81）,组织学结果显示胰岛细胞在小鼠肝内存活良好。结论 采用本方法可建立小鼠胰岛经门静脉移植到肝内的模型,为下一步进行胰岛移植相关药物筛选及免疫学等方面研究奠定了基础。     

两种不同肝脏灌注方法对大鼠肝脏保护作用的比较

目的探讨两种不同的肝脏灌注方法对冷保存的离体大鼠肝脏保护作用的差异,为后期离体大鼠肝脏保存实验提供简单而有效的方法。方法制备两种不同的单纯冷保存的离体大鼠肝脏灌注模型,分为实验组（经门静脉和腹主动脉灌注法,10只）;对照组（经门静脉、腹主动脉及肝动脉灌注法,10只）,灌注后取出肝脏置于4℃威斯康星大学保存液保存,12h后切取肝组织。行苏木素-伊红（HE）染色,观察肝组织病理损伤情况,并且用免疫组织化学染色法检测核因子（NF）-κB（SABC法）的表达情况。结果经12h冷保存后,两组大鼠肝脏组织病理示：肝脏小叶结构紊乱,肝细胞变性、肿胀及空泡形成,肝血窦和中央静脉有不同程度的淤血,部分肝窦内皮细胞坏死并脱落,但两组之间无明显差别。两组之间NF-κB阳性表达率差异无统计学意义（P〉0．05）。结论经门静脉和腹主动脉灌注法与经门静脉、腹主动脉及肝动脉灌注法,对离体大鼠肝脏的保护作用无差异,但前者操作相对简单,更容易在今后研究大鼠离体肝脏的实验中应用。     

肝胰岛细胞联合移植的临床应用(附1例报告)

目的 评估肝胰岛细胞联合移植治疗终末期肝病和糖尿病的可行性和疗效. 方法 对1例原有2型糖尿病史的原发性肝癌、肝炎肝硬化患者,施行肝移植的同时进行胰岛细胞经门静脉肝内移植,观察移植肝功能的变化,移植前后C肽、糖化血红蛋白的变化. 结果 患者胰岛素的注射量术前40~60 U/d,术后第45天降至20-30U/d,术后7月完全脱离胰岛素,血糖保持在正常范围. 结论 对肝脏功能衰竭的糖尿病患者施行肝胰岛细胞联合移植是可行而有效的.     

部分门静脉动脉化对大鼠肝脏血管铸型变化的影响

目的：探讨用部分门静脉动脉化重建肝血流后对肝脏微血管和组织学的影响。方法：建立大鼠部分门静脉动脉化重建肝脏血流的实验模型，观察该模型大鼠肝脏微血管和组织学的变化。结果：行门静脉动脉化手术后1个月动物肝脏组织未见明显异常。血管铸型标本显示肝窦略变粗，较正常充盈，肝窦无明显变形，仍呈放射状分布于中央静脉的周围。墨汁灌注标本显示肝窦内墨汁灌注规整，略显增宽，颜色均匀并加深。结论：部分门静脉动脉化后在近期内不影响肝脏的微血管及组织学结构。     

肝细胞、胰岛细胞肝内移植对肝硬变影响的研究

目的避免脾切除、门腔分流术后所致向肝血流减少造成的肝功能损害。方法白蛋白将分离纯化的肝细胞、胰岛细胞经肝动脉灌注肝内移植。结果细胞组与对照组比较血总蛋白、白蛋白、胆红素改善程度,差异有极显著意义（Ｐ＜０．０１）。肝纤维化光镜检查及Ⅰ、Ⅲ型胶原免疫组织化学染色及图象分析结果,差异有显著意义（Ｐ＜０．０５）。结论肝细胞、胰岛细胞经肝动脉灌注肝内移植,不但能改善肝功能,还能促进肝脏胶原纤维降解,逆转肝纤维化、肝硬变的程度。     

大鼠胰岛转染人血红素氧合酶-1基因对同种胰岛移植的影响

目的探讨大鼠胰岛转染人血红素氧合酶-1(HO-1)基因在胰岛移植中的潜在应用价值。方法采用携带人HO-1基因的腺病毒对新分离的SD大鼠胰岛细胞进行转染;对体外培养的胰岛细胞使用重组人肿瘤坏死因子α及放线菌酮诱导凋亡。使用流式细胞术检测凋亡率;每只链脲霉素诱导的糖尿病模型大鼠门静脉内置入约1200个胰岛当量的胰岛后,观察糖尿病大鼠血糖及体重变化;免疫组织化学法检测肝内移植胰岛细胞的胰岛素、HO-1蛋白表达及表达CD3抗原的淋巴细胞浸润情况。结果转染人HO-1基因的胰岛细胞凋亡率明显低于对照组(P<0.05);单纯胰岛细胞移植后移植物存活时间为(5.33±4.18)d,转染人HO-1基因的胰岛细胞移植后移植物存活时间为(10.56±4.33)d,两组比较,P<0.05;转染人HO-1基因的胰岛细胞培养48 h即见人HO-1蛋白表达;肝内移植7 d后移植物有人HO-1蛋白表达;移植胰岛周边及胰岛内淋巴细胞浸润程度较单纯胰岛移植组明显减轻。结论大鼠胰岛转染人HO-1基因能够增加体外培养胰岛细胞的抗凋亡能力,并能延长体内移植胰岛的存活时间,减轻淋巴细胞对胰岛的浸润。     

大鼠同种骨髓间充质细胞门静脉输注对肝大部分切除肝衰模型术后肝功能、生存率的影响

目的 观察大鼠同种骨髓间充质细胞(BMSCs)经门静脉肝内移植对80％肝切除肝衰竭模型术后肝功能、生存率的影响.方法 通过80％肝切除建立大鼠边缘肝衰竭模型,经门静脉输注绿荧光蛋白(GFP)标记的第5代(P5)大鼠同种MSC,观察BMSCs肝内移植对肝大部分切除边缘肝衰竭模型术后生存率、肝功能的影响.结果 术后7d内的存活率:MSC组72.2％( 13/18)、对照组44.4％ (8/18).MSC组的存活率明显高于对照组(72.2％比44.4％,P＜0.05;MSC组大鼠术后肝功能恢复情况明显优于对照组(P＜0.05);MSC组肝组织中可观察到移植的细胞.结论 经门静脉的BMSCs移植可促进80％肝切除边缘肝衰竭大鼠的肝功能恢复和提高生存率.     

猪肝脏灌注异常现象的相关机制

目的通过对猪正常肝脏建立灌注异常模型,探讨肝灌注异常的发生因素。方法9头实验用小型猪进入研究,随机平均分为A、B、C3组,分别采用明胶海绵碎屑进行肝内门静脉、肝动脉、肝静脉分支栓塞,术后即刻及1周后进行CT增强扫描,观察是否存在肝灌注异常现象。结果术后即刻CT扫描,全部研究对象出现肝灌注异常现象,表现为动脉期肝实质内出现楔形或不规则形一过性强化,门静脉期恢复正常。A组出现灌注异常的部位与栓塞血管所在肝段、叶一致;B组栓塞区出现低灌注区,非栓塞区出现一过性强化现象。C组2头出现灌注异常的部位与栓塞区域一致,1头比栓塞区域大。1周时CT复查,除C组存在部分灌注异常现象外,A、B组灌注异常现象消失。结论肝内门静脉、肝动脉、肝静脉分支堵塞是造成肝灌注异常现象的因素。     

肝内移植微囊对肝纤维化的影响:经门静脉及肝动脉途径对比

目的通过比较经门静脉及肝动脉两种途径肝内移植海藻酸钠微囊对肝纤维化影响的差别,探求安全、简便的肝内胰岛移植途径。方法实验犬30只随机分为V、A两组,每组15只,分别作为经门静脉移植组及经肝动脉移植组,V、A两组分别随机分为三个小组,每小组5只,分别移植海藻酸钠微囊8000个/kg、16000个/kg、32000个/kg,观察移植前、后血清肝纤维化标志物透明质酸(HA)的变化,并对肝脏进行病理组织学检查。结果血清纤维化指标:V组:血清HA值在移植后逐渐升高,移植前、后有明显差别(P<0.01);且各移植量组间存在显著性差异(P<0.01)。A组:血清HA值在移植后轻度升高,但移植前后无显著差异(P>0.05),且各移植量组间的差异无统计学意义(P>0.05)。相同移植量Vn及An组间血清HA值的比较差异有显著性(P<0.01)。肝脏病理组织学检查:移植术后12周时,V3组肝脏组织学检查发现汇管区少量胶原纤维沉积,肝细胞浊肿,间质见炎性细胞浸润;而A组肝脏组织学检查未见明显异常。结论经肝动脉移植微囊对肝脏损伤小,有望成为一种相对简单、安全的肝内胰岛移植途径。     

骨髓间充质干细胞移植途径对胰岛移植物免疫排斥反应的影响

目的:探讨骨髓间充质干细胞(MSC)与胰岛共移植对诱导胰岛移植物免疫耐受的作用,并比较MSC不同途径移植的效果。方法:SD大鼠和Lewis大鼠分别作为供、受体。取SD大鼠股骨,贴壁培养法分离和扩增MSC,胶原酶V分离胰岛。应用链脲佐菌素制备Lewis大鼠糖尿病模型后,将其随机均分为A组(将BrdU标记的MSC与胰岛经门静脉混合输入),B组(将胰岛经门静脉输入,BrdU标记的MSC经尾静脉输入),C组(胰岛经门静脉输入,联合应用环孢素A)和D组(单纯胰岛门静脉移植)。观察各组术后血糖变化,比较各组胰岛移植物存活时间。术后第7天切取各组部分存活大鼠肝脏、胸腺、脾脏行免疫组化染色观察MSC归巢位置。结果:A,B两组大鼠术后正常血糖维持时间最长,C组次之,D组最短;各组胰岛存活时间A组为(12.1±2.3)d,B组为(8.6±1.4)d,C组为(13.2±1.9)d,D组为(2.2±0.6)d;MSC归巢部位观察显示,A组BrdU阳性的MSC主要分布于肝脏,并在植入胰岛周围形成"类微囊化效应",B组BrdU阳性的MSC主要分布于胸腺、脾脏。结论:MSC与胰岛共移植能诱导胰岛移植物免疫耐受,且MSC和胰岛混合经门静脉移植效果优于胰岛门静脉移植联合MSC外周静脉移植。     

大鼠肝脏胆管及门静脉铸型扫描图像分析

目的 建立大鼠去胆管肝叶和去门静脉肝叶自身对照模型,观察两肝叶之间胆管及门静脉是否存在交通支及其大体形态变化.方法 SD大鼠40只,分为S、BL、PL和BPL共4组,分别应用氰基丙烯酸酯对肝右叶胆管进行栓塞结扎制备去胆管肝叶;对肝方叶行门静脉结扎制备去门静脉肝叶.通过测量肝重/体重和方叶重/右叶重及对各组大鼠胆管和门静脉分别灌注硫酸钡明胶混悬液制备铸型标本,并运用Micro-CT扫描来观察两叶肝脏胆道和门静脉形态变化.结果 (1)大鼠手术后在本观察期内存活率达到100%,无黄疸表现.肝叶大体形态观察和两叶肝重量比指标显示,S、BL、PL组肝重/体重为3.5%,与BPL组比较差异有统计学意义(P<0.01).S、BL组方叶/右叶重量比为60%～70%,PL及BPL组则为20%左右,提示去胆管和去门脉肝叶之间的重量比差异有统计学意义(P<0.05或P<0.01).(2)Micro-CT铸型扫描可以直观地显示胆管和门静脉形态变化,未发现两个肝叶之间存在交通支或侧枝循环.结论 去胆管肝叶无明显萎缩.胆管及门静脉灌注造影显示两叶胆管及门静脉无明显的侧枝循环.Micro-CT扫描可以直观地显示胆管及门静脉形态变化,硫酸钡明胶灌注铸型为小动物肝脏Glissons系统形态学研究提供了一种借鉴方法.

Abstract：

Objective To establish a rat self-control model with the bile duct deprived (BDD) and the portal vein deprived (PVD) hepatic lobe and to observe whether there were communicated branches between the two lobes.Methods Forty SD rats were divided into four groups: group S with sham operation as an undisposed blank control, group BL with the right lobe bile duct embolized and ligated, group PL with the quadrate lobe portal vein ligated, and group BPL with the right lobe bile duct embolized and ligated and meanwhile the quadrate lobe portal vein ligated. The right hepatic bile ducts were embolized with cyanoacrylate and then ligated to prepare the BDD lobe. The portal vein of quadrate hepatic lobes was ligated as the PVD lobes. The observation period was 1 month after the bile duct or portal vein ligated. The values of liver weight/body weight and the quadrate lobe weight/the right lobe weight were recorded. The bile duct and portal vein casting specimens of these four groups were prepared by a perfusion with barium and gelatin solution. Three-dimensional micro-computerized tomography (Micro-CT) data sets were acquired to observe the morphological changes of bile duct and portal vein of the livers and whether there were communicated branches between the right and quadrate lobes in order to estimate the feasibility of the model.Results (1) The survival rate of rats after operation was 100%. No jaundice was observed. The ratio of liver/body weight in groups S, BL and PL was about 3.5%, significantly lower than that in group BPL (P<0.01). The ratio of quadrate/right lobe weight in groups S and BL was about 60%-70%, while that was about 20% in groups PL and BPL (P<0.05, or P<0.01); (2) Micro-CT images exhibited directly the morphological changes of the hepatic bile duct and portal vein, and no communicated branches or side circulation situation were observed between the two lobes.Conclusion No collateral branches were found between the two lobes and the model was successfully established. The barium casting liver specimen scanned by micro-CT provided a useful method for the morphological observation of rat liver Glissons system.     

Portal vein thrombosis after intraportal hepatocytes transplantation in a liver transplant recipient

Hepatocytes transplantation is viewed as a possible alternative or as a bridge therapy to liver transplantation for patients affected by acute or chronic liver disorders. Very few data regarding complications of hepatocytes transplantation is available from the literature. Herein we report for the first time a case of portal vein thrombosis after intraportal hepatocytes transplantation in a liver transplant recipient. A patient affected by acute graft dysfunction, not eligible for retransplantation, underwent intraportal infusion of 2 billion viable cryopreserved ABO identical human allogenic hepatocytes over a period of 5 h. Hepatocytes were transplanted at a concentration of 14 million/ml for a total infused volume of 280 ml. Doppler portal vein ultrasound and intraportal pressure were monitored during cell infusion. The procedure was complicated, 8 h after termination, by the development of portal vein thrombosis with liver failure and death of the patient. Autopsy showed occlusive thrombosis of the intrahepatic portal vein branches; cells or large aggregates of epithelial elements (polyclonal CEA positive), suggestive for transplanted hepatocytes, were co-localized inside the thrombus.     

Intraportal pig islet xenotransplantation into athymic mice as an in vivo model for the study of the instant blood-mediated inflammatory reaction

Abstract:  One of the main obstacles to successful intraportal islet transplantation is the instant blood-mediated inflammatory reaction (IBMIR) elicited by the isolated islets when exposed to fresh human blood. In the present study, we investigated whether intraportal transplantation of pig islets into diabetic athymic mice could be used as a small animal model to study xenogeneic IBMIR in vivo.
Adult porcine islets (APIs) or rat islets were implanted into the portal vein or under the renal subcapsular space of diabetic athymic mice. Graft survival and morphology were evaluated by measuring blood glucose levels and by performing immunohistochemical staining, respectively. Transplantation of rat islets, irrespective of implantation site, cured all diabetic athymic mice. APIs transplanted subcapsularly also cured all diabetic athymic mice, while none of the animals transplanted with an equivalent amount of APIs via the portal vein remained normoglycemic for more than 10 days after transplantation. Immunohistochemical staining on day 7 showed that most of intraportally transplanted APIs were entrapped in clots and infiltrated with CD11b+ leukocytes. Intraportal transplantation of APIs into athymic mice induced IBMIR, thus providing a small animal model for studying xenogeneic IBMIR.     

Effects of intrahepatic canine islet autotransplantation on the portal vein pressure and liver function

Currently, the most common method used for human islet transplantation is intrahepatic implantation via the portal vein, which may affect portal vein pressure and liver function. The aim of this study was to investigate the effects of intrahepatic canine islet autotransplantation on portal vein pressure and liver function. After total pancreatectomy was performed in 30 mongrel dogs, islets were isolated and transplanted back into the portal vein of the same dog. In our series, 12 dogs achieved normoglycemia (fasting glucose <200 mg/dL) without exogenous insulin after transplantation. The portal vein pressure increased from 4.6 +/- 1.5 to 7.7 +/- 2.9 cm H(2)O after islet infusion (P < .05). Alanine transferase amino transferase (ALT) levels gradually increased after pancreatectomy with the peak at 4 weeks after islet infusion. But the changes of portal vein pressure and ALT were not significantly different between successful and failed islet transplantation. In summary, elevation of portal vein pressure and liver enzymes were noted after intrahepatic canine islet autotransplantation. However, they did not influence the transplant outcome.     

Portal versus systemic transplantation of dispersed neonatal pancreas.

It is not known whether the advantage of the portal vein as a transplant site for islet transplantation is caused by the immediate availability of a blood supply or by the localization of the islets in the portal circulation. We transplanted minimal quantities of islet tissue from neonatal rat donors to isogeneic adult rats with streptozotocin-induced diabetes. Transplants were performed to three sites, i.p., portal vein, and systemic vein (i.v.). When four neonatal donors were used for each recipient there were no i.p. cures but 90% i.v. and 100% portal vein cures, which suggests that access to a blood supply is important. As the amount of tissue transplanted was decreased, there were significantly more cures with the portal vein route, which suggests that localization of the islets in the portal circulation is also important to graft survival.     

Transplantation of fetal rat islets into the cerebral ventricles of alloxan-diabetic rats. Amelioration of diabetes by syngeneic but not allogeneic islets

Syngeneic fetal rat islets were isolated and transplanted into alloxan-diabetic inbred male Lewis rats. The effect of transplantation of islets into the cerebral ventricles on the diabetic state of the recipients was compared with that of the conventional transplantation of islets homeotypically into the liver via the portal vein. Fourteen of fourteen rats surviving after stereotaxic implantation of islets into the ventricles returned to normoglycemia; normoglycemia has been maintained for up to 34 wk. Glucose tolerance tests revealed an improved, although not completely normalized, pattern. Histologic examination of the brains of these recipients revealed clusters of intact islets in the ventricle. These data provided a physiologic basis for further investigation of the immunologically privileged nature of the intraventricular space as a site for implantation of allogeneic pancreatic islets. Islets from Wistar-Furth rats (major histocompatibility difference) or Fischer 344 rats (minor histocompatibility difference) were transplanted into the ventricles of alloxan-diabetic Lewis rats. There were only small and unsustained changes in body weight and blood and urine glucose of any of the rats receiving the allogeneic islets. Histologic examination of the ventricles of these rats 3 wk after transplantation revealed only glial scar tissue. These data suggest that the cerebral ventricles cannot serve as a privileged site for islet transplantation, at least using the type of islet preparation employed in these experiments.     

Influence of the numbers of islets on the models of rat syngeneic-islet and allogeneic-islet transplantations

One of the main barriers to widespread application of islet transplantation is the limited availability of human pancreatic islets. The reduction of graft islet mass for transplantation to a recipient is one of the strategies in islet transplantation. However, transplantation of only a small number of islets may result in primary nonfunction. To optimize the sites and numbers of islets for transplantation, we analyzed these factors using pancreatic islets from Lewis or F344 rats transplanted into rats rendered diabetic by streptozotocin (50 mg/kg IV) and confirmed as such prior to transplantation (>300 mg/dL blood glucose). Approximately 500 to 1500 islets were injected via the portal vein or under the renal capsule into the diabetic F344 rats. The blood glucose level of all animals bearing 1500 syngeneic or allogeneic islets transplanted to the liver or under the kidney capsule exhibited restored normoglycemia (<200 mg/dL) at 1 day after transplantation. Graft function deteriorated after only 3 days in three animals (5.8%). The loss of graft function after 3 days occurred in 10 of 28 rats transplanted with 1000 to 1200 syngeneic islets, 4 of 19 rats transplanted with 800 to 900 syngeneic islets, and 7 of 17 rats transplanted with 500 to 600 syngeneic islets. There was no significant difference in the loss of graft function between the sites of transplantation via portal vein or under the kidney capsule. In conclusion, higher frequencies of primary nonfunction occurred with less than 1500 islets transplanted. They were independent of the sites in the rat-islet transplantation model.     

Hepatocyte transplantation through the hepatic vein: a new route of cell transplantation to the liver

The efficiency of hepatocyte transplantation into the liver varies with the method of administration. This study investigated whether retrograde infusion via the hepatic vein provides a sufficient number of donor cells for the liver. Donor hepatocytes were isolated from dipeptidyl peptidase IV (DPPIV(+)) rats and transplanted into DPPIV(-) rat livers either by antegrade portal vein infusion or retrograde hepatic vein infusion. Hepatocyte engraftment ratios and localization were evaluated by histological DPPIV enzymatic staining at 1 week and 8 weeks after the transplantation. No significant differences in engraftment efficiency were observed at either 1 week or 8 weeks after transplantation by either route. However, the localization of the transplanted hepatocytes differed with the administration route. Portal vein infusion resulted in predominantly periportal engraftment, whereas hepatic vein infusion led to pericentral zone engraftment. Immunohistochemical analysis showed that the transplanted hepatocytes engrafted in the pericentral zone after retrograde infusion displayed intense CYP2E1 staining similar to the surrounding native hepatocytes. CYP2E1 staining was further enhanced by administration of isosafrole, an inducing agent for various cytochrome P450 enzymes, including CYP2E1. This study demonstrates a novel approach of transplanting hepatocytes into the liver through retrograde hepatic vein infusion as the means to target cell implantation to the pericentral zone.     

应用硝普钠促进经门静脉移植的骨髓间充质干细胞在肝内弥散分布

目的探讨硝普钠是否可促进经门静脉移植的骨髓间充质干细胞(MSCs)在肝内弥散分布。方法分离SD大鼠的骨髓MSCs,进行培养、传代,流式细胞仪鉴定MSCs表面标记,以Fe2O3-PLL标记供移植的MSCs。以彩色多普勒超声检查大鼠注射硝普钠后10～20 min时门静脉内径及血流速度变化,并计算其血流量。受者(SD大鼠)背部皮下注射四氯化碳,制成肝损伤模型,将模型随机分为实验组和对照组,实验组经门静脉注射以Fe2O3-PLL标记的MSCs悬液0.5 ml及含硝普钠的生理盐水(硝普纳的注入量为24 nmol/100g),对照组仅注射Fe2O3-PLL标记的MSCs悬液0.5 ml。分别于MSCs注射前及注射后3 h、1周、2周、4周行磁共振扫描,测定肝脏信噪比(SNR)。最后1次磁共振扫描后,处死全部受者,取各叶肝组织,行HE染色和普鲁士蓝染色,进行组织学观察。结果分离获得的MSCs经培养、传代,高表达CD29(99.88%)和CD90(99.84%),CD45阳性细胞极少(0.13%)。注射硝普钠后10～20 min,大鼠门静脉内径明显扩张(P<0.01),门静脉血流量明显增多(P<0.01)。移植后随着时间延长,两组肝脏SNR均逐渐增高,实验组移植后3 h、1周、2周时的肝脏SNR明显低于对照组(P<0.05),至移植后4周,两组肝脏SNR的差异无统计学意义;两组移植后3 h、1周、2周时的肝脏SNR均明显低于移植前(P<0.05),至移植后4周,肝脏SNR恢复至移植前水平。实验组肝组织普鲁士蓝染色阳性细胞多于对照组(P<0.01)。结论门静脉注入硝普钠后,其分支内径扩大,血流量增加,在经门静脉移植MSCs的同时注入硝普钠可促进MSCs向末梢分支与血窦弥散分布。