Implanted adipose-derived stem cells attenuate small-for-size liver graft injury by secretion of VEGF in rats

Graft injury after small-for-size liver transplantation impairs graft function and threatens the survival of the recipients. The use of adipose-derived stem cells (ADSCs) for liver injury protection and repair is promising. Our aim was to investigate the role of vascular endothelial growth factor (VEGF) secreted by ADSCs in the treatment of small-for-size liver graft injury. Studies were performed using ADSCs with VEGF secretion blocked by RNA interference. In vitro, ADSCs prevented apoptosis of freshly isolated liver sinusoidal endothelial cells (LSECs) by secretion of VEGF. Syngeneic 35% orthotopic liver transplantation followed by implantation of syngeneic ADSCs through the portal vein system was performed using Wistar rats. We found VEGF secreted by implanted ADSCs improved graft microcirculatory disturbances, serum liver function parameters and survival. The improved microcirculatory status was also reflected by reduced hepatocellular damage, especially LSEC apoptosis and improved liver regeneration. These effects were accompanied by decreased expression of endothelin receptor type A, increased Bcl-2/Bax ratio, decreased expression of Bad and elevated proportion of phosphorylated Bad. In conclusion, implanted syngeneic ADSCs attenuated small-for-size liver graft injuries and subsequently enhanced liver regeneration in a rat 35% liver transplantation model. The VEGF secreted by implanted ADSCs played a crucial role in this process.     

梗阻性黄疸肝内增生胆管上皮细胞凋亡的实验研究

目的：研究梗阻性黄疸肝内胆管上皮细胞凋亡对增生胆管的影响。方法：应用末端脱氧苷酸转移酶介导的脱氧三磷酸尿苷（dUTP）缺口末端标记技术（TUNEL）观察大鼠胆道梗阻及胆肠内引流术后肝内胆管上皮细胞凋亡与胆管增生的关系。结果：胆道梗阻后，肝内胆管明显增生，胆管上皮细胞凋亡明显增加；早期胆肠内引流术后，增生的胆管及胆管上皮细胞凋亡明显减少。结论：胆道梗阻胆管上皮细胞凋亡增加可能与清除过度增生的胆管有关，是机体维持自身组织稳定的一种重要机制；早期胆肠内引流术后，随着增生的胆管减少，胆管上皮细胞凋亡明显减少。     

Embryonic stem cells attenuate viral myocarditis in murine model

We used mice to test our hypothesis that in response to viral invasion, stem cells may migrate into the heart and attenuate the effect of viral myocarditis. Male BALB/c mice were divided into three groups: mouse embryonic stem (ES) cell control, encephalomyocarditis virus (EMCV), and EMCV + ES cells. After administration of ES cells via tail vein, mice were immediately inoculated with EMCV. Mice were sacrificed at different days after EMCV inoculation. Mortality was recorded. Inflammatory cell infiltration and necrosis (major pathological changes of viral myocarditis) were evaluated by hematoxylin-eosin staining. ES cell migration and differentiation were identified by immunofluorescence. The survival rate in the EMCV + ES cell group (80%) was significantly increased (p < 0.05) over the EMCV-alone group (64%). Also, the incidence of inflammatory cell infiltration and myocardial lesions was lower in the EMCV + ES cell mice. Furthermore, the result of green fluorescent protein (GFP) and alpha-actinin analysis indicated that ES cells migrated into the heart and differentiated into myocytes after virus inoculation. In conclusion, ES cells significantly increased the survival of viral myocarditis mice and also decreased the necrosis and infiltration of inflammatory cells. These results demonstrated the ability of stem cells to mitigate the effects of viral infection on the heart and illustrated their potential therapeutic application to other mammalian species, including humans.     

Nitric oxide-mediated liver injury in the presence of experimental bile duct obstruction

We investigated the possible mechanism of common bile duct (CBD) obstruction-related liver cell necrosis in a guinea pig model during a 24-hour period of biliary occlusion. A total of 30 male albino guinea pigs were randomly and equally assigned to two groups. Group 1 underwent sham laparotomy (SL), and group 2 underwent common bile duct ligation (CBDL). All the animals were followed for the first 24-hours after operation. The liver antioxidant defense was examined by measuring liver total superoxide dismutase (TSOD), copper/zinc-containing superoxide dismutase (Cu-ZnSOD), manganese superoxide dismutase (MnSOD), and glutathione peroxidase (GPx) activities as well as the reduced glutathione (GSH) concentration. Severity of necrosis was assessed by blind quantitation of liver specimens using a histologic scoring system. Histologic evidence of grade +2 hepatocellular necrosis was observed in the CBDL group, as was a more than fourfold increase in plasma nitrite plus nitrate [NOx] concentrations in these animals. Although no significant difference was found between the two groups for liver Cu-ZnSOD activity, the CBDL group showed a marked decrease in MnSOD activity. Concomitant increases in liver GPx activity and the GSH level were measured in the CBDL group. These data supported the hypothesis that excessive production of [NOx] and its derivative peroxynitrite contribute to a coexisting MnSOD deficiency in the mitochondria and lead to liver cell necrosis in cholestatic animals.     

急性胆道梗阻肝脏损伤的实验研究

目的:探讨大鼠急性胆道梗阻后肝脏损伤的变化.方法:雄性Wistar大鼠,随机分为实验组(胆总管结扎)和对照组(假手术),观察术后不同时间点动物的一般情况、黄疸、生化指标和肝脏病理学变化.结果:对照组术后各项观察指标均无明显异常.实验组术后出现体质量降低、活动减少和明显的黄疸,但3d后均有所改善;术后血清总胆红素(TBIL)和直接胆红素(DBIL)水平明显升高,3d达到高峰,之后缓慢降低;术后谷丙转氨酶(ALT)迅速增加,1～2 d达到高峰,3d后迅速下降;碱性磷酸酶( ALP)于术后2d达到高峰,3d后逐渐下降;术后肝组织Metavir评分逐渐增加,3d时开始出现明显胶原纤维增生.结论:肝脏急性损伤期在急性胆道梗阻后1周内期间2～3 d进展达高峰,并出现纤维化.     

Injection of amniotic fluid stem cells delays progression of renal fibrosis

Injection of amniotic fluid stem cells ameliorates the acute phase of acute tubular necrosis in animals by promoting proliferation of injured tubular cells and decreasing apoptosis, but whether these stem cells could be of benefit in CKD is unknown. Here, we used a mouse model of Alport syndrome, Col4a5(-/-) mice, to determine whether amniotic fluid stem cells could modify the course of progressive renal fibrosis. Intracardiac administration of amniotic fluid stem cells before the onset of proteinuria delayed interstitial fibrosis and progression of glomerular sclerosis, prolonged animal survival, and ameliorated the decline in kidney function. Treated animals exhibited decreased recruitment and activation of M1-type macrophages and a higher proportion of M2-type macrophages, which promote tissue remodeling. Amniotic fluid stem cells did not differentiate into podocyte-like cells and did not stimulate production of the collagen IVa5 needed for normal formation and function of the glomerular basement membrane. Instead, the mechanism of renal protection was probably the paracrine/endocrine modulation of both profibrotic cytokine expression and recruitment of macrophages to the interstitial space. Furthermore, injected mice retained a normal number of podocytes and had better integrity of the glomerular basement membrane compared with untreated Col4a5(-/-) mice. Inhibition of the renin-angiotensin system by amniotic fluid stem cells may contribute to these beneficial effects. In conclusion, treatment with amniotic fluid stem cells may be beneficial in kidney diseases characterized by progressive renal fibrosis.     

HB-EGF augments the ability of mesenchymal stem cells to attenuate intestinal injury

Background

We have previously demonstrated that heparin-binding EGF-like growth factor (HB-EGF) and mesenchymal stem cell (MSC) administration protect the intestines from ischemia/reperfusion (I/R) injury in vivo, with amniotic fluid-derived MSC (AF-MSC) being more efficacious than bone marrow-derived MSC (BM-MSC). The goal of the current study was to determine whether the protective effects of HB-EGF were from direct effects on MSC or via alternative mechanisms.

Methods

Murine MSC were transfected with an HB-EGF plasmid or control plasmid by electroporation. Mice were subjected to segmental intestinal I/R injury and received either BM-MSC or AF-MSC either with or without exogenous HB-EGF, or BM-MSC or AF-MSC that endogenously over-expressed HB-EGF. MSC engraftment, intestinal histologic injury, and intestinal permeability were quantified.

Results

There was increased MSC engraftment into injured compared to uninjured intestine. HB-EGF increased AF-MSC engraftment into injured intestine. Administration of HB-EGF and MSC improved intestinal histology and intestinal permeability after I/R injury, with AF-MSC being most efficacious. The effect of HB-EGF on MSC was similar when the growth factor was administered exogenously, or when it was overexpressed endogenously.

Conclusions

The effect of HB-EGF on AF-MSC was similar with both exogenous administration and endogenous overexpression of the growth factor, implying that HB-EGF has a direct effect on AF-MSC. This information may assist in guiding potential future AF-MSC-based therapies for patients at risk of intestinal ischemic injuries.     

The role of bile salt toxicity in the pathogenesis of bile duct injury after non-heart-beating porcine liver transplantation

BACKGROUND: Intrahepatic bile duct strictures are a serious complication after non-heart-beating (NHB) liver transplantation. Bile salt toxicity has been identified as an important factor in the pathogenesis of bile duct injury and cholangiopathies. The role of bile salt toxicity in the development of biliary strictures after NHB liver transplantation is unclear. METHODS: In a porcine model of NHB liver transplantation, we studied the effect of different periods of warm ischemia in the donor on bile composition and subsequent bile duct injury after transplantation. After induction of cardiac arrest in the donor, liver procurement was delayed for 0 min (group A), 15 min (group B), or more or equal to 30 min (group C). Livers were subsequently transplanted after 4 hr of cold preservation. In the recipients, bile flow was measured, and bile samples were collected daily to determine the bile salt-to-phospholipid ratio. Severity of bile duct injury was semiquantified by using a histologic grading scale. RESULTS: Posttransplantation survival was directly related to the duration of warm ischemia in the donor. The bile salt-to-phospholipid ratio in bile produced early after transplantation was significantly higher in group C, compared with group A and B. Histopathologic condition showed the highest degree of bile duct injury in group C. CONCLUSION: Prolonged warm ischemia in NHB donors is associated with the formation of toxic bile after transplantation, with a high biliary bile salt-to-phospholipid ratio. These data suggest that bile salt toxicity contributes to the pathogenesis of bile duct injury after NHB liver transplantation.     

Defective renal autoregulation in the chronic bile duct ligation model of liver failure

Background

Cirrhosis of the liver is often associated with an impairment of renal function that is usually not associated with consistent structural abnormalities of the renal parenchyma, but is thought to be the functional consequence of arterial underfilling and reduced arterial blood pressure.

Method

We have used the cirrhosis model of chronic bile duct ligation (BDL) to assess the response of renal blood flow to a change of blood pressure. We have measured renal haemodynamics in BDL rats.

Result

Three weeks after BDL, rats showed elevated levels of total bilirubin, AST, and ALT as well as reduced arterial blood pressure. Creatinine clearance was significantly reduced, and plasma creatinine and urea nitrogen were elevated. Renal blood flow at baseline blood pressure was significantly lower in the BDL group than in the sham group. Clamp-induced reductions of renal perfusion pressure caused significantly greater changes of renal blood flow in BDL than control rats. The autoregulatory index over a comparable blood pressure range averaged 0.28?±?0.35 in control rats and 1.26?±?0.6 in BDL rats (p?=?0.0004) indicating impairment of renal autoregulation in liver cirrhosis.

Conclusion

Tubuloglomerular feedback (TGF) responses were significantly attenuated in BDL rats, especially in the subnormal flow range. Impairment of renal blood flow autoregulation, to some extent mediated by reduced TGF-mediated vasodilatation, may contribute to the renal vascular constrictor state in liver cirrhosis by preventing the full dilatory response to the blood pressure reduction.

     

医源性胆管损伤的治疗进展

胆管损伤一直是普外科倍受关注的问题.随着人们对胆管损伤后果严重性认识的提高,并采取了许多积极有效的预防措施,使胆管损伤的发病率有所下降,并随着医学技术的发展和大量的临床实践总结使已经造成胆管损伤的患者在治疗方法和临床疗效上都有了很大改善.同时又面临许多新的困难.本文概述了医源性胆管损伤的治疗现状.     

小切口胆囊切除术中胆道损伤的预防

目的探讨预防小切口胆囊切除术（MC）中胆道损伤的方法。方法对本组15131例MC中发生的18例胆道损伤进行回顾分析。结果1994年12月前施行MC2321例，发生胆道损伤9例（0．39％），1995年以来实施MC12810例，发生胆道损伤9例（0．07％）。结论思想上高度重视、规范手术适应证、规范手术操作是预防MC中胆道损伤的关键。     

Human liver stem cells alleviate Con-A induced liver injury by regulating the balance of Treg/Th17 cells

BackgroundLiver injury is a serious threat to human health that has become a worldwide problem. To date, there is still no effective treatment strategy. In the present study, we examined the protective effects of Human liver stem cells (HLSCs) against concanavalin A (Con A)-induced acute liver injury.MethodsIsolated HLSCs were characterized by microscopy, functional assays, and gene expression. HLSCs or HLSCs culture medium were transplanted in mice for 12 h and subsequently challenged with Con A via tail-vein injection. The effects were evaluated through survival rate, histology, blood tests, TUNEL assay, quantitative RT-PCR and flow cytometry. CellTracker™ CM-Dil labled HLSCs were tracked by fluorescence microscope.ResultsTransplantation of HLSCs reduced the mortality rate, reduced the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL), narrowed the area of liver necrosis, and inhibited hepatocyte apoptosis induced by Con A. Injection of HLSCs culture medium could also alleviate Con A-induced liver injury. Of note, HLSCs-transplanted mice exhibited lower frequencies of Th17 cells and higher frequencies of Tregs in their liver and spleen following Con A injection. Moreover, transplantation of HLSCs significantly reduced the expression of IL-17A, IL-17F and ROR-γt induced by Con A, while reversed Con A-induced downregulation of Foxp3 expression and IL-10.ConclusionsHLSCs protect mice from immune-mediated liver injury by regulating the balance of Treg/Th17 cells, suggesting that transplantation of HLSCs is a potential and effective therapeutic method for amelioration of liver injury.     

单中心腹腔镜胆囊切除术预防胆管损伤的体会

目的:探讨如何预防腹腔镜胆囊切除术(LC)胆管损伤。方法:回顾分析37 781例LC的临床资料。结果:胆管损伤25例(0.066%),其中术中发现8例,术后发现17例。胆管横断伤12例,其中离断+缺损8例(1例是中转开腹损伤),钛夹夹闭无胆管缺损4例;胆总管部分剪切伤4例;肝总管电损伤2例,分离损伤2例;右肝管损伤3例;副肝管损伤2例。胆管修补(端端吻合)+T管支撑引流5例、胆肠Roux-en-Y吻合16例,腹腔穿刺+鼻胆管引流1例(ENBD)、损伤胆管修复,置管引流3例。无死亡病例。结论:熟悉肝门解剖,仔细处理Calot三角,适时中转开腹,避免盲目自信可以有效的降低胆管损伤的发生率。     

胆囊床胆管的损伤与处理对策

目的　提高对胆囊床胆管及其损伤的认识和处理对策。方法　回顾性分析了胆囊切除后胆囊床胆管损伤 37例的诊治情况。结果　 37例中 ,证实为Luschka胆管 17例、胆囊肝管 4例、右前叶肝管及其分支 3例 ;其余 13例仅发现胆瘘而未见损伤胆管。本组 30例经胆瘘处缝合引流或单纯引流治愈 ,4例未置引流者因局限性胆汁积聚经皮置管或穿刺引流治愈 ,另 3例因病情恶化而再次剖腹手术。结论　胆囊切除时易致胆囊床胆管损伤 ,术中仔细检查裸露胆囊床有无胆瘘应列为常规步骤 ,一旦发现有胆瘘 ,不论对胆漏处如何处理均应放置引流     

腹腔镜下胆囊切除术胆管损伤的预防

我院近5年来施行腹腔镜胆囊切除术1560例，术中采取以肝十二指肠韧带右缘作为手术操作范围的右缘线，有助于术中避免胆管损伤。对解剖关系不清者及时中转开腹手术，其中13例（0．83％）因胆囊三角区冷冻样粘连而中转开腹手术。本组无胆管损伤或死亡病例。