Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system.

回顾性分析南京大学医学院附属鼓楼医院肝胆胰中心手术后病理诊断的2例胰腺内异位副脾的临床资料，术前均误诊为胰腺神经内分泌肿瘤。本研究提示对于胰尾部的单发圆形实质性肿块，如果直径较小，CT强化方式和CT值与脾脏类似，应警惕胰腺内异位副脾的可能。可以结合超声造影、CT、MRI、核素扫描显像、生长抑素受体显像等加以诊断。如果仍然难以确诊，可以选择内镜超声下穿刺活检。

Background:Prognosis of hepatocellular carcinoma (HCC) is closely related to residual tumor cells and tissues after tumor resection. Thus, close monitoring to ensure complete removal of residual tumor is fundamental. In this regard, intraoperative near-infrared fluorescence (NIRF) imaging has been of great assistance to surgeons for precision cancer surgeries. However, up to now, the identification of tiny lesions has not been reported. Herein, we report our findings on the case of an ultra-small HCC focus of about 430 µm that was successfully detected using NIRF during real-time monitored liver cancer surgery. The patient had a background of hepatitis B cirrhosis, which is the most phenomenon in China. Surgeons usually unable to distinguish sclerotic nodules from small tumor tissue with the naked eyes.Case Description:A 55-year-old man with chronic hepatitis B infection was preoperatively diagnosed with a space-occupying liver lesion. A fluorescence signal was detected on the surface of the liver through the NIRF imaging system which had not been found by preoperative computed tomography (CT) and ultrasound examination. We subsequently tested the residual liver surface and observed a high signal point, less than 1 mm in the right anterior lobe of the liver. Histopathological examination revealed that the tiny fluorescent spot belong to an early HCC focus.Conclusions:Based on these results, we think indocyanine green (ICG)-NIRF imaging may be used as a routine intraoperative detection method for liver cancer surgery in order to remove any residual tumor cells and tissue, hence minimizing further risk of remnant tumor regrowth.

目的 比较机器人与传统开腹手术行肝叶切除术治疗肝内胆管细胞癌(ICC)患者的安全性和短期疗效.方法 2019年1月～2020年12月我院诊治的ICC患者27例,其中9例接受机器人手术,18例接受传统开腹肿瘤根治术,比较两组手术情况.结果 两组均完成肿瘤根治术;机器人组和开腹组患者手术时间[(198±32)分对(215±74)分]、术中出血量[200(100,250)ml对(275(200,300)ml]和术中输血次数(0次对2次)均无统计学差异(P>0.05);机器人组和开腹组患者肿瘤直径[4.0(2.5,5.5)cm对6.3(3.9,6.5)cm]、R0切除率(100.0％对88.0％)和淋巴结转移率(44.4％对38.9％)比较,差异无统计学意义(P>0.05);机器人组术后卧床时间和住院日分别为2(1,2.5)d和7(4,8)d,显著短于开腹组[分别为3(1.8,3.5)d和11(8,12)d,P<0.05],机器人组住院费用为11.3(9.1,13.5)万,与开腹组的10.1(8.8,11.5)万比,无统计学差异(P>0.05);两组术后并发症发生率为11.1％和27.8％,无统计学差异(P>0.05).结论 开展机器人肿瘤根治术治疗ICC患者安全,术后恢复快.

Suicide is the most severe consequence of depression which has become a leading cause of disability and a global disease burden. Recent evidence indicates a central role of small molecules in the pathogenesis of depression and associated suicidal behaviors. However, there lacks a systemic exploration of small molecules in the development of depression-associated suicide, and it remains unclear how they affect an individual's behavior. In order to compare the metabonomic profiles between drug-naïve patients with depression-associated suicidal behaviors and healthy individuals, we conducted a systemic database search for studies of metabolic characteristics in depression-associated suicidal behavior. Manual data curation and statistical analysis and integration were performed in Excel. We further performed an enrichment analysis of signaling pathway prediction using the Reactome Pathway Analysis tool. We have identified 17 metabolites that expressed differently between drug-naïve patients with depression-associated suicidal behaviors and healthy controls. We have integrated these metabolites into biological signaling pathways and provided a visualized signaling network in depressed suicidal patients. We have revealed that "transport of small molecules", "disease", "metabolism" and "metabolism of proteins" were the most relevant signaling sections, among which "transport of inorganic cations/anions and amino acids/oligopeptides", "SLC-mediated transmembrane transport", and "metabolism of amino acids and derivatives" should be further studied to elucidate their potential pathogenic mechanism in the development of depression and associated suicidal behavior. In conclusion, our findings of these 17 metabolites and associated signaling pathways could provide an insight into the molecular pathogenesis of depression-associated suicidal behavior and potential targets for new drug inventions.

Acetaminophen (APAP) induced liver injury is a leading cause for drug-induced liver damage. Glabridin is an isoflavonoid that is reported to possess anti-inflammatory and oxidative stress preventing effects. The aim of this study was to evaluate the protective effects of glabridin against APAP-induced liver injury in rats. Rats were pre-administered with glabridin (30 mg/kg, p.o.; 7 days) followed by single dose of APAP (500 mg/kg, i.p.; 7th day) one hour after glabridin administration. The resulting levels of liver enzymes (AST, ALT, ALP), lipid peroxidation, antioxidant enzymes (CAT, GPx, SOD, GST), reduced glutathione (GSH), inflammatory mediators (TNF-α, IL-6, IL-1β), and apoptosis regulator proteins (casp-3, casp-9, Bcl2, Bax) were determined. Oxidative stress, inflammation and apoptosis triggered due to APAP-induced liver injury were significantly alleviated (p < 0.05) by glabridin. Western blot analysis showed the regulatory effect of glabridin on phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and Nrf2/HO-1 protein expressions against APAP-induced toxicity. Histopathological (H&E) data provided evidence for the protective effect of glabridin on rat liver tissue from injury caused by APAP. In conclusion, the protective effect of glabridin on APAP-induced liver injury was exerted by activating the PI3K/Akt/Nrf2/HO-1 pathway, inhibiting oxidative stress, inflammation, and apoptosis.

Background Boot camp can enable residents to acquire surgical skills and confidence, but they can lose these skills over time if they do not use them. The purpose of this study was to explore whether boot camp and subsequent repetitive practice could strengthen residents’ clinical skills and self-confidence. Methods This is a comparative study of surgical residents who were enrolled in our institution from 2016 to 2017. The residents in the experimental group (enrolled in 2017) received boot camp training and a year of repetitive practice. The control group (enrolled in 2016) only received routine residency training. The rotation assessment pass rates of the two groups during the first year of the residency training were compared. A survey was conducted at different points in time to investigate the influence of boot camp and repetitive practice on the confidence of the residents. Results The assessment pass rate of the experimental group was significantly higher than that of the control group ( p < 0.05). The residents’ confidence in themselves improved significantly after the boot camp, and it was comparable to that of the residents in the control group after their first year of residency. The level of self-confidence of the experimental group was further improved after repetitive practice. Finally, residents in the experimental group received better evaluations by their colleagues than the control group received. Conclusions This study showed that boot camp can improve the surgical skills and confidence of residents and that repetitive practice can further strengthen them. Residents in the experimental group developed their self-confidence in boot camp, and it increased after repetitive practice.

肝功能衰竭病情凶险、进展迅速、预后较差,严重危害人民健康.肝移植是治疗肝衰竭最有效方法,但由于器官短缺,仅有少数患者能及时接受移植.干细胞相关技术(包括干细胞移植、生物人工肝等)因具有解毒、合成、代谢、分泌等功能,为这一问题的解决带来了希望.此项目在干细胞移植和生物人工肝2个领域取得了多项原创性成果,开创了肝衰竭治疗的新领域,具有显著的经济和社会效益.文中主要从3方面进行阐述:干细胞移植治疗肝功能衰竭的机制探讨和临床转化研究;骨髓间充质干细胞与猪肝细胞共培养体系的建立和基于共培养细胞的生物人工肝临床转化研究;人源化肝样诱导细胞的建立和基于人源化种子细胞的生物人工肝的临床转化研究.

In recent years, transplantation of mesenchymal stem cells (MSCs) has attracted much attention as a potential cell-based therapy for acute liver failure (ALF). As an inducible enzyme, heme oxygenase 1 (HO-1) has been reported to have cytoprotective, anti-apoptotic and immunoregulatory effects. Autophagy, a conserved catabolic process in cells, may be an important pathway for MSCs to treat ALF. In this study, we aimed to explore whether MSCs treat ALF by regulating autophagy and whether HO-1 was involved in the same pathway. Bone marrow-derived MSCs were isolated from Sprague-Dawley rats and cultured according to an established protocol. Co-culture systems of MSCs and hepatocytes were used to assess autophagy in the treatment of ALF. Meanwhile, MSCs were transplanted into rats with d-galactosamine (Gal)-induced ALF. Autophagy inhibitor (3-methyladenine, 3-MA), HO-1 inhibitor (zinc protoporphyrin, ZnPP) and PI3K specific inhibitor (LY294002) were employed in the study. Blood samples and liver tissues were collected before euthanasia. Survival rate, liver function, inflammatory factors, histology, Ki67 and TUNEL staining were determined. MSCs transplantation alleviated ALF both in vivo and in vitro. Autophagy and autophagy-related proteins were significantly up-regulated during MSCs treatment. 3-MA attenuated the therapeutic effect of MSCs. Administration of LY294002 before ALF induction inhibited hepatocyte autophagy. During the MSCs treatment, the HO-1 expression was increased, while inhibiting HO-1 attenuated the therapeutic effect of MSCs as well as hepatocyte autophagy. These findings suggested MSCs could alleviate ALF by increasing the HO-1 expression, which played an important role in activating autophagy through PI3K/AKT signaling pathway.

Aim: Recently, the benefit ofmesenchymal stemcells (MSCs) as a cell-based therapy for acute liver failure (ALF) has gained much attention, although the mechanism of action of MSCs in the treatment of ALF remains elusive. Pyroptosis is a novel form of programmed cell death with an intense inflammatory response. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects through inhibiting pyroptosis in ALF. Methods: Mesenchymal stem cells obtained from C57BL/6 mice were isolated and cultured according to an established protocol. The MSCs were transplanted into mice with D-galactosamine (D-Gal)-induced ALF. Liver function, survival rate, histology, and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)-10, ShIL-RNA, and MCC950 (NLRP3 inhibitor) were given to the mice to explore the therapeutic mechanism of MSCs. Statistical analyses were carried out with SPSS version 19.0, and all data were analyzed by independent-samples t-test. Results: Injection of IL-10 or MSC transplantation ameliorated D-Gal-induced increase in alanine aminotransferase, aspartate aminotransferase, total bilirubin, NH3, and inflammatory cytokines. Blockage of IL-10 confirmed the therapeutic significance of this cytokine. Conclusion: Pyroptosis was inhibited after IL-10 infusion and inhibition of NLRP3 by MCC950 reversed liver dysfunction.

Mesenchymal stem cells (MSCs) have been studied for the treatment of acute liver failure (ALF) for several years. MSCs may exert their effect via complex paracrine mechanisms. Heme oxygenase (HO) 1, a rate-limiting enzyme in heme metabolism, exerts a wide range of anti-inflammatory, anti-apoptotic and immunoregulatory effects in a variety of diseases. However, the relationship between MSCs and HO-1 in the treatment of ALF is still unclear. We investigated the preventive and therapeutic potential of intravenously administered BMSCs.

Objective To study the synergetic effect and possible mechanism of transplanting mesenchymal stem cells (MSCs) in combination with interleukin-1 receptor antagonist (IL-1Ra) on acute liver failure (ALF).Methods MSCs transplantation combined with IL-1Ra was used for a swine model of ALF induced by 85％ total hepatectomy.The living conditions,blood samples and survival time were recorded or collected for analysis of hepatic function.Liver injury histology was analyzed.Hepatic cell regeneration and apoptosis were studied by immunohistochemistry staining of Ki67 and TUNELassays respectively.The expression levels of AKT and NF-κB were analyzed by Western blotting.Results The difference on the survival time between the model group and combined therapy group was statistically significant (P ＜ 0.05).Combined therapy displayed improvement not only in the serum biochemical conditions but also in the serum inflammatory cytokines.Furthermore,the observed hepatic histopathological score was significantly less compared to model group.In addition,the combined therapy group significantly inhibited the liver cell apoptosis and increased hepatic cell regeneration.Finally,a significant increase in AKT expression and decrease of NF-κB expression (P ＜ 0.05) were observed,which was consistent with their important roles in liver regeneration.Conclusion The combined therapy displayed a synergistic effect on liver regeneration,by promoting restoration and reconstruction of ALF,through regulation of inflammation and apoptosis signaling network.

Poly (ADP-ribose) polymerase 1 (PARP-1) is a crucial contributor to exacerbate ischemia and reperfusion (IR) injury and cancer process. However, there is little research into whether PARP-1 affects the hepatocellular carcinoma (HCC) recurrence after liver transplantation. In this study, we investigated the influence of PARP-1 on hepatic neutrophil mobilizing and phenotype shifting which may lead to HCC recurrence after liver transplantation. We found that rats received the grafts with warm ischemic injury had higher risk of HCC recurrence, which was markedly prevented by pharmacological inhibition of PARP-1 after liver transplantation. In mouse models, the up-regulation of PARP-1 was closely related to the greater tumor burden and increased hepatic susceptibility to recurrence after IR injury. The reason was that high hepatic PARP-1 led to increased liver CXCL1 levels, which in turn promoted recruitment of neutrophils. Both blocking CXCL1/CXCR2 signaling pathway and depleting neutrophils decreased tumor burden. Moreover, these infiltrating neutrophils were programmed to a proangiogenic phenotype under the influence of PARP-1 in vivo after hepatic IR injury. In conclusion, IR-induced PARP-1 up-regulation increased the hepatic recruitment of neutrophils through regulation of CXCL1/CXCR2 signaling and polarized hepatic neutrophils to proangiogenic phenotype, which further promoted HCC recurrence after transplantation.

Objective To establish a porcine model of liver failure after different percent hepatectomy.Methods The porcine models of liver failure 75%,85%,95% hepatectomy were developed and the living conditions and survival time were recorded.The blood samples of pre-surgery,post-hepatectomy d1,d3,d5 and post-hepatectomy 1 week,2 weeks,and 3 weeks were collected for hepatic function analysis.Histological examination of liver tissues was performed using HE staining.Liver injury histology was interpreted and scored in the terminal samples.Results The average survival time of pigs with post-hepatectomy liver failure after 75%,85%,95% hepatectomy was 19.0±5.6 days,17.3±5.5 days,1.3±1.5 days,respectively.Their pathological scores were 5.67±0.52,8.17±0.82 and 8.50±0.71,respectively.With the increase of percent hepatic resection,the incidence of hepatic failure was increasing.ALT,AST,ALP,LDH and TBA were dramatically increased in the pigs after 85% hepatectomy.Conclusions The pig model of acute liver failure by 85% hepatectomy is successfully established,which can cause typical acute liver failure in Bama miniature pigs.

Objective To explore the therapeutic effects of soluble cytokines secreted by mesenchymal stem cells (MSCs) on acute liver failure (ALF).Methods MSCs isolated from Sprague-Dawley rats were determined by FACS analysis.Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray.SD rats were divided into 3 groups:(1) ALF + dulbecco's modified eagle medium (DMEM) group:1 ml DMEM was injected into SD rats after D-Gal administration;(2) ALF + MSCs group:1 ml MSCs (1 × 106) was injected into SD rats after D-Gal administration;(3) ALF + MSCs-CM group:1 ml MSCs-CM was injected into SD rats after D-Gal administration.Biochemical indicators,survival rate,histology and inflammatory factors were studied.Exogenous recombinant rat IL-10,antirat IL-10 antibody and AG490 (STAT3 signaling pathway inhibitor) were administrated to explore the therapeutic mechanism of MSCs-CM.Results The respective serum biochemical indexes of ALF + DMEM group,ALF + MSCs group,and ALF + MSCs-CM group were:ALT (1 709.8 ± 372.1,865.5 ± 52.8,964.7 ± 414.6 U/L),AST (4234.0 ± 807.3,2440.8 ± 511.9,2739.8 ± 587.3 U/L),andTBil (79.3 ± 10.9,43.8 ± 7.0,61.2 ± 6.7 μg/L).The survival rates of the three groups were 10.0％,80.0％,and 70.0％,respectively.The levels of inflammatory factors in each group were IFN-γ (69.8 ± 4.7,46.4 ± 4.3,54.6 ± 2.4pg/ml),IL-1β (58.5 ± 7.6,40.5 ± 6.9,44.1 ± 6.0pg/ml),IL-6 (71.9 ± 16.1,38.4 ± 7.7,45.3 ± 9.0),and IL-10 (38.3 ± 6.0,75.4 ± 11.1,59.6 ± 11.9 pg/ml).Protein microarray results suggested that MSCs-CM expresses a variety of inflammatory-related cytokines,with IL-10 levels being most pronounced.IL-10 (ALT 1 126.9 ± 419.3 U/L,AST2370.8 ± 561.2 U/L) alone significantly reduced transaminase levels compared with ALF group (ALT 1 709.8 ± 372.1 U/L,AST 4234.0 ± 807.3 U/L),while anti-IL-10 antibody (ALT 1 568.5 ± 325.4 U/L,AST4043.7 ± 819.0 U/L) neutralized the therapeutic effect of MSCs-CM (ALT 964.7 ± 414.6 U/L,AST 2 739.8 ± 587.3 U/L).IL-10 could significantly increase the level of pSTAT3 in ALF rats (0.93 ± 0.03 vs 0.68 ± 0.01),while STAT3 inhibitor AG490 (0.84 ± 0.04) could decrease the expression of pSTAT3 and reverse the therapeutic effect of IL-10.Conclusion The factors released by MSCs,especially IL-10,have the potential therapeutic effect on ALF,and STAT3 signaling pathway may mediate the anti-inflammation effects of IL-10.

Background: Uncontrolled hapatic inflammatory response is regarded as the primary pathological mechanism of acute liver failure and impairs the regeneration of hepatocytes and stem cell grafts. Interleukin-1 plays a key role for activating immune and inflammatory response. Recently, siRNA has made quite a few progresses in treating inflammatory response.Aim. To assess the effect of IL-1β siRNA adenovirus on MSC and the therapeutic effect of MSC combined with IL-1β siRNA adenovirus in ALF.Material and methods. We implanted MSC or/and IL-1β siRNA adenovirus via the tail vein, using CCl4-induced ALF in a mice model. Mice were sacrificed at different time points. Blood samples and liver tissues were collected. Hepatic injury, liver regeneration, cytokines (CXCL1, IL-1β, IL-10, IL-6, VEGF and HGF), animal survival and vital MSC were assessed after cell transplantation.Results. MSC combined with IL-1β siRNA reduced the inflammatory levels and prevented liver failure. These animals administrated with MSC and IL-1β siRNA also exhibited improved liver regeneration and increased survival rates. Immunohistochemistry and fluorescence microscopy revealed the number of vital MSC in ALF + MSC + IL-1β siRNA group were significantly more than that in ALF + MSC group.Conclusion. IL-1β siRNA adenovirus could enhance MSC ability of tissue regeneration through increasing its survival rate. Accordingly, combination of IL-1β siRNA adenovirus and MSC had a synergistic effect on acute liver failure.

BACKGROUND:Mesenchymal stem cells (MSCs) transplantation has been proven to have therapeutic potential for acute liver failure (ALF). However, the mechanism remains controversial. Recently, modulation of inflammation by MSCs has been regarded as a crucial mechanism. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects in ALF.METHODS:MSCs isolated from Sprague-Dawley rats were identified by fluorescence-activated cell sorting analysis. Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray. MSCs and MSCs-CM were transplanted into rats with D-galactosamine-induced ALF. Liver function, survival rate, histology, and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)-10, anti-rat IL-10 antibody, and AG490 (signal transducer and activator of transcription 3 [STAT3] signaling pathway inhibitor) were administered to explore the therapeutic mechanism of MSCs-CM. Statistical analysis was performed with SPSS version 19.0, and all data were analyzed by the independent-sample t-test.RESULTS:There are statistical differences of the survival curve between ALF+MSCs group and ALF+Dulbecco's modified Eagle's medium (DMEM) group, as well as ALF+MSCs-CM group and ALF+DMEM group (all P < 0.05). Serum alanine aminotransferase (ALT) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (865.53±52.80 vs. 1709.75±372.12 U/L and 964.72±414.59 vs. 1709.75±372.12 U/L, respectively, all P < 0.05); meanwhile, serum aspartate aminotransferase (AST) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (2440.83±511.94 vs. 4234.35±807.30 U/L and 2739.83±587.33 vs. 4234.35±807.30 U/L, respectively, all P < 0.05). Furthermore, MSCs or MSCs-CM treatment significantly reduced serum interferon-γ (IFN-γ), IL-1β, IL-6 levels and increased serum IL-10 level compared with DMEM (all P < 0.05). Proteome profile analysis of MSCs-CM indicated the presence of anti-inflammatory factors and IL-10 was the most distinct. Blocking of IL-10 confirmed the therapeutic significance of this cytokine. Phosphorylated STAT3 was upregulated after IL-10 infusion and inhibition of STAT3 by AG490 reversed the therapeutic effect of IL-10.CONCLUSIONS:The factors released by MSCs, especially IL-10, have the potential for therapeutic recovery of ALF, and the STAT3 signaling pathway may mediate the anti-inflammatory effect of IL-10.

Liver fibrosis, which is the pathophysiologic process of the liver due to sustained wound healing in response to chronic liver injury, will eventually progress to cirrhosis. Puerarin, a bioactive isoflavone glucoside derived from the traditional Chinese medicine pueraria, has been reported to have many anti-inflammatory and anti-fibrosis properties. However, the detailed mechanisms are not well studied yet. This study aimed to investigate the effects of puerarin on liver function and fibrosis process in mice induced by CCl4. C57BL/6J mice were intraperitoneally injected with 10% CCl4 in olive oil(2mL/kg) with or without puerarin co-administration (100 and 200mg/kg intraperitoneally once daily) for four consecutive weeks. As indicated by the ameliorative serum hepatic enzymes and the reduced histopathologic abnormalities, the data collected showed that puerarin can protect against CCl4-induced chronic liver injury. Moreover, CCl4-induced development of fibrosis, as evidenced by increasing expression of alpha smooth muscle actin(α-SMA), collagen-1, transforming growth factor (TGF)-β and connective tissue growth factor(CTGF) in liver, were suppressed by puerarin. Possible mechanisms related to these suppressive effects were realized by inhibition on NF-κB signaling pathway, reactive oxygen species(ROS) production and mitochondrial dysfunction in vivo. In addition, these protective inhibition mentioned above were driven by down-regulation of PARP-1 due to puerarin because puerarin can attenuate the PARP-1 expression in CCl4-damaged liver and PJ34, a kind of PARP-1 inhibitor, mimicked puerarin's protection. In conclusion, puerarin played a protective role in CCl4-induced liver fibrosis probably through inhibition of PARP-1 and subsequent attenuation of NF-κB, ROS production and mitochondrial dysfunction.