制备导管大鼠模型观察置管手术对肝功能的影响

【目的】观察开腹置管手术对大鼠肝功能的影响。【方法】制备导管大鼠模型 ,在术后不同时点收集胆汁标本检测胆汁流量 (bileflow ,BF)、总胆酸 (totalbileacid ,TBA) ,谷丙转氨酶 (ALT)和谷胱甘肽 (glutathione ,GSH/GSSG)等重要肝功能指标。【结果】BF各时点值间无显著性差异 (P均 >0 0 5 ) ;TBA在术后 2 4h内明显降低 ,各时点值间的两两比较 ,除 48与 72h之外 ,均有显著性差异 (P <0 0 5或 0 0 1) ;ALT以术后 6h最高 ,但各组数值仍在正常范围 ;GSH和GSSG在术后即时测值最高 ,6h最低 ,二者之间有正相关关系。【结论】开腹置管手术明显降低肝脏分泌胆酸的功能 ,但对胆汁分泌量、ALT、GSH以及GSSG等其他肝功能的影响较小。

Potential risk factors for nonalcoholic steatohepatitis related to pancreatic secretions following pancreaticoduodenectomy

AIM: To identify risk factors for nonalcoholic steatohepatitis following pancreaticoduodenectomy, with a focus on factors related to pancreatic secretions. METHODS: The medical records of 228 patients who had a pancreaticoduodenectomy over a 16-mo period were reviewed retrospectively. The 193 patients who did not have fatty liver disease preoperatively were included in the final analysis. Hepatic steatosis was diagnosed using the differences between splenic and hepatic attenuation and liver-to-spleen attenuation as measured by non-enhanced computed tomography. RESULTS: Fifteen patients (7.8%) who showed postoperative hepatic fatty changes were assigned to Group A, and the remaining patients were assigned to Group B. Patient demographics, preoperative laboratory findings (including levels of C-peptide, glucagon, insulin and glucose tolerance test results), operation types, and final pathological findings did not differ significantly between the two groups; however, the frequency of pancreatic fistula (P = 0.020) and the method of pancreatic duct stenting (P = 0.005) showed significant differences between the groups. A multivari- ate analysis identified pancreatic fistula (HR = 3.332, P = 0.037) and external pancreatic duct stenting (HR = 4.530, P = 0.017) as independent risk factors for the development of postoperative steatohepatitis. CONCLUSION: Pancreatic fistula and external pancreatic duct stenting were identified as independent risk factors for the development of steatohepatitis following pancreaticoduodenectomy.

Endocrine and paracrine role of bile acids

Bile acids are not only important for the absorption of dietary lipids and fat soluble vitamins but are signalling molecules with diverse endocrine and paracrine functions. Bile acids regulate bile acid, lipid and glucose metabolism and modulate temperature and energy homeostasis. Furthermore, bile acids can not only promote cell proliferation and liver regeneration but can also induce programmed cell death. Bile acid functions are mediated through different pathways which comprise the activation of nuclear hormone receptors, of intracefular kinases and of the plasma membranebound, G-protein coupled bile acid receptor TGRS/Gpbar-1.

Heterogeneity in predisposition of hepatic cells to be induced into pancreatic endocrine cells by PDX-1

AIM: The role of Pancreatic and Duodenal Homeobox-1(PDX-1) as a major regulator of pancreatic development determines the function and phenotype of β cell. In this study, potential plasticity of liver cells into pancreatic endocrine cells induced by PDX-1 was evaluated.METHODS: Human hepatoma cell line HepG2 was stably transfected with mammalian expression plasmid pcDNA3-PDX encoding human PDX-1 gene. Ectopic expression of PDX-1 and insulin were detected by RT-PCR,Western blot and/or immunostaining. PDX-1+ HepG2 cells were transplanted under renal capsule of STZ-induced diabetic nude mice (n = 16) to examine the inducing effect in vivo.RESULTS: Exogenous PDX-1 transgene was proved to express effectively in HepG2 cell at both mRNA and protein levels. The expression of endogenous insulin and some βcell-specific differentiation markers and transcription factors were not induced in PDX-1+ HepG2 cells. When transplanted under renal capsule of STZ-induced diabetic nude mice, PDX-1+ HepG2 cells did not generate insulinproducing cells. These data indicated that stable transfected PDX-1 could not convert hepatoma cell line HepG2 to pancreatic cells in vitro or in vivo. Mature hepatocytes might need much more complicated or rigorous conditions to be shifted to insulin-producing cells.CONCLUSION: The expression of exogenous PDX-1 is not sufficient to induce relatively mature hepatocytes differentiating into insulin-producing cells.

Taurolithocholate impairs bile canalicular motility and canalicular bile secretion in isolated rat hepatocyte couplets

AIM: To investigate the effects of taurolithocholate (TLC)on the canalicular motility in isolated rat hepatocyte cou-plets (IRHC).METHODS: TLC was added to IRHC at concentrationsof 10 and 50 μmol/L, respectively. In each group, fi vetime-lapse movies containing 3 representative bile cana-liculi were taken under phase-contrast microscopy for12 h. The number of bile canalicular contractions andthe intervals between consecutive canalicular contrac-tions were calculated. Furthermore, the effects of TLC onIRHC were examined by transmission electron micros-copy.RESULTS: The bile canalicular contractions were spon-taneous and forceful in the controls. Active vesicularmovement was observed in the pericanalicular region.Immediately after the addition of TLC, the bile canaliculiwere deformed, and canalicular bile was incorporatedinto the vacuoles. The canaliculi were gradually dilated,and canalicular contractions were markedly inhibited byTLC. The vesicular movements became extremely slowin the pericanalicular region. The number of canalicularcontractions significantly decreased in the TLC-treatedgroups, as compared with that in the controls. The timeintervals were prolonged, as the TLC dosage increased,indicating that bile secretion into the canaliculi wasimpaired with TLC. Transmission electron microscopyrevealed the lamellar transformation of the canalicularmembranes in IRHC treated with TLC.CONCLUSION: TLC impairs both the bile canalicularcontractions and the canalicular bile secretion, possiblyby acting directly on the canalicular membranes in TLC-induced cholestasis.

Liver transplantation for metastatic neuroendocrine tumor: A case report and review of the literature

Neuroendocrine tumors are divided into gastrointestinal carcinoids and pancreatic neuroendocrine tumors. The WHO has updated the classification of these lesions and has abandoned the term ＂carcinoid＂. Both types of tumors are divided into functional and non-functional tumors. They are characterized by slow growth and frequent metastasis to the liver and may be limited to the liver for long periods. The therapeutic approach to hepatic metastases should consider the number and distribution of the liver metastases as well as the severity of symptoms related to hormone production and tumor bulk. Surgery is generally considered as the first line therapy. In patients with unresectable liver metastases, alternative treatments are dependent on the type and the growth rate. Initial treatments consist of long acting somatostatin analogs and/or interferon. Streptozocin-based chemotherapy is usually reserved for symptomatic patients with rapidly advancing disease, but generally the therapy is poorly tolerated and its effects are short-lived. Locoregional therapy directed such as hepatic-artery embolization and chemoembolization, radiofrequency thermal ablation and cryosurgery, is often used instead of systemic therapy, if the disease is limited to the liver. However, liver transplantation should be considered in patients with neuroendocrine metastases to the liver that are not accessible to curative or cytoreductive surgery and if medical or Iocoregional treatment has failed and if there are life threatening hormonal symptoms. We report a case of liver transplantation for metastatic neuroendocrine tumor of unknown primary source and provide a detailed review of the world literature on this controversial topic.

Macrophage secretory products induce an inflammatory phenotype in hepatocytes

AIM:To investigate the influence of macrophages on hepatocyte phenotype and function.METHODS:Macrophages were differentiated from THP-1 monocytes via phorbol myristate acetate stimulation and the effects of monocyte or macrophageconditioned medium on HepG2 mRNA and protein expression determined.The in vivo relevance of these findings was confirmed using liver biopsies from 147 patients with hepatitis C virus(HCV)infection.RESULTS:Conditioned media from macrophages,but not monocytes,induced a transient morphological change in hepatocytes associated with upregulation of vimentin(7.8±2.5-fold,P=0.045)and transforming growth factor(TGF)-β1(2.6±0.2-fold,P<0.001)and downregulation of epithelial cadherin(1.7±0.02-fold,P=0.017)mRNA expression.Microarray analysis revealed significant upregulation of lipocalin-2(17-fold,P <0.001)and pathways associated with inflammation,and substantial downregulation of pathways related to hepatocyte function.In patients with chronic HCV,realtime polymerase chain reaction and immunohistochemistry confirmed an increase in lipocalin-2 mRNA(F0 1.0 ±0.3,F1 2.2±0.2,F2 3.0±9.3,F3/4 4.0±0.8,P= 0.003)and protein expression(F1 1.0±0.5,F2 1.3± 0.4,F3/4 3.6±0.4,P=0.014)with increasing liver injury.High performance liquid chromatography-tandem mass spectrometry analysis identified elevated levels of matrix metalloproteinase(MMP)-9 in macrophageconditioned medium,and a chemical inhibitor of MMP-9 attenuated the change in morphology and mRNA expression of TGF-β1(2.9±0.2 vs 1.04±0.1,P<0.001) in macrophage-conditioned media treated HepG2 cells.In patients with chronic HCV infection,hepatic mRNA expression of CD163(F0 1.0±0.2,F1/2 2.8±0.3,F3/4 5.3±1.0,P=0.001)and MMP-9(F0 1.0±0.4,F1/2 2.8±0.3,F3/4 4.1±0.8,P=0.011)was significantly associated with increasing stage of fibrosis.CONCLUSION:Secreted macrophage products alter the phenotype and function of hepatocytes,with increased expression of inflammatory mediators,suggesting that hepatocytes actively participate in liver injury.

Neuroendocrine liver metastases: Contributions of endoscopy and surgery to primary tumor search

Neuroendocrine tumors (NETs) are diagnosed with increasing frequency and patients often present with liver metastases at the time of diagnosis. Apart from treatment of the metastases, resection of the primary tumor at an early phase is recommended to prevent complications, although it may be difficult to locate, especially in patients with functionally inactive NETs. Small and multifocal tumors in the jejunum and ileum represent a particular challenge. Primary hepatic neuroendocrine carcinoma is extremely rare and is diagnosed only after exclusion of other primary tumors. Therefore, some uncertainty may remain, as small non-hepatic primary tumors may escape detection. Diagnostic work-up in these patients includes biochemical assays and imaging modalities (also comprising specific techniques of scintigraphy and positron emission tomography). This editorial highlights the contributions of endoscopy and operative exploration to the search for the primary tumor. Besides esophagogastro-duodenoscopy, endoscopic ultrasonography, colonoscopy and bronchoscopy, special endoscopic techniques such as balloon enteroscopy or capsule endoscopy are used with growing experience. Compared with balloon enteroscopy, capsule endoscopy is noninvasive and better tolerated, but it cannot localize a lesion precisely and does not allow biopsy or removal of lesions. Before proceeding to surgery, a discussion of the findings by a tumor board should be a standard procedure. Improvements in diagnostic tools have created new perspectives for the detection of obscure primary tumors in patients with neuroendocrine liver metastases, and these searches are best coordinated by a multidisciplinary team.

Clinicopathological and prognostic implications of endoglin (CD105) expression in hepatocellular carcinoma and its adjacent non-tumorous liver

AIM: The expression pattern of endoglin (CD105) in hepatocellular carcinoma (HCC) has not been reported so far. We hypothesized that CD105 could differentially highlight a subset of microvessels in HCC, and intratumoral microvessel density (IMVD) by CD105 immunostaining (IMVD-CD105) could provide better prognostic information than IMVD by CD34 immunostaining (IMVD-CD34).METHODS: Paraffin blocks of tumor and adjacent nontumorous liver tissues from 86 patients who underwent curative resection of HCC were used for this study. Serial sections were stained for CD105 and CD34, respectively,to highlight the microvessels. IMVD was counted according to a standard protocol.RESULTS: In the HCC tissues, CD105 was either negatively or positively stained only in a subset of microvessels. In contrast, CD34 showed positive and more extensive microvessel staining in all cases examined. However, in the adjacent non-tumorous liver sections, CD105 showed a diffuse pattern of microvessel staining in 20 of 86 cases,while CD34 showed negative or only focal staining of the sinusoids around portal area. Correlation with clinicopathological data demonstrated that lower scores of IMVD-CD105 were found in larger sized tumors [mean 41.4/0.74 mm2 (＞5 cm tumor) vs 65.9/0.74 mm2(≤ 5 cm tumor), P = 0.043] and more aggressive tumors,as indicated by venous infiltration [36.8/0.74 mm2 (present)vs 64.2/0.74 mm2 (absent), P = 0.020], microsatellite nodules [35.1/0.74 mm2 (present) vs 65.9/0.74 mm2(absent), P = 0.012], and advanced TNM tumor stage [38.8/0.74 mm2 (stage 3 or 4) vs 68.3/0.74 mm2 (stage 1or 2), P = 0.014]. No prognostic significance was observed when median values were used as cut-off points using either IMVD-CD105 or IMVD-CD34. However, the presence of the diffuse pattern of CD105 expression in the adjacent non-tumorous liver tissues predicted a poorer disease-free survival (median 8.6 vs 21.5 mo, P = 0.026).CONCLUSION: Our data demonstrate that a lower IMVDCD105 is associated with larger and more aggressive tumors. In this study, IMVD-CD105 did not provide significant prognostic information. However, active angiogenesis as highlighted by diffuse CD105 staining of the microvessels in the adjacent non-tumorous liver tissues is predictive of early recurrence.

Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris

AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coilyeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GSl15 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Hut ＋ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor. RESULTS： The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g （DCW）/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION： The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.

Effect of Different Diet Level on the Physiological Adaptability, Biochemical and Endocrine Responses and Relative Hepatic HSP70 and HSP90 Genes Expression in Osmanabadi Kids

The study was conducted to investigate the impact of different levels of feed on the adaptive capability based on physiological, blood biochemical, endocrine and molecular mechanisms in growing Osmanabadi kids. The primary objective of the study was to identify if HSP70 and HSP90 can be a nutritional stress marker for goat. The study was conducted for a period of two months. The animals were randomly divided into three groups as GI （n = 6; ad libitum feeding）, GII （n = 6; 20% less than ad libitum） and GIII （n = 6; 40% less than ad libitum）. The animals were fed with feed consisting of 50% roughage and 50% concentrate. Blood collection was carried out at fortnightly intervals. Body weights were recorded at weekly interval. Physiological responses, biochemical responses, plasma tri-iodo-thyronine （T3）, thyroxin （＇1＂4） and cortisol were recorded at fortnightly interval. At the end of study period, only GI and Gill animals were slaughtered and different organs were collected for histopathological studies as well as for hepatic HSP70 and HSP90 mRNA transcript expression. Body weight recorded showed significant （P 〈 0.01） differences between the groups. Physiological responses showed significant （P 〈 0.01） variation among the groups. Among the biochemical parameters, plasma glucose and total plasma protein and globulin showed significant （P 〈 0.01） differences between the groups. Plasma T3 （P 〈 0.01）, T4 （P 〈 0.01） and cortisol （P 〈 0.05） also differed significantly between the groups. The relative hepatic HSP70 mRNA transcript expression was significantly （P 〈 0.05） higher in Gill （2.8 fold） as compared to GI （1 fold） kids. Similar result was obtained for hepatic HSP90 mRNA transcript expression. From the results, it can be concluded that Osmanabadi kids possessed the ability to alter their adaptive mechanisms to maintain homeostasis. Further, the study revealed the significance of providing the optimum nutrition for these animals to adapt to existing environmental conditions. The study also established that respiration rate （RR）, rectal temperature （RT）, T3, T4 and cortisol are considered as nutritional stress markers for goat. Further, the results revealed that probably this is the first study to establish the nutritional stress impact on heat shock protein （HSP） expression in goats. The study identified both HSP70 and HSP90 to be the ideal molecular markers for feed deficit in goats.

Perioperative insulin therapy using a closed-loop artificial endocrine pancreas after hepatic resection

Postoperative hyperglycemia is common in critically ill patients, even in those without a prior history of diabetes mellitus. It is well known that hyperglycemia induced by surgical stress often results in dysregulation of liver metabolism and immune function, impairing postoperative recovery. Current evidence suggests that maintaining normoglycemia postoperatively improves surgical outcome and reduces the mortality and morbidity of critically ill patients. On the basis of these observations, several large randomized controlled studies were designed to evaluate the benefit of postoperative tight glycemic control with intensive insulin therapy. However, intensive insulin therapy carries the risk of hypoglycemia, which is linked to serious neurological events. Recently, we demonstrated that perioperative tight glycemic control in surgical patients could be achieved safely using a closed-loop glycemic control system and that this decreased both the incidence of infection at the site of the surgical incision, without the appearance of hypoglycemia, and actual hospital costs. Here, we review the benefits and requirements of perioperative intensive insulin therapy using a dosed-loop artificial endocrine pancreas system in hepatectomized patients. This novel intensive insulin therapy is safe and effectively improves surgical outcome after hepatic resection.

An Investigation into Disease Progression and the Correlation between Diabetes Mellitus and Hepatocellular Carcinoma

Diabetes mellitus has been associated with anincreased risk of hepatocellular carcinoma in recent studies ofpatients. At the same time, advanced hepatocellular carcinomaitself can cause glucose intolerance and can aggravate diabetes.Diabetes mellitus inducing hepatocellular carcinoma may resultin changes in the following aspects: dysfunction of organism,endocrine hormone balance and interactions, endothelins andso on. One way, diabetes mellitus may induce hepatocellularcarcinoma through the effects of chemotherapeutics and otheradjuvant drugs. This review outlines the relationship betweendiabetes mellitus and the risk of hepatocellular carcinoma as wellas treatments for hepatocellular carcinoma, which may be helpfulfor clinicians.

Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.

Xuebijing injection alleviates liver injury by inhibiting secretory function of Kupffer cells in heat stroke rats

OBJECTIVE： To evaluate the effects of Xuebijing （XBJ） injection in heat stroke （HS） rats and to inves- tigate the mechanisms underlying these effects. METHODS： Sixty anesthetized rats were random- ized into three groups and intravenously injected twice daily for 3 days with 4 mL XBJ （XBJ group） or phosphate buffered saline （HS and Sham groups） per kg body weight. HS was initiated in the HS and XBJ groups by placing rats in a simulated climate chamber （ambient temperature 40℃：, humidity 60% ）. Rectal temperature, aterial pressure, and heart rate were monitored and recorded. Time to HS onset and survival were determined, and serum concentrations of tumor necrosis factor （TNF）-α, in-terleukin （IL）-1β, IL-6, alanine-aminotransferase （ALT）, and aspartate-aminotransferase （AST） were measured. Hepatic tissue was harvested for patho- logical examination and electron microscopic ex- amination. Kupffer cells （KCs） were separated from liver at HS initiation, and the concentrations of se- creted TNF-α, IL-1β3 and IL-6 were measured. RESULTS： Time to HS onset and survival were signif- icantly longer in the XBJ than in the HS group. Moreover, the concentrations of TNF-α, IL-1β, IL-6, ALT and AST were lower and liver injury was milder in the XBJ than in the HS group. Heat-stress in- duced structural changes in KCs and hepatic cells were more severe in the HS than in the XBJ group and the concentrations of TNF-α, IL-1β and IL-6 se- creted by KCs were lower in the XBJ than in the HS group. CONCLUSION： XBJ can alleviate HS-induced sys- temic inflammatory response syndrome and liver injury in rats, and improve outcomes. These protec- tive effects may be due to the ability of XBJ to inhib- it cytokine secretion by KCs.

Comparison of alternative extraction methods for secretome profiling in human hepatocellular carcinoma cells

Secreted proteins are important sources for early detection and diagnosis of disease, and as such have received considerable attention. The extraction of low concentration proteins from large volumes of culture media, which are rich in salts and other compounds that interfere with most proteomics techniques, presents a problem for secretome studies. Ultrafiltration, precipitation, and dialysis are three major extraction methods that can be used to overcome this problem. The present study for the first time, compared the merits and shortcomings of these three methods, without bias. Centrifugal ultrafiltration provided the best extraction efficiency, and precipitation provided the highest number of identifiable proteins. The three methods yielded closely related, but different, information on the secretome; thus, they should be considered complementary or, at least, supplementary methods. Three hundred and sixty unique proteins were identified, including 211 potential secreted proteins. Compared with previous studies, this study also identified 42 new secreted proteins. The present study not only offers a reference for the selection of secretome extraction methods, but also expands the secretome database for the investigation of hepatocellular carcinoma.

Establishment of a transgenic mouse model with liver-specific expression of secretory immunoglobulin D

Mutation of mevalonate kinase （MVK） is thought to account for most cases of hyperimmunoglobulinemia D syndrome （HIDS） with recurrent fever. However, its mechanism and the relationship between elevated serum immunoglobulin D （IgD） and the clinical features of HIDS are unclear. In this study, we generated by fusion PCR a vector to express high levels of chimeric secretory IgD （cslgD） specifically in the liver. We then generated seven founder lines of transgenic mice by co-microinjection, and verified them using genomic PCR and Southern blotting. We detected the expression of csIgD by reverse transcription PCR, quantitative PCR, western blotting, and enzyme-linked immunosorbent assays. We demonstrated that csIgD could be specifically and stably expressed in the liver. We used flow cytometry to show that overexpression of csIgD in the bone marrow and spleen cells had no effect on B cell development. Morphologic and anatomical observation of the transgenic mice revealed skin damage, hepatosplenomegaly, and nephromegaly in some transgenic mice; in these mice, pathological sections showed high levels of cell necrosis and protein-like sediments in the liver, spleen, and kidney. We demonstrated that the genomic insertion sites of the transgeues did not disrupt the MVK gene on mouse chromosome 5. This transgenic mouse will be useful to explore the pathogenesis of HIDS.