lncRNA NEAT1 promotes the proliferation and metastasis of hepatocellular carcinoma by regulating the FOXP3/PKM2 axis.

Objective: Hepatocellular carcinoma (HCC) is a malignant tumor. The occurrence of HCC is involved in the alteration of a variety of oncogenes or tumor suppressor genes, but the specific molecular mechanism remains unknown. This research proved the effects of long non-coding RNA NEAT1 (lncRNA NEAT1) on the viability, proliferation, migration, and invasion of hepatocellular carcinoma cells and explored the mechanism behind these effects. Methods: NEAT1 in 97H and Huh7 cell lines was overexpressed or knocked down, respectively. The expression of FOXP3 and its target gene PKM2 was hinged on qRT-PCR and Western blot, respectively. RNA pulldown and RNA immunoprecipitation experiments were carried out to detect the interaction between NEAT1 and proteins. Finally, the effect of NEAT1 on the tumor volume of HCC was verified by animal experiments. Results: A series of experiments have shown that NEAT1 knockdown can inhibit the viability, proliferation, migration, and invasion of HCC cells; NEAT1 can bind FOXP3 to promote PKM2 transcription; PKM2 knockdown can inhibit the viability, proliferation, migration, and invasion of HCC cells; and PKM2 knockdown reversed the function of NEAT1. Conclusion: lncRNA NEAT1 can promote the malignant behavior of HCC cells, while silencing of NEAT1 can inhibit that behavior of HCC cells. Mechanically, NEAT1 promotes the transcriptional activation of PKM2 by binding FOXP3, and PKM2 knockout reverses the function of NEAT1.

Neoadjuvant chemoradiotherapy followed by liver transplantation is a promising treatment for patients with unresectable hilar cholangiocarcinoma: A case report.

Hilar cholangiocarcinoma is a highly malignant tumor and is currently treated by surgical resection or liver transplantation; however, these treatments result in poor patient prognosis accompanied with high recurrence and low patient mortality rates. Neoadjuvant therapy with liver transplantation is a novel treatment that exhibits promising clinical application, with a reported 5-year survival rate of 82%. However, transplantation centers conducting research into this treatment are limited due to its length and complexity. In the current study, the effects of brachytherapy and chemoradiotherapy followed by orthotopic liver transplantation (OLT) were investigated in a patient with unresectable hilar cholangiocarcinoma. Following treatment, the liver function of the patient normalized and physical status significantly improved. Furthermore, tomographic evaluation demonstrated no sign of recurrence 8 months later following continued adjunct chemotherapy. Therefore, neoadjuvant therapy followed by OLT may be an effective novel therapeutic strategy to treat patients with unresectable hilar cholangiocarcinoma.

The up-regulation of P62 levels is associated with resistance of sorafenib in hepatocarcinoma cells.

BACKGROUND: Sorafenib is one of the most commonly used systemic therapies for hepatocellular carcinoma (HCC), but the acquired resistance towards sorafenib found in HCC patients usually led to failure of treatment and poor prognosis. Therefore, there is an urgent need to study the molecular mechanism caused by the acquired resistance. Previous studies demonstrated that P62 plays an important role in tumor cell resistance towards systemic therapies including chemotherapy and targeted therapy. However, the role of P62 in acquired resistance to sorafenib in HCC has not been clearly investigated. MATERIALS AND METHODS: In this study we screened the most sensitive HCC cell lines towards sorafenib using CCK8. Then on this cell line, we analyzed the relationship between P62 expression level and the sensitivity towards sorafenib by western blot and CCK8. After knockdown and overexpression of P62 in HCC cells, cells were then treated with sorafenib. After that, we detect changes of sensitivity towards sorafenib. HCC samples were used to investigate the expression of P62 and their survival time. RESULTS: Among four HCC cell lines in our lab, HepG2 cell line with the highest sensitivity to sorafenib was screened and selected. After treatment with sorafenib, the expression of P62 was significantly increased. In HCC cells, we found that significant up-regulation of P62 was correlated with the reduction of sorafenib sensitivity. In HCC samples, we found that the expression of P62 was associated with sorafenib resistance and a shorter survival time. CONCLUSION: The up-regulation of P62 could reduce the sensitivity of HCC towards sorafenib. Thus, P62 could be therapeutic target to overcome sorafenib acquired resistance in the future.

Risk Factors for Post-Transplant Death in Donation after Circulatory Death Liver Transplantation.

PURPOSE: In spite of the increasing success of liver transplantation, there remains inevitable risk of postoperative complications, re-operations, and even death. Risk factors that correlate with post-transplant death have not been fully identified. MATERIALS AND METHODS: We performed a retrospective analysis of 65 adults that received donation after circulatory death liver transplantation. Binary logistic regression and Cox's proportional hazards regression were employed to identify risk factors that associate with postoperative death and the length of survival period. RESULTS: Twenty-two recipients (33.8%) deceased during 392.3 ± 45.6 days. The higher preoperative Child-Pugh score (p = .007), prolonged postoperative ICU stay (p = .02), and more postoperative complications (p = .0005) were observed in deceased patients. Advanced pathological staging (p = .02) with more common nerve invasion (p = .03), lymph node invasion (p = .02), and para-tumor satellite lesion (p = .01) were found in deceased group. The higher pre-transplant Child-Pugh score was a risk factor for post-transplant death (OR = 4.38, p = .011), and was correlated with reduced post-transplant survival period (OR = 0.35, p = .009). Nerve invasion was also a risk factor for post-transplant death (OR = 13.85, p = .014), although it failed to affect survival period. CONCLUSIONS: Our study emphasizes the impact of recipient's pre-transplant liver function as well as pre-transplant nerve invasion by recipient's liver cancer cells on postoperative outcome and survival period in patients receiving liver transplantation.

USP39 promotes the growth of human hepatocellular carcinoma in vitro and in vivo.

Ubiquitin specific protease 39 (USP39) plays an important role in mRNA splicing. In the present study, we investigated the role of USP39 in regulating the growth of hepatocellular carcinoma (HCC). We detected USP39 expression in more than 100 HCC clinical samples. The USP39 expression was significantly higher in the tumor tissues compared to the adjacent normal tissues, and was strongly associated with the pathological grade of HCC. USP39 knockdown inhibited cell proliferation and colony formation in vitro in the HepG2 cells, while upregulation of USP39 promoted tumor cell growth. FCM assay showed that USP39 knockdown led to G2/M arrest and induced apoptosis in the HepG2 cells. USP39 knockdown by shRNA inhibited xenograft tumor growth in nude mice. Moreover, USP39 knockdown led to the upregulation of p-Cdc2 and downregulation of p-Cdc25c and p-myt1, while the expression of total Cdc2, Cdc25c and myt1 was not changed in the USP39-knockdown cells. We also found that p-Cdc2 was decreased in the USP39-overexpressing cells and was upregulated in the xenografted tumors derived from the HepG2/KD cells from nude mice. Meanwhile, the expression levels of FoxM1 and its target genes PLK1 and cyclin B1 were decreased in the USP39-knockdown cells. These results suggest that USP39 may contribute to FoxM1 splicing in HCC tumor cells. Our data indicate that USP39 knockdown inhibited the growth of HCC both in vitro and in vivo through G2/M arrest, which was partly achieved via the inhibition of FoxM1 splicing.

Comparative analysis of the efficacy and complications of nasojejunal and jejunostomy on patients undergoing pancreaticoduodenectomy.

BACKGROUND: The efficacy and feeding-related complications of a nasojejunal feeding tube and jejunostomy after pancreaticoduodenectomy (PD) was investigated with a randomized, controlled clinical trial at the Affiliated Drum Tower Hospital. METHODS: Sixty-eight patients who underwent PD in the Department of Hepatobiliary Surgery were randomly divided into 2 groups: 34 patients received enteral feeding via a nasojejunal tube (NJT group) and 34 patients received enteral feeding via a jejunostomy tube (JT group). The assessment of clinical outcome was based on postoperative investigation of complications. The second part of the assessment included tube related complications and an index on catheter efficiency. RESULTS: There were 15 cases with infectious complications in the JT group and 13 cases in the NJT group, and there was no significant difference in the rate of infectious complications between the 2 groups. The rate of intestinal obstruction and delayed gastric emptying was significantly decreased in the NJT group (P < .05). Catheter-related complications were more common in the JT group as compared with the NJT group (35.3% vs 20.6%, P < .05). The time for removal of the feeding tube and nasogastric tube was significantly decreased in the NJT group. The postoperative hospital stay in the NJT group was significantly decreased (P < .05), and there was no hospital mortality in this study. CONCLUSION: Nasojejunal feeding is safer than jejunostomy, and it is associated with only minor complications. Nasojejunal feeding can significantly decrease the incidence of delayed gastric emptying and shorten the postoperative hospital stay.

Effect of early enteral combined with parenteral nutrition in patients undergoing pancreaticoduodenectomy.

AIM: To investigate the effect of early enteral nutrition (EEN) combined with parenteral nutritional support in patients undergoing pancreaticoduodenectomy (PD). METHODS: From January 2006, all patients were given EEN combined with parenteral nutrition (PN) (EEN/PN group, n = 107), while patients prior to this date were given total parenteral nutrition (TPN) (TPN group, n = 67). Venous blood samples were obtained for a nutrition-associated assessment and liver function tests on the day before surgery and 6 d after surgery. The assessment of clinical outcome was based on postoperative complications. Follow-up for infectious and noninfectious complications was carried out for 30 d after hospital discharge. Readmission within 30 d after discharge was also recorded. RESULTS: Compared with the TPN group, a significant decrease in prealbumin (PAB) (P = 0.023) was seen in the EEN/PN group. Total bilirubin (TB), direct bilirubin (DB) and lactate dehydrogenase (LDH) were significantly decreased on day 6 in the EEN/PN group (P = 0.006, 0.004 and 0.032, respectively). The rate of grade I complications, grade II complications and the length of postoperative hospital stay in the EEN/PN group were significantly decreased (P = 0.036, 0.028 and 0.021, respectively), and no hospital mortality was observed in our study. Compared with the TPN group (58.2%), the rate of infectious complications in the EEN/PN group (39.3%) was significantly decreased (P = 0.042). Eleven cases of delayed gastric emptying were noted in the TPN group, and 6 cases in the EEN/PN group. The rate of delayed gastric emptying and hyperglycemia was significantly reduced in the EEN/PN group (P = 0.031 and P = 0.040, respectively). CONCLUSION: Early enteral combined with PN can greatly improve liver function, reduce infectious complications and delayed gastric emptying, and shorten postoperative hospital stay in patients undergoing PD.

Effects of ω-3 fish oil lipid emulsion combined with parenteral nutrition on patients undergoing liver transplantation.

BACKGROUND: The effect of parenteral nutrition (PN) support supplemented with ω-3 fatty acids was investigated in a randomized, controlled clinical trial at the Affiliated Drum Tower Hospital, Medical School of Nanjing University. MATERIALS AND METHODS: Ninety-eight patients with the diagnosis of end-stage liver disease or hepatic cellular carcinoma were admitted for orthotopic liver transplantation at the Affiliated Drum Tower Hospital. The patients were randomly divided into 3 groups: diet group (n = 32), PN group (n = 33), and polyunsaturated fatty acid (PUFA) group (n = 33). Patients in the PN and PUFA groups received isocaloric and isonitrogenous PN for 7 days after surgery. Venous heparin blood samples were obtained for assay on days 2 and 9 after surgery. A pathological test was performed after reperfusion of the donor liver and on day 9. RESULTS: Alanine aminotransferase levels were improved significantly by PUFA treatment compared with traditional PN support (P < .05). Compared with the results on day 9 in the PN group, a significant difference was seen in the extent of increase of the prognostic nutrition index and prealbumin in the PUFA group. The pathological results also showed that ω-3 fatty acid supplementation reduced hepatic cell injury. PUFA therapy also decreased the incidence of infectious morbidities and shortened the posttransplant hospital stay significantly. CONCLUSION: Posttransplant PN support can greatly improve metabolism of protein and nutrition states of patients. ω-3 fatty acid-supplemented PN significantly reduces injury of the transplanted liver, decreases the incidence of infectious morbidities, and shortens posttransplant hospital stay.

Effect of parenteral fish oil lipid emulsion in parenteral nutrition supplementation combined with enteral nutrition support in patients undergoing pancreaticoduodenectomy.

BACKGROUND: The effect of parenteral fish oil lipid emulsion in parenteral nutrition (PN) supplementation combined with enteral nutrition (EN) support on pancreaticoduodenectomy (PD) was investigated with a randomized controlled clinical trial at the Affiliated Drum Tower Hospital. MATERIALS AND METHODS: Seventy-six patients who underwent PD in the Department of Hepatobiliary Surgery were randomly divided into 2 groups: the polyunsaturated fatty acid (PUFA) group (n = 38) and control group (n = 38). Patients in the PUFA group received parenteral fish oil lipid emulsion supplementation combined with early EN support for 5 days after PD. Venous blood samples were obtained for assay on the day before surgery and on day 6 after surgery. RESULTS: Compared with the results of the control group, a significant difference was seen in the extent of the decrease in total protein and prealbumin in the PUFA group (P < .05). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were significantly decreased on day 6 in the PUFA group (P < .01), and a significant difference was seen in the extent of decrease in ALT, AST, and LDH in the PUFA group (P < .05). The ratio of infectious complications in the PUFA group was significantly decreased, as well as the postoperative hospital stay (P < .05), and there was no hospital mortality in this study. CONCLUSION: Parenteral fish oil lipid emulsion in PN supplementation combined with EN support can greatly improve the nutrition state and liver function of patients, decrease the incidence of infectious morbidities, and shorten the postoperative hospital stay.

Effects of warm ischemia time on biliary injury in rat liver transplantation.

AIM: To investigate the effect of different secondary warm ischemia time (SWIT) on bile duct injury in liver-transplanted rats. METHODS: Forty-eight male inbred Sprague-Dawley rats were randomly assigned into four groups: a sham-operation group and three groups with secondary biliary warm ischemia time of 0 min, 10 min and 20 min. A rat model of autologous liver transplantation under ether anesthesia was established, and six rats were killed in each group and blood samples and the median lobe of the liver were collected for assay at 6 h and 24 h after hepatic arterial reperfusion. RESULTS: With prolongation of biliary warm ischemia time, the level of vascular endothelial growth factor-A was significantly decreased, and the value at 24 h was higher than that at 6 h after hepatic arterial reperfusion, but with no significant difference. The extended biliary SWIT led to a significant increase in bile duct epithelial cell apoptosis, and a decrease in the number of blood vessels, the bile duct surrounding the blood vessels and bile duct epithelial cell proliferation in the early postoperative portal area. Pathologic examinations showed that inflammation of the rat portal area was aggravated, and biliary epithelial cell injury was significantly worsened. CONCLUSION: A prolonged biliary warm ischemia time results in aggravated injury of the bile duct and the surrounding vascular plexus in rat autologous orthotopic liver transplantation.

Liver-protecting effects of omega-3 fish oil lipid emulsion in liver transplantation.

AIM: To investigate the liver-protecting effect of parenteral nutrition (PN) support with omega-3 fatty acids in a randomized controlled clinical trial. METHODS: Sixty-six patients with the diagnosis of end-stage liver disease or hepatic cellular carcinoma were admitted to the Affiliated Drum Tower Hospital, Nanjing University, China for orthotopic liver transplantation. The patients were randomly divided into two groups: PN group (n = 33) and polyunsaturated fatty acid (PUFA) group (n = 33). All patients received isocaloric and isonitrogenous PN for seven days after surgery, and in PUFA group omega-3 fish oil lipid emulsion replaced part of the standard lipid emulsion. Liver function was tested on days 2 and 9 after surgery. Pathological examination was performed after reperfusion of the donor liver and on day 9. Clinical outcome was assessed based on the post-transplant investigations, including: (1) post-transplant mechanical ventilation; (2) total hospital stay; (3) infectious morbidities; (4) acute and chronic rejection; and (5) mortality (intensive care unit mortality, hospital mortality, 28-d mortality, and survival at a one-year post-transplant surveillance period). RESULTS: On days 2 and 9 after operation, a significant decrease of alanine aminotransferase (299.16 U/L ± 189.17 U/L vs 246.16 U/L ± 175.21 U/L, P = 0.024) and prothrombin time (5.64 s ± 2.06 s vs 2.54 s ± 1.15 s, P = 0.035) was seen in PUFA group compared with PN group. The pathological results showed that omega-3 fatty acid supplement improved the injury of hepatic cells. Compared with PN group, there was a significant decrease of post-transplant hospital stay in PUFA group (18.7 d ± 4.0 d vs 20.6 d ± 4.6 d, P = 0.041). Complications of infection occurred in 6 cases of PN group (2 cases of pneumonia, 3 cases of intra-abdominal abscess and 1 case of urinary tract infection), and in 3 cases of PUFA group (2 cases of pneumonia and 1 case of intra-abdominal abscess). No acute or chronic rejection and hospital mortality were found in both groups. The one-year mortality in PN group was 9.1% (3/33), one died of pulmonary infection, one died of severe intra-hepatic cholangitis and hepatic dysfunction and the other died of hepatic cell carcinoma recurrence. Only one patient in PUFA group (1/33, 3.1%) died of biliary complication and hepatic dysfunction during follow-up. CONCLUSION: Post-transplant parenteral nutritional support combined with omega-3 fatty acids can significantly improve the liver injury, reduce the infectious morbidities, and shorten the post-transplant hospital stay.

Establishment of a rat liver transplantation model with prolonged biliary warm ischemia time.

AIM: To investigate the impact of different time points of secondary warm ischemia on bile duct in a rat autologous liver transplantation model with external bile drainage. METHODS: One hundred and thirty-six male inbred SD rats were randomly assigned to one of four groups (I-IV) according to the secondary warm ischemia time of 0, 10, 20 and 40 min. A rat model of autologous liver transplantation with continuous external biliary drainage under ether anesthesia was established. Ten rats in each group were used to evaluate the one-week survival rate. At 6 h, 24 h, 3 d and 7 d after reperfusion of the hepatic artery, 6 rats were killed in each group to collect the blood sample via the infrahepatic vena cava and the median lobe of liver for assay. Warm ischemia time of liver, cold perfusion time, anhepatic phase, operative duration for biliary external drainage and survival rates in the four groups were analyzed for the establishment of models. RESULTS: No significant difference was shown in warm ischemia time, anhepatic phase and operative duration for biliary external drainage among the four groups. Five of the 40 rats in this study evaluated for the one-week survival rate died, including three deaths of severe pulmonary infection in group IV. A significant decrease of one-week survival rate in group IV was noted compared with the other three groups. With the prolongation of the biliary warm ischemia time, the indexes of the liver function assessment were significantly elevated, and biliary epithelial cell apoptosis index also increased. Pathological examinations showed significantly aggravated inflammation in the portal area and bile duct epithelial cell injury with the prolonged secondary warm ischemia time. Microthrombi were found in the micrangium around the biliary tract in some sections from groups III and IV. CONCLUSION: The relationship between secondary warm ischemia time and the bile duct injury degree is time-dependent, and 20 min of secondary warm ischemia time is feasible for the study of bile duct injury.

Efficacy and safety of liver transplantation in patients with cholangiocarcinoma: a systematic review and meta-analysis.

The aim of our study was to evaluate the efficacy and safety of liver transplantation in patients with cholangiocarcinoma. According to the requirements of Cochrane systematic review, a thorough literature search was performed in PubMed/Medline, Embase and Cochrane electronic databases between 1995 and 2009 in terms of the key words "liver transplantation" and "cholangiocarcinoma," "cholangiocellular carcinoma" or "bile duct cancer," with restricted articles for the English language. Data were processed for a meta-analysis by Stata 10 software. Altogether 14 clinical trials containing 605 transplanted patients of bile duct cancers were finally enrolled in our study. The overall 1-, 3- and 5-year pooled survival rates were 0.73 [95% confidence interval (CI) = 0.65-0.80], 0.42 (95% CI = 0.33-0.51) and 0.39 (95% CI = 0.28-0.51), respectively. Of note, preoperative adjuvant therapies [orthotopic liver transplantation (OLT)-PAT group] rendered the transplanted individuals with comparably favorable outcomes with 1-, 3- and 5-year pooled survival rates of 0.83 (95% CI = 0.57-0.98), 0.57 (95% CI = 0.18-0.92) and 0.65 (95% CI = 0.40-0.87). In addition, the overall pooled incidence of complications was 0.62 (95% CI = 0.44-0.78), among which that of OLT-PAT group (0.58; 95% CI = 0.20-0.92) was relatively acceptable compared to those of liver transplantation alone (0.61; 95% CI = 0.33-0.85) and liver transplantation with extended bile duct resection (0.78; 95% CI = 0.55-0.94). In comparison to curative resection of cholangiocarcinoma with the 5-year survival rate reported from 20 to 40%, the role of liver transplantation alone is so limited. In the future, attention will be focused on liver transplantation following neoadjuvant radiochemotherapy, which requires a well-designed, prospective randomized controlled study.

[Meta-analysis of efficacy and safety of liver transplantation in treating cholangiocarcinoma].

OBJECTIVE: To evaluate the therapeutic efficacy and safety of liver transplantation for patients with cholangiocarcinoma. METHODS: According to the requirements of Cochrane systematic review, a thorough literature search was performed in Pubmed/Medline, Embase and Cochrane Central Register electronic databases ranged between 1995 and 2009 in terms of the key words "liver transplantation", and "cholangiocarcinoma" or "cholangiocellular carcinoma" or "bile duct cancer". And restricted the articles published in the English language. Two reviewers independently screened the studies for eligibility, evaluated the quality and extracted the data from the eligible studies with confirmation by cross-checking. Data were processed for a meta-analysis by Stata 10 software with 1-, 3-, 5-year survival rates and incidence of complications. RESULTS: A total of 14 clinical trials containing 605 patients were finally enrolled in this study. The overall 1-, 3-, 5-year pooled survival rates were 73% (95%CI: 0.65 - 0.80), 42% (95%CI: 0.33 - 0.51) and 39% (95%CI: 0.28 - 0.51), respectively. Of note, preoperative adjuvant therapies (OLT-PAT group) rendered the transplanted individuals comparably favorable outcomes with 1-, 3-, 5-year pooled survival rates of 83% (95%CI: 0.57 - 0.98), 57% (95%CI: 0.18 - 0.92) and 65% (95%CI: 0.40 - 0.87), respectively. In addition, the overall pooled incidence of complications was 62% (95%CI: 0.44 - 0.78), among which that of OLT-PAT group (58%, 95%CI: 0.20 - 0.92) was relatively acceptable compared to those of liver transplantation alone (61%, 95%CI: 0.33 - 0.85) and liver transplantation with extended bile duct resection (78%, 95%CI: 0.55 - 0.94). CONCLUSIONS: In comparison to curative resection of cholangiocarcinoma with the 5-year survival rate reported from 20% to 40%, the role of liver transplantation alone is so limited, but neoadjuvant radiochemotherapy combined with liver transplantation can bring better short- and long-term prognosis.

[The prevention and treatment of post-resectional live failure for hepatocellular carcinoma].

OBJECTIVE: To investigate the cause of liver failure after hepatectomy for hepatocellular carcinoma and explore its prevention and treatment. METHODS: The clinical data of 1000 patients with hepatocellular carcinoma undergone hepatectomy from July 2000 to June 2008 were retrospectively analyzed. There were 922 male and 78 female, aging from 21 to 89 years old. RESULTS: Among the 1000 patients, there were 18 patients with liver failure after hepatectomy (1.8%). Among the 18 patients with liver failure, 13 patients were more than 65 years old, 14 patients were with more than 20% of indocyanine green retention rate at 15 minutes, 14 patients were with more than 1000 ml blood loss during operation, 6 patients were with F4/F3 liver fibrosis (Metavir Scores), and 9 patients were with less than 40.0% liver volume of residue liver. CONCLUSIONS: Patients with hepatocellular carcinoma with less than volume of residue liver, much more blood loss or transfusion, more than 20% of ICGR15, F4/F3 liver cirrhosis are prone to be with liver failure after hepatectomy. Artificial liver or liver transplantation may be the important alternative for liver failure after hepatectomy.

In vitro analysis of cryopreserved alginate-poly-L-lysine-alginate-microencapsulated human hepatocytes.

BACKGROUND: The availability of well-characterized human hepatocytes that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after microencapsulated cryopreservation and investigated whether these cryopreserved microencapsulated hepatocytes can be used for clinical applications. METHODS: Adult hepatocytes of 18 separate donors were isolated with a two-step extracorporeal collagenase perfusion technique. After pre-incubation at 4 degrees C for 12-24 h in HepatoZYME-SFM, hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules. The microencapsulated hepatocytes were transferred to a complete medium containing 10% dimethyl sulphoxide. They were immediately placed into an isopropanol progressive freezing container at -80 degrees C overnight and immersed in liquid nitrogen the next day. During the post-thawing culture period, albumin secretion, urea synthesis, cell cycle, mRNA and protein levels, as well as the morphology and pathology structure of pre-incubation before microencapsulated cryopreservation (PMC) groups were analysed. RESULTS: Compared with the immediate cryopreservation (IC) groups, we found significant improvement in the mRNA and protein levels in the attached cells, and higher secretion of albumin and urea levels after thawing. In the attached cultured human cryopreserved/thawed hepatocytes from the PMC group, albumin production was not significantly different from those of the direct culture groups on days 2, 3 and 4. The preserved morphology in the PMC group compared with the IC group was obvious. CONCLUSIONS: The results of the present study suggested recovery of the functional and morphological integrity of human hepatocytes after pre-incubation at 4 degrees C for 12-24 h before microencapsulated cryopreservation. These studies offer the possibility for clinical applications in pharmacotoxicology, bioartificial liver and cell therapy in humans.

A simple isolation and cryopreservation method for adult human hepatocytes.

OBJECTIVE: The objective of this study was to establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for the treatment of acute and chronic liver diseases by hepatocyte transplantation and bioartificial liver support systems, and for the use of hepatocytes as an in vitro model of the liver. METHODS: Adult hepatocytes of 20 separate donors were isolated with a two-step extracoporeal collagenase perfusion technique. Seven preincubation time points (2h, 6h, 12h, 24h, 36h, 48h and 72h) were selected, then the hepatocytes were transferred to HepatoZYME-SFM medium containing 10% FBS and 10% DMSO, and were immediately put into an isopropanol progressive freezing container at -80 degrees C overnight and immersed in liquid nitrogen the next day. During the postthawing culture period, viability, plating efficiency, albumin secretion and urea synthesis were analyzed. RESULTS: The viability and plating efficiency of hepatocytes after partial hepatectomy using two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0% respectively. Preincubation at 4? for 12 hours or 24 hours proved to be the optimal time at which the albumin secretion was higher than at other time points (p<0.05). Compared to the immediate cryopreservation groups (IC), we also found significant improvement in viability (61.4+/-4.8%/62.0+/-5.6% vs. 53.4+/-4.2%, p<0.05), plating efficiency (63.2+/-5.8%/62.6+/-3.6% vs. 55.2+/-4.6%, p<0.05), albumin secretion and urea synthesis (p<0.05) at these time points. CONCLUSIONS: The two-step extracorporeal collagenase perfusion technique after partial hepatectomy provides a novel, simple, and reliable method for hepatocyte isolation. The results of the present study suggest that recovery of human hepatocytes after isolation preincubation at 4 degrees C for 12 hours to 24 hours prior to cryopreservation can obtain hepatocytes ideal for use in pharmacotoxicology, bioartificial liver and cell therapy research purposes.

Identification and clinical significance of mobilized endothelial progenitor cells in tumor vasculogenesis of hepatocellular carcinoma.

PURPOSE: To investigate the distribution, frequency, and clinical significance of mobilized endothelial progenitor cells (EPC) in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: In healthy controls and patients with HCC, the frequency of circulating EPCs was determined by colony-forming assays, fluorescence-activated cell sorting, and real-time PCR. One hundred sixty-five--amino acid form of vascular endothelial growth factor and platelet-derived growth factor-BB in plasma and tissue were quantified by ELISA. The distribution and frequency of EPCs were evaluated by immunofluorescence, immunohistochemistry, and real-time PCR in normal liver (n = 8), and tumor tissue (TT), adjacent nonmalignant liver tissue (AT), and tumor-free tissue 5 cm from the tumor edge (TF) from 64 patients with HCC. Clinicopathologic data for these patients were evaluated. RESULTS: Compared with values for healthy controls, colony-forming unit scores were higher in the peripheral blood of patients with HCC. Plasma 165-amino acid form of vascular endothelial growth factor and platelet-derived growth factor-BB correlated with the expression level of the AC133 gene, which was also higher in the peripheral blood of patients with HCC. Immunohistochemical analysis showed that EPCs were incorporated into the microvessels in cirrhotic and tumor tissue. Compared with normal liver (9.00), increased AC133(+) microvessel density (microvessels/0.74 mm(2)) was found in TT (53.56), AT (84.76), and TF (48.33). The levels of AC133 gene expression and AC133-microvessel density in AT, which were the highest among four groups, correlated with clinicopathologic variables (the absence of tumor capsule, venous invasion, proliferating cell nuclear antigen intensity, and early recurrence). CONCLUSIONS: Mobilized EPCs participate in tumor vasculogenesis of HCC. AC133 gene or antigen in peripheral blood and liver tissue could be used as a biomarker for predicting the progression of HCC.

Diagnosis and treatment of gastrointestinal stromal tumors of the stomach: report of 28 cases.

We retrospectively analyzed 28 patients with gastric gastrointestinal stromal tumors (GISTs) at our hospital to investigate their clinical features, diagnosis, and treatment. All patients underwent surgical resection. There were 18 cases with subtotal gastrectomy, 8 cases with partial gastrectomy, 1 case with total gastrectomy, and 1 case with subtotal gastrectomy combined with right hemihepatectomy. Based on pathological findings, the tumors were benign in 16 cases (57%), borderline in 2 cases (7%), and malignant in 10 cases (36%). The tumor diameter was significantly correlated with the malignancy of gastric GISTs (3.5+/-1.6 cm in benign tumors, vs 7.5+/-1.3 cm in malignant tumors, p<0.05). Immunohistochemical staining for CD117 and CD34 was positive in 26 (93%) and 17 (61%), respectively. The mean follow-up period ranged from 6 to 60 mo in 23 of the patients, and the other 5 patients (all with benign tumors) were lost to follow-up. No recurrence or metastasis was found in patients with benign gastric GISTs. Four cases of malignant GISTs (17%, 4/23) had liver metastasis or intra-abdominal dissemination, and 2 of them received a second resection for liver metastasis. Four cases of malignant gastric GISTs died with tumors more than 10 cm in maximum diameter, 3 of them died of liver metastasis and multiple organ failure, and 1 died of myocardial infarction. Excluding 2 patients with benign tumors that were followed for <3 yr, the 3-yr survival rate was 81% (17/21) in this group, and 60% (6/10) in the patients with malignant gastric GISTs. In conclusion, the prognosis is related to the tumor size and the number of mitoses seen on histological examination. Positive detection of CD117, combined with other markers and pathological features, is of great importance in the differential diagnosis of gastric GISTs. Complete resection with negative margins remains the fundamental objective in the surgical management of gastric GISTs.