[In-vitro amplification of oval cells with preservation of stem cell phenotype].

OBJECTIVE: To explore cell culture techniques for amplification of oval cells with preservation simultaneously of the stem cell characteristics. METHODS: Oval cell line OC3 was cultured in RPMI 1640 supplemented with 15% fetal bovine serum and 20 µg/L EGF. Cells were harvested every 5 passages and were examined with biomarkers including OV-6, c-kit, gamma-glutamyl transpeptidase, placental form of glutathione-S-transferase (GST-P), pyruvate kinase M2, pyruvate kinase L and albumin using techniques including RT-PCR, immunocytochemistry, and enzymo-cytochemistry. RESULTS: OC3 cell lines could be amplified abundantly in-vitro associating with expression of infant liver cell markers at various level, including OV-6, c-kit, gamma-glutamyl transpeptidase, GST-P, pyruvate kinase M2, but no expression of mature hepatocyte markers detected including pyruvate kinase L and albumin. CONCLUSIONS: Amplification of OC3 cells with preservation of the stem cell phenotype and high proliferation index can be achieved up to the 79(th) passages by culturing in RPMI 1640 supplemented with 15% fetal bovine serum and 20 µg/L EGF.

[Clinical and pathological analysis of chronic rejection following orthotopic liver transplantation].

OBJECTIVE: To investigate the clinical manifestation and pathological features of chronic rejection (CR) and the management of CR after orthotopic liver transplantation (OLT). METHODS: From January 2004 to December 2006, there were 516 patients who had undergone OLT. All the clinical and pathological data were collected and retrospectively studied. Clinical manifestation, histopathological feature, diagnosis and anti-rejection treatment of CR were summarized and analyzed. RESULTS: The incidence of CR was 2.3% (12/516), including 7 cases with early phases of CR and 5 cases with late phases of CR. The main pathological changes of CR were the vanishing bile duct syndrome and obliterative arteriopathy;and the early stage of CR were the damage of inter lobular bile duct, necrotic inflammation in central lobule, and inflammatory cells infiltration in portal area. Among 12 patients with CR, 7 cases with early CR were reversed by methylprednisolone (MP) pulse treatment and adjusting immunosuppressant dose, including 2 cases of whom were prescribed OKT3 treatment and 2 cases treated by ATG, and 5 cases with late CR underwent liver retransplantation (re-LT). Two patients died from infection, 1 case died from multiple organ failure in perioperative period after re-LT, another 2 cases were cured by re-LT, and the CR related mortality was 25.0% (3/12). CONCLUSIONS: Chronic rejection following OLT is lack of typical clinical manifestation and pathological features, and the pathological changes can overlap and coexist. Post-transplant liver biopsy and graft specimen after re-LT is still "gold standard" to CR diagnosis. Some of early CRs can be reversed by early diagnosis and early treatment; for late CR recipient, re-LT should be considered.

Clinical and pathological analysis of acute rejection following orthotopic liver transplantation.

BACKGROUND: Acute rejection is one of the most important factors for prognosis following liver transplantation. With the use of potent immunosuppressants, acute rejection does not always present typical manifestations. Moreover, other complications often occur concomitantly after liver transplantation, which makes early diagnosis of acute rejection more difficult. Acute rejection is best diagnosed by liver biopsy. Differentiation of clinical manifestations and pathological features plays an important role in achieving individualized immunosuppressive treatment and prolonging long term survival of patients given orthotopic liver transplants. METHODS: From January 2004 to December 2006, 516 orthotopic liver transplantations were performed at the First Affiliated Hospital, Sun Yat-sen University. For patients who suffered acute rejection, clinical manifestations, histopathological features, diagnosis and anti-rejection treatment were summarized and analyzed. RESULTS: In 86 cases (16.7%), of the 516 recipients, 106 episodes of acute rejection occurred, which included 9 with histopathological borderline changes, 36 Banff I rejections, 48 Banff II and 13 Banff III. Among these, 36 were cured by adjusting the dose of immunosuppressant and 65 were reversed by methylprednisolone pulse treatment. Five were methylprednisolone resistant, 3 of whom were given OKT3 treatment and 2 underwent liver retransplantation. CONCLUSIONS: Due to potent immunosuppressive agents, acute rejection following an orthotopic liver transplantation lacks typical clinical manifestations and pathological features. Acute rejection is best diagnosed by liver biopsy. Designing rational individualized immunosuppressive regimen based on clinical and pathological features of acute rejection plays an important role in prolonging long term survival of patients.

[Induced differentiation of rat hepatic oval cells in-vitro by combined hepatocyte growth factor and epidermal growth factor treatment].

OBJECTIVE: To characterize the biologic featrues of hepatic oval cells and their protein expression profiles during induced differentiation in vitro. METHODS: Rat hepatic oval cells were treated with epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in vitro, followed by morphological and molecular marker assessment by electromicroscopy, immunocytochemistry, RT-PCR and protein expression chip technology. RESULTS: Ten weeks after induction, the levels of GST-P mRNA and M2-PK mRNA were significantly reduced, whereas those of ALB and CK18 were elevated. Significant variations of expression was seen in 8 protein species during the course of the induced differentiation. CONCLUSION: Combined EGF and HGF treatment in vitro induces cell differentiation of hepatic oval cells, a process in which 8 protein species may play some regulatory roles.

Energy metabolism and survival of liver grafts from non-heart-beating donor rats with warm ischemia injury.

BACKGROUND: The shortage of donor livers is a critical limiting factor for the use of liver transplantation in treatment of end-stage liver diseases. Organs from non-heart-beating donors seem to be an effective option to alleviate this problem. Warm ischemia injury, however, directly influences the grafts' activity and functional recovery after operation. We investigated the energy metabolism and post-transplant survival of liver grafts after different warm ischemia times (WITs) in rats and determined the maximum limit for liver grafts with warm ischemia. METHODS: Rats were randomized into 7 groups with WITs of 0 (control), 10, 15, 20, 30, 45 or 60 minutes. The indices of energy metabolism were measured by reversed-phase high performance liquid chromatograpy and all liver graft specimens were subjected to ultrastructural observation. After orthotopic liver transplantation (OLT), the recovery of energy metabolism in liver grafts after 24 and 48 hours and the survival of the rats were assessed. RESULTS: The levels of adenosine triphosphate (ATP) and energy charge (EC) decreased gradually after different WITs in a time-dependent manner, and this was especially significant within 30 minutes. The levels of ATP and EC in liver grafts with 30 minutes of warm ischemia largely recovered 24 hours after OLT, with 45 minutes of warm ischemia partially recovered 48 hours after OLT, and with 60 minutes of warm ischemia, hardly recovered even 48 hours after OLT. The survival time after OLT did not significantly change with up to 30 minutes of WIT, while long-term survival was reduced with 45 and 60 minutes of WIT. CONCLUSIONS: The levels of ATP and EC after OLT may be important criteria for evaluating the quality of a liver graft. The WIT of a liver graft is closely related to the recovery of hepatic energy metabolism and the graft survival.

Dynamical changing patterns of histological structure and ultrastructure of liver graft undergoing warm ischemia injury from non-heart-beating donor in rats.

AIM: To investigate the histological and ultra-structural characteristics of liver graft during different of warm ischemia time (WIT) in rats and to predict the maximum limitation of liver graft to warm ischemia. METHODS: The rats were randomized into 7 groups undergoing warm ischemia injury for 0, 10, 15, 20, 30, 45 and 60 min, respectively. All specimens having undergone warm ischemia injury were investigated dynamically by light and electron microscopy, and histochemistry staining. After orthotopic liver transplantation (OLT), the recovery of morphology of liver grafts after 6, 24 and 48 h was observed. RESULTS: The donor liver from non-heart-beating donors (NHBD) underwent ischemia injury both in the warm ischemia period and in the reperfusion period. Morphological changes were positively related to warm ischemia injury in a time-dependent manner during the reperfusion period. The results demonstrated that different degrees of histiocyte degeneration were observed when WIT was within 30 min, and became more severe with the prolongation of WIT, no obvious hepatocyte necrosis was noted in any specimen. In the group undergoing warm ischemia injury for 45 min, small focal necrosis occurred in the central area of hepatic lobule first. In the group undergoing warm ischemia injury for 60 min, patchy or diffused necrosis was observed and the area was gradually extended, while hepatic sinusoid endothelial cells were obviously swollen. Hepatic sinusoid was obstructed and microcirculation was in disorder. CONCLUSION: The rat liver graft undergoing warm ischemia injury is in the reversible stage when the WIT is within 30 min. The 45 min WIT may be a critical point of rat liver graft to endure warm ischemia injury. When the WIT is over 60 min, the damage is irreversible.

[Features of morphometry of ultrasound and endometrial histology in the anovulatory polycystic ovary syndrome women].

OBJECTIVE: To investigate the relationship between ultrasonographic features of endometrium and the relation of histological staging of the endometrium and sexual hormone levels in anovulatory polycystic ovary syndrome (PCOS) women. METHODS: Seventy-six anovulatory PCOS patients and 32 women with normal ovulation were enrolled in this study. Ultrasonographic examination, and transmission electron microscope were used to observe endometrium. The expressions of nulear antigen associated with cell proliferation Ki-67 and calcitonin were analyzed by immunohistochemistry. The sexual hormone levels were measured by chemiluminescent microparticle immunoassay. RESULTS: In 11 patients the endometrium showed secretory change out of 76 anovulatory PCOS patients. The frequency of secretory change of the endometrium was not increased with the increase of menses-biopsy interval (P > 0.05). The frequency of abnormal stroma was significantly lower in tripleline endometria than those in non-tripleline endometria (9% vs 43%, P < 0.05). Compared with the control group, the anovulatory PCOS group showed a significant higher expression of Ki-67 in the glandular cell of the secretory phase endometrium (P < 0.05). In the proliferative endometrium, anovulatory PCOS group had more cell organelles than those of the control group. The endometrium showed insufficient secretory changes in the anovulatory PCOS patients. CONCLUSIONS: Proliferative and secretory stage of the endometrium in the anovulatory PCOS group show abnormal features. The abnormal stroma may contribute to the hyperechonic images of the endometrium in the anovulatory PCOS patients.

[Effects of enhanced external counterpulsation in atherosclerosis and NF-kappaB expression: a pig model with hypercholesterolemia].

OBJECTIVE: To study the effects of enhanced external counterpulsation (EECP) on the vascular morphology, and endothelial function using experimentally induced hypercholesterolemic pigs. METHODS: Thirty five male pigs were randomly divided into three groups: 7 normal control animals, 11 hypercholesterolemic animals, and 17 hypercholesterolemic animals receiving EECP. Serum cholesterol was measured. The coronary arteries and aortas were sampled for histopathologic and ultrastructural examination. The NF-kappaB protein expression of porcine coronary arteries was investigated by immunofluorescence. RESULTS: Compared with the normal controls, serum cholesterol levels were significantly higher in the hypercholesterolemic animals with or without EECP. The plaque/intimal area ratio of the aorta decreased significantly in animals receiving EECP [(3.33 +/- 2.40)%, versus (12.03 +/- 7.12)% in those without EECP, P < 0.05]. Lipid deposition, endothelial damage and proliferation of smooth muscle cells were less severe in animals receiving EECP than those not. Moreover, activation and expression of NF-kappaB also decreased significantly (P < 0.05) in animals receiving EECP. CONCLUSIONS: EECP improves the morphology and function of vascular endothelium, and retards the development and progression of atherosclerosis, likely through the inhibition of NF-kappaB signaling pathway.

Dynamical changing patterns of glycogen and enzyme histochemical activities in rat liver graft undergoing warm ischemia injury.

AIM: To investigate the changing patterns of glycogen and enzyme histochemical activities in rat liver graft under a different warm ischemia time (WIT) and to predict the tolerant time limitation of the liver graft to warm ischemia injury. METHODS: The rats were randomized into five groups, WIT was 0, 15, 30, 45, 60 min, respectively, and histochemical staining of liver graft specimens was observed. The recovery changes of glycogen and enzyme histochemistry activities were measured respectively 6 and 24 h following liver graft implantation. RESULTS: The activities of succinic dehydrogenase, cytochrome oxidase, apyrase (Mg++-ATPase) and content of glycogen were decreased gradually after different WIT in a time-dependent manner. The changes were significant when WIT was over 30 min. CONCLUSION: Hepatic injury is reversible within 30 min of warm ischemia injury. Glycogen and enzyme histochemistry activities of liver grafts and their recovery potency after reperfusion may serve as criteria to evaluate the quality of liver grafts.

Safe time to warm ischemia and posttransplant survival of liver graft from non-heart-beating donors.

AIM: To explore the dynamical changes of histology, histochemistry, energy metabolism, liver microcirculation, liver function and posttransplant survival of liver graft in rats under different warm ischemia times (WIT) and predict the maximum limitation of liver graft to warm ischemia. METHODS: According to WIT, the rats were randomized into 7 groups, with WIT of 0, 10, 15, 20, 30, 45, 60 min, respectively. The recovery changes of above-mentioned indices were observed or measured after liver transplantation. The graft survival and postoperative complications in each subgroup were analyzed. RESULTS: Liver graft injury was reversible and gradually resumed normal structure and function after reperfusion when WIT was less than 30 min. In terms of graft survival, there was no significant difference between subgroups within 30 min WIT. When WIT was prolonged to 45 min, the recipients' long-term survival was severely insulted, and both function and histological structure of liver graft developed irreversible damage when WIT was prolonged to 60 min. CONCLUSION: The present study indicates that rat liver graft can be safely subjected to warm ischemia within 30 min. The levels of ATP, energy charge, activities of glycogen, enzyme-histochemistry of liver graft and its recovery potency after reperfusion may serve as the important criteria to evaluate the quality of liver graft.

Dynamic microcirculatory changes in liver graft from non-heart-beating donor with warm ischemia injury in rat.

BACKGROUND: Since the 1990s, liver grafts from non-heart-beating donor (NHBD) have become an alternative because of the deficiency of grafts from heart-beating-donors (HBDs). Warm ischemia injury, however, directly influences the grafts' activity and functional recovery after operation. We investigated the microcirculatory change of liver graft at different warm ischemia time (WIT) in rats and determined the maximum limitation of liver graft to warm ischemia. METHODS: According to WIT, 120 rats were divided randomly into 5 groups of 0, 15, 30, 45, 60 minutes respectively. The microcirculatory changes of their liver grafts were measured including serum level of hyaluronic acid (HA) and ultrastructural changes. After orthotopic liver transplantation (OLT), the recovery of microcirculation of the liver grafts after 24 hours, 48 hours and 3 days was observed. RESULTS: Microcirculatory changes and function of the liver grafts became normal after reperfusion when the WIT was less than 30 minutes. In the 45-minute WI group, part of blood sinusoids was full of cytoplasmic blebs stemming from the microvilli of hepatocytes and hemocytes. The serum level of HA in each group after 45 minutes of WI recovered after reperfusion. CONCLUSIONS: The microcirculatory change of rat liver graft is reversible when the WIT is less than 30 minutes: rat liver graft could be safely subject to warm ischemia within 30 minutes. The maximal 45 minutes of WI can be tolerated by the microcirculatory function of liver graft. After 60 minutes of WI, irreversible disturbance of microcirculation may appear.

Influence of warm ischemia injury on hepatic functional status and survival of liver graft in rats.

OBJECTIVE: To investigate the changing patterns of functional and histological status, observe the posttransplantation survival of liver graft under different warm ischemia time (WIT) in rats, and determine the maximum limitation of liver graft to warm ischemia. METHODS: According to WIT, the rats were randomized into 7 groups, with WIT of 0, 10, 15, 20, 30, 45, 60 minutes respectively. Serum concentrations of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase were measured at 1, 2, 3 and 5 days after orthotopic liver transplantation respectively. Liver graft specimens were observed histopathologically at the same interval. The rats' survival in each subgroup was observed. RESULTS: In terms of graft survival, there was no significant difference between subgroups within 30-minute WIT. In the group with 30-minute WIT, the recipient rats' survival rate was 83.3% (10/12) at one week, 58.3% (7/12) at one month, and 50.0% (6/12) at 3 months. In the group with 45-minute WIT, the recipient rats' survival rate was 66.7% (8/12) at one week, 33.3% (4/12) at one month, and 8.3% (1/12) at 3 months, whereas only 8.3% (1/12) of the rats had one-week survival in the group with 60-minute WIT. CONCLUSIONS: These results indicate that rat liver graft could be safely subject to warm ischemia within 30 minutes. When WIT is prolonged to 45 minutes, the recipients long-term survival is severely insulted, and both function and histological structure of liver graft may develop irreversible damage when WIT is prolonged to 60 minutes.

[The influence of warm ischemia injury on viability and posttransplantative outcome of liver graft from non-heart-beating donor in rats].

OBJECTIVE: To investigate the dynamical changes of histology, histochemistry, energy metabolism, liver function and posttransplantive survival of liver graft under different warm ischemia times (WIT) in rats and determine the maximum limitation of liver graft to warm ischemia. METHODS: According to WIT, the rats were randomized into 7 groups, WIT were 0, 10, 15, 20, 30, 45, 60 minutes respectively. The recovery changes of above-mentioned index were observed or measured after liver transplantation. The graft survival and postoperative complications in each subgroup were analyzed. RESULTS: Liver graft injury was reversible and gradually resumed normal structure and function after reperfusion when WIT was less than 30 minutes. In terms of graft survival, there was no significant difference between subgroups within 30 WIT. When WIT was prolonged to 45 minutes, the recipients long-term survival was severely insulted, and both function and histological structure of liver graft would develop irreversible damage when WIT was prolonged to 60 minutes. CONCLUSION: These results indicate that rat liver graft could be safely subject to warm ischemia within 30 minutes. The levels of adentriphos (ATP) and energy charge (EC) and the activities of glycogen and enzyme-histochemistry of liver graft and its recovery potency after reperfusion may serve as the important criteria to evaluate the quality of liver graft.

In situ hybridization assay of androgen receptor gene in hepatocarcinogenesis.

AIM:To determine the correlation between expression of androgen receptor (AR) gene and hepatocarcinogenesis.METHODS:Male SD rats were used as experimental animals and the animal model of experimental hepatocarcinoma was established by means of 3'-me-DAB administration. Androgen receptor mRNA was detected by a non-radioactive in situ hybridization assay in neoplastic and non-neoplastic liver tissues.RESULTS:The expression of androgen receptor mRNA was observed only in neoplastic cells and some atypical hyperplastic cells. In the liver tissue of control animal and the remaining normal liver cells adjacent to the carcinoma tissue, no positive signal was seen.CONCLUSION:Androgen has an important correlation with hepatocarcinogenesis and the expression of androgen receptor gene might be a mark event during hepatocarcinogenesis.