[Expression and its clinicopathological features of tumor rejective antigen 1 in human tissues of hepatocellular carcinoma and liver cirrhosis].

OBJECTIVE: To investigate the expression of tumor rejective antigen 1 in hepatocellular carcinoma (HCC) and liver cirrhosis(LC) tissues, and the relationship between clinicopathological feature and HCC. METHODS: The expressions of TRA1 mRNA and its protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively. Immunohistochemical staining was used to further examine the expression of TRA1 protein in LC, HCC and control tissues. The relationship between clinicopathological feature and HCC was analyzed. Data of RT-PCR and Western blot were analyzed by One-way ANOVA; results of immunohistochemical staining were analyzed by Fisher's exact test and correlation analysis using Spearman rank correlation. RESULTS: RT-PCR data showed that the expression of TRA1 mRNA was higher in HCC and LC tissues than that in the normal liver tissues (F values were 20.821 and 12.311 respectively, P is less than 0.05). The expression of TRA1 protein in HCC and LC tissues was signifIcantly higher than that in control by Western blot (F values were 21.231 and 20.125 respectively, P < 0.05). The immunohistochemical data showed the expression of TRA1 protein was gradually increased in HCC group than that in the LC group and control group, and the positive expression rate of TRA1 was 57.14%, 78.95% and 93.75% respectively. The expression of TRA1 protein was negatively correlated with HCC differentiation (r = -0.4655, P = 0.0073) and positively correlated with HCC TNM staging (r = 0.5157, P = 0.0025). CONCLUSION: The over-expression of TRA1 in hepatocirrhosis and HCC is correlated with the formation and development of HCC. It may be a prognostic marker for the diagnosis of HCC and be associated with the degree of differentiation and HBV infection. It can be used as a marker for prognostic prediction of HCC.

Apoptosis of human pancreatic cancer cells induced by Triptolide.

AIM: To investigate apoptosis in human pancreatic cancer cells induced by Triptolide (TL), and the relationship between this apoptosis and expression of caspase-3' bcl-2 and bax. METHODS: Human pancreatic cancer cell line SW1990 was cultured in DMEM media for this study. MTT assay was used to determine the cell growth inhibitory rate in vitro. Flow cytometry and TUNEL assay were used to detect the apoptosis of human pancreatic cancer cells before and after TL treatment. RT-PCR was used to detect the expression of apoptosis-associated gene caspase-3' bcl-2 and bax. RESULTS: TL inhibited the growth of human pancreatic cancer cells in a dose-and time-dependent manner. TL induced human pancreatic cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of human pancreatic cancer cells with 40 ng/mL TL for 12 h and 24 h, the apoptotic rates of human pancreatic cancer cells increased significantly. RT-PCR demonstrated that caspase-3 and bax were significantly up-regulated in SW1990 cells treated with TL while bcl-2 mRNA was not. CONCLUSION: TL is able to induce the apoptosis in human pancreatic cancer cells. This apoptosis may be mediated by up-regulating the expression of apoptosis-associated caspase-3 and bax gene.

Diagnosis of pancreatic cancer by cytology and telomerase activity in exfoliated cells obtained by pancreatic duct brushing during endoscopy.

BACKGROUND: Telomerase activity is reported to be specific and frequent in human pancreatic cancer. We conducted this study to assess the usefulness of monitoring telomerase activity in exfoliated cells obtained by pancreatic duct brushing during endoscopic retrograde cholangiopancreatography (ERCP) for the diagnosis of pancreatic cancer. METHODS: Exfoliated cells obtained by pancreatic duct brushing during ERCP from 21 patients (18 with pancreatic cancer, 3 with chronic pancreatitis) were examined. Telomerase activity was detected by polymerase chain reaction and telomeric repeat amplification protocol assay (PCR-TRAP-ELISA). RESULTS: D450 values of telomerase activity were 0.446+/-0.2700 in pancreatic cancer and 0.041+/-0.0111 in chronic pancreatitis. 77.8% (14/18) of patients with pancreatic cancer had cells with telomerase activity. None of the samples from patients with chronic pancreatitis showed telomerase activity, when the cutoff value of telomerase activity was set at 2.0. Cytological examination showed cancer cells in 66.7% (12/18) of the patients. CONCLUSIONS: Telomerase activity may be an early malignant event in pancreatic cancer development. Cytology and telomerase activity in cells obtained by pancreatic duct brushing may complement each other for the diagnosis of pancreatic cancer.

[Effects of antisense RNA of connective tissue growth factor expressing plasmid on rat liver fibrosis].

OBJECTIVE: To observe the effects of antisense RNA of connective tissue growth factor (CTGF) on rat liver fibrosis. METHODS: Gene recombinant techniques were used to construct a rat antisense RNA of CTGF recombinant plasmid which could be expressed in eukaryotic cells. The recombinant plasmids were encapsulated with lipofectamine and then transducted into a carbon tetrachloride (CCl4) induced rat liver fibrosis model. Expression of CTGF was assessed by RT-PCR, Western blot and immunohistochemistry. Immunohistochemistry was used to identify type I and III collagens. HE stained liver slides were used for pathological study. RESULTS: The mRNA and protein expression of CTGF in the fibrotic liver transfected with antisense-CTGF were significantly decreased compared with those of the controls (P<0.01). The depositions of type I and type III collagens were also decreased (P<0.05). Antisense-CTGF also minimized the pathological fibrosis in the rat livers (P<0.01). CONCLUSION: The results demonstrate that the antisense RNA of CTGF recombinant plasmid has certain effects in preventing liver fibrosis and makes it a possible candidate for use in future gene therapy.

Anti-hepatoma effect of arsenic trioxide on experimental liver cancer induced by 2-acetamidofluorene in rats.

AIM: To study the anti-hepatoma efficiency of arsenic trioxide (As(2)O(3)) in the treatment of experimental rat hepatocellular carcinoma (HCC) induced by 2-acetamidofluorene (2-FAA) and to elucidate the possible mechanisms. METHODS: SD rats (2 mo old) had been fed with 2-FAA for 8 wk to induce HCC, and then they were treated with As(2)O(3) or matrine. On d 29, the rats were killed and the liver was weighed and liver tumors were counted. The histological changes of liver tissue were observed under microscope, and the cellular dynamic parameters were studied by flow cytometry. Immunohistochemistry (two-step method) was used to observe the expression of vascular endothelial growth factor (VEGF) and micro-vessel density (MVD) on consecutive sections. The pathological parameters were also analyzed, the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBi), and direct bilirubin (DBi). RESULTS: The number of liver tumors decreased significantly in groups treated with As(2)O(3), especially in medium-dose (1 mg/kg) group (t = 2.80, P<0.01). As(2)O(3) caused HCC cell death via apoptosis; necrosis was seen and apoptosis was common when the dose was 1 mg/kg. Proliferation index decreased sharply in medium-dose (1 mg/kg) group (7.87+/-4.11 vs 24.46+/-6.49, t = 2087, P<0.01), but not in 0.2 mg/kg group. However, S-phase fraction decreased dramatically in both groups, it reached the bottom level only when the dose was 1 mg/kg compared with control (0.40+/-0.13 vs 3.01+/-0.51, t = 2.97, P<0.01), and it was obviously accompanied with accumulation of cells in G(0)/G(1) (G(0)/G(1) restriction). The expressions of VEGF and MVD in medium-dose (1 mg/kg) group were significantly lower than normal saline group (0.63+/-0.74 vs 2.44+/-0.88, P<0.05; 15.75+/-3.99 vs 47.44+/-13.41, t = 2.80, P<0.01). Compared with normal saline group, medium- and low-dose groups As(2)O(3) and matrine lowered the levels of ALT in serum (61.46+/-9.46, 63.75+/-20.40, 61.18+/-13.00 vs 108.98+/-29.86, t = 2.14, P<0.05), but had no effect on the level of serum AST, TBi, and DBi. CONCLUSION: As(2)O(3) had inhibitory effect on growth of experimental HCC in rats induced by 2-FAA, but had no obvious effect on normal hepatic cells. The mechanisms may involve decrease of cell division, accumulation of cells in G(0)/G(1) phase, apoptosis of tumor cells, and inhibitory effect on angiogenesis through blocking VEGF.

Glycylproline dipeptidyl aminopeptidase isoenzyme in diagnosis of primary hepatocellular carcinoma.

AIM: To investigate the role of glycylproline dipeptidyl aminopeptidase (GPDA) isoenzyme in the diagnosis of primary hepatocellular carcinoma (PHC), especially in patients with negative alpha-fetoprotein (AFP). METHODS: A stage gradient polyacrylamide gel discontinuous electrophoresis system was developed to separate serum GPDA isoenzymes, which were determined in 102 patients with PHC, 45 cases with liver cirrhosis, 24 cases with chronic hepatitis, 35 cases with benign liver space-occupying lesions, 20 cases with metastatic liver cancer and 50 cases with extra-hepatic cancer, as well as 80 healthy subjects. The relationships between GPDA isoenzymes and AFP, the sizes of tumors, as well as alanine aminotransferase (ALT) were also analyzed. RESULTS: Serum GPDA was separated into two isoenzymes, GPDA-S and GPDA-F. The former was positive in all subjects, while the latter was found mainly in majority of PHC (85.3 %) and a few cases with liver cirrhosis (11.1 %), chronic hepatitis (33.3 %), metastatic liver cancer (15.0 %) and non-hepatic cancer (16.0 %). GPDA-F was negative in all healthy subjects and patients with benign liver space-occupying lesions, including abscess, cysts and angioma. There was no correlation between GPDA-F and AFP concentration or tumor size. GPDA-F was consistently positive and not correlated with ALT in PHC, but GPDA-F often converted to negative as decline of ALT in benign liver diseases. The electrophoretic migration of GPDA-F became sluggish after the treatment of neuraminidase. CONCLUSION: GPDA-F is a new useful serum marker for PHC. Measurement of serum GPDA-F is helpful in diagnosis of PHC, especially in patients with negative AFP. GPDA-F is one kind of glycoproteins rich in sialic acid.

Detection of K-ras point mutation and telomerase activity during endoscopic retrograde cholangiopancreatography in diagnosis of pancreatic cancer.

AIM: To study the value of monitoring K-ras point mutation at codon 12 and telomerase activity in exfoliated cells obtained from pancreatic duct brushings during endoscopic retrograde cholangiopancreatography (ERCP) in the diagnosis of pancreatic cancer. METHODS: Exfoliated cells obtained from pancreatic duct brushings during ERCP were examined in 27 patients: 23 with pancreatic cancers, 4 with chronic pancreatitis. K-ras point mutation was detected with the polymerase chain reaction and restriction fragment-length polymorphism (PCR-RFLP). Telomerase activity was detected by PCR and telomeric repeat amplification protocol assay (PCR-TRAP-ELISA). RESULTS: The telomerase activities in 27 patients were measured in 21 exfoliated cell samples obtained from pancreatic duct brushings. D450 value of telomerase activities in pancreatic cancer and chronic pancreatitis were 0.446+/-0.27 and 0.041+/-0.0111, respectively. Seventy-seven point eight percent (14/18) of patients with pancreatic cancer and none of the patients with chronic pancreatitis showed telomerase activity in cells collected from pancreatic duct brushings when cutoff value of telomerase activity was set at 2.0. The K-ras gene mutation rate (72.2%) in pancreatic cancer was higher than that in chronic pancreatitis (33.3%) (P<0.05). In considering of both telomerase activities and K-ras point mutation, the total positive rate was 83.3%(15/18), and the specificity was 100%. CONCLUSION: Changes of telomerase activities and K-ras point mutation at codon 12 may be an early event of malignant progression in pancreatic cancer. Detection of telomerase activity and K-ras point mutation at codon 12 may be complementary to each other, and is useful in diagnosis of pancreatic cancer.

[The expression of platelet-derived growth factor (PDGF) receptor-beta and its correlation with extracellular matrix in hepatic tissue in hepatic fibrosis rats].

OBJECTIVE: To investigate the expression of PDGF receptor-beta and its correlation with extracellular matrix in hepatic tissue during hepatic fibrosis. METHODS: The model of hepatic fibrosis in rats was induced by carbon tetrachloride. PDGF receptor-beta subunit, collagen I, collagen III and a-SMA in hepatic tissues of these rats were examined using immunohistochemistry. The correlation between PDGF receptor-beta subunit and collagen I, III was analyzed using SAS software after the results of immunohistochemistry were semi-quantified. RESULTS: PDGF receptor-beta subunit and a-SMA were not detected in normal controls. Collagen I and III were distributed in the portal tracts and beneath the endothelia of the central veins and of the Disse spaces. Two weeks after CCl4 injection, the PDGF receptor-beta and a-SMA were detected, and the expression of collagen I and III increased. At the end of 4 and 6 weeks, the above four proteins were further increased. Two weeks after CCl4 injection, PDGF receptor-beta had no apparent correlation with collagen I and III. However, PDGF receptor-beta had a significant correlation with collagen I and III 2 weeks later, and the correlation coefficient was 0.74 and 0.60 respectively at 4 weeks, and 0.83 and 0.67 respectively at 6 weeks. PDGF receptor-beta had a significant correlation with a-SMA during the whole process of hepatic fibrosis and the correlation coefficient was 0.62, 0.69 and 0.81, respectively at the time of 2, 4 and 6 weeks after CCl4 injection. CONCLUSION: The PDGF receptor-beta was overexpressed during the process of hepatic fibrosis development, and it significantly correlated with collagen I and collagen III.

[Detection of GGT-II by dot-ELISA with monoclonal antibody in the diagnosis of hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Hepatoma-specific gamma- glutamyltransferase isoenzyme II(GGT-II) is considered as the best hepatoma marker except alpha-fetoprotein (AFP), but there is no simple and easy method to determine it now. The purpose of this study was to explore the value of detection of GGT-II by dot-ELISA with monoclonal antibody in the diagnosis of hepatocellular carcinoma (HCC). METHODS: GGT-II was purified and then BALB/c mouse was immunized. The monoclonal antibody against GGT-II was raised by the hybridoma technique. Serum GGT-II was detected in 123 cases with hepatocellular carcinoma and 164 cases with various benign liver diseases using both dot-ELISA and electrophoresis simultaneously. RESULTS: The positive rate of serum GGT-II in HCC by dot-ELISA was 71.5%, which was not significantly different from that by electrophoresis (76.4%). However, the false positive rates of GGT-II by dot-ELISA in liver cirrhosis (23.7%) and chronic hepatitis (27.1%) were significantly higher than those by electrophoresis (10.0% and 8.4% for liver cirrhosis and chronic hepatitis, respectively). CONCLUSION: Detection of GGT-II by dot-ELISA with monoclonal antibody is helpful for the diagnosis of HCC, but its diagnostic specificity deserves to be improved.

Expression and kinetic changes of alkaline phosphatase and its isoenzymes in experimental rat hepatoma.

AIM:To explore the expression and changes of hepatoma specific alkaline phospatase (ALP) in rats during canceration.METHODS:The ALPs and isoenzymes of rat livers and sera were investigated in SD hepatomas induced with 0.05% 2-fluoenylacetamide (2-FAA).RESULTS: By pathological examination and biochemical analysis. ALPs were overexpressed in rat livers during canceration and then were secreted into blood. Serum total ALP activities, liver ALP specific activities (U/g) including soluble and membrane-combined ALP activities of each group were all significantly higher (P < 0.01)than those of control group. The average ratios of soluble ALP to membrane-combined ALP were increased significantly after 6 weeks. ALP isoenzymes of rat sera and livers showed 5 bands on PAGE:ALP-I and ALP-II were specific for normal liver and rat hepatoma tissues, the ALP-II appeared in rat liver after 6 weeks and in sera after 8 weeks.CONCLUSION:ALP with carcino-embryonic protein was overexpressed in hepatoma tissues; the abnormal ALP-II of ALP isoenzymes in sera and liver of rats can be used as a tumor marker for early diagnosis of rat hepatoma.

[Factors related to acute upper gastrointestinal bleeding after transcatheter arterial chemoembolization in patients with hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Acute upper gastrointestinal bleeding (UGIB) often occurs after transcatheter arterial chemoembolization (TACE) in the patients with hepatocellular carcinoma (HCC). The authors studied the factors associated with UGIB for better prevention and management of the complication. METHODS: Epirubicin, cisplatin, mitomycin, 5-fluorouracil, lipidol and/or gelfoam were infused via catheters inserted in ciliac artery, common hepatic artery, arteria hepatica propria, or left or right hepatic artery by Seidinger method in 208 cases of advanced HCC confirmed by image techniques, alpha-fetoprotein (AFP) and/or pathology. Factors related to UGIB (vomiting of blood and/or melena, or positive fecal occult blood) were analyzed with reference to endoscopy, biochemical parameters of liver function, selection of blood vessels, and the amount of drugs. RESULTS: Of 208 patients, 31 cases were complicated with UGIB. Acute gastric mucosal lesion was confirmed in 18 cases; acute ulcer in 3 cases; Mallory-Weiss syndrome in 3 cases; and esophageal varices bleeding in 2 cases. Positive correlation was found between B grade of Child-Pugh hepatic functional reserve and bleeding (r = 0.59, P < 0.005). The incidence of UGIB in patients in whom drugs were infused via ciliac artery (7/18, 38.9%); or common hepatic artery (18/38, 47.4%) was significantly higher than in those via arteria hepatica propria, left, or right hepatic artery (5/146, 3.4%; P < 0.005). Patients with larger amount of chemotherapy drug and embolization agent had higher bleeding rate. CONCLUSION: Many factors may be associated with UGIB after TACE in patients with HCC, such as higher scores of hepatic functional reserve in Child-Pugh grading, selection of blood vessels, and amount of drugs. In order to reduce the incidence of UGIB, these factors should be necessarily considered in improvement of TACE procedure, in inspection and management after TACE.