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Background: Lymphovascular invasion (LVI) and perineural invasion (PNI) are important prognostic factors for gastric cancer (GC) that indicate an increased risk of metastasis and poor outcomes. Accurate preoperative prediction of LVI/PNI status could help clinicians identify high-risk patients and guide treatment decisions. However, prior models using conventional computed tomography (CT) images to predict LVI or PNI separately have had limited accuracy. Spectral CT provides quantitative enhancement parameters that may better capture tumor invasion. We hypothesized that a predictive model combining clinical and spectral CT parameters would accurately preoperatively predict LVI/PNI status in GC patients., Aim: To develop and test a machine learning model that fuses spectral CT parameters and clinical indicators to predict LVI/PNI status accurately., Methods: This study used a retrospective dataset involving 257 GC patients (training cohort, n = 172; validation cohort, n = 85). First, several clinical indicators, including serum tumor markers, CT-TN stages and CT-detected extramural vein invasion (CT-EMVI), were extracted, as were quantitative spectral CT parameters from the delineated tumor regions. Next, a two-step feature selection approach using correlation-based methods and information gain ranking inside a 10-fold cross-validation loop was utilized to select informative clinical and spectral CT parameters. A logistic regression (LR)-based nomogram model was subsequently constructed to predict LVI/PNI status, and its performance was evaluated using the area under the receiver operating characteristic curve (AUC)., Results: In both the training and validation cohorts, CT T3-4 stage, CT-N positive status, and CT-EMVI positive status are more prevalent in the LVI/PNI-positive group and these differences are statistically significant ( P < 0.05). LR analysis of the training group showed preoperative CT-T stage, CT-EMVI, single-energy CT values of 70 keV of venous phase (VP-70 keV), and the ratio of standardized iodine concentration of equilibrium phase (EP-NIC) were independent influencing factors. The AUCs of VP-70 keV and EP-NIC were 0.888 and 0.824, respectively, which were slightly greater than those of CT-T and CT-EMVI (AUC = 0.793, 0.762). The nomogram combining CT-T stage, CT-EMVI, VP-70 keV and EP-NIC yielded AUCs of 0.918 (0.866-0.954) and 0.874 (0.784-0.936) in the training and validation cohorts, which are significantly higher than using each of single independent factors ( P < 0.05)., Conclusion: The study found that using portal venous and EP spectral CT parameters allows effective preoperative detection of LVI/PNI in GC, with accuracy boosted by integrating clinical markers., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: Contrast-enhanced ultrasound (CEUS) can be used to diagnose focal liver lesions (FLLs) in children. The America College of Radiology developed the CEUS liver imaging reporting and data system (LI-RADS) for standardizing CEUS diagnosis of FLLs in adult patients. Until now, no similar consensus or guidelines have existed for pediatric patients to improve imaging interpretation as adults., Aim: To evaluate the performance of CEUS LI-RADS combined with alpha-fetoprotein (AFP) in differentiating benign and malignant FLLs in pediatric patients., Methods: Between January 2011 and January 2021, patients ≤ 18 years old who underwent CEUS for FLLs were retrospectively evaluated. The following criteria for diagnosing malignancy were proposed: Criterion I considered LR-4, LR-5, or LR-M lesions as malignancies; criterion II regarded LR-4, LR-5 or LR-M lesions with simultaneously elevated AFP (≥ 20 ng/mL) as malignancies; criterion III took LR-4 Lesions with elevated AFP or LR-5 or LR-M lesions as malignancies. The sensitivity, specificity, accuracy and area under the receiver operating characteristic curve (AUC) were calculated to determine the diagnostic value of the aforementioned criteria., Results: The study included 63 nodules in 60 patients (mean age, 11.0 ± 5.2 years; 26 male). There were no statistically significant differences between the specificity, accuracy, or AUC of criterion II and criterion III (95.1% vs 80.5%, 84.1% vs 87.3%, and 0.794 vs 0.902; all P > 0.017). Notably, criterion III showed a higher diagnostic sensitivity than criterion II (100% vs 63.6%; P < 0.017). However, both the specificity and accuracy of criterion I was inferior to those of criterion II and criterion III (all P < 0.017). For pediatric patients more than 5 years old, the performance of the three criteria was overall similar when patients were subcategorized by age when compared to all patients in aggregate., Conclusion: CEUS LI-RADS combined with AFP may be a powerful diagnostic tool in pediatric patients. LR-4 with elevated AFP, LR-5 or LR-M lesions is highly suggestive of malignant tumors., Competing Interests: Conflict-of-interest statement: There are no conflicts of interest to report., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: Combined hepatocellular-cholangiocarcinoma (CHC) is a rare type of primary liver cancer. Due to its complex histopathological characteristics, the imaging features of CHC can overlap with those of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC)., Aim: To investigate the possibility and efficacy of differentiating CHC from HCC and ICC by using contrast-enhanced ultrasound (CEUS) Liver Imaging Reporting and Data System (LI-RADS) and tumor biomarkers., Methods: Between January 2016 and December 2019, patients with histologically confirmed CHC, ICC and HCC with chronic liver disease were enrolled. The diagnostic formula for CHC was as follows: (1) LR-5 or LR-M with elevated alpha-fetoprotein (AFP) and carbohydrate antigen 19-9 (CA19-9); (2) LR-M with elevated AFP and normal CA19-9; or (3) LR-5 with elevated CA19-9 and normal AFP. The sensitivity, specificity, accuracy and area under the receiver operating characteristic curve were calculated to determine the diagnostic value of the criteria., Results: After propensity score matching, 134 patients (mean age of 51.4 ± 9.4 years, 108 men) were enrolled, including 35 CHC, 29 ICC and 70 HCC patients. Based on CEUS LI-RADS classification, 74.3% (26/35) and 25.7% (9/35) of CHC lesions were assessed as LR-M and LR-5, respectively. The rates of elevated AFP and CA19-9 in CHC patients were 51.4% and 11.4%, respectively, and simultaneous elevations of AFP and CA19-9 were found in 8.6% (3/35) of CHC patients. The sensitivity, specificity, positive predictive value, negative predictive value, accuracy and area under the receiver operating characteristic curve of the aforementioned diagnostic criteria for discriminating CHC from HCC and ICC were 40.0%, 89.9%, 58.3%, 80.9%, 76.9% and 0.649, respectively. When considering the reported prevalence of CHC (0.4%-14.2%), the positive predictive value and NPV were revised to 1.6%-39.6% and 90.1%-99.7%, respectively., Conclusion: CHCs are more likely to be classified as LR-M than LR-5 by CEUS LI-RADS. The combination of the CEUS LI-RADS classification with serum tumor markers shows high specificity but low sensitivity for the diagnosis of CHC. Moreover, CHC could be confidently excluded with high NPV., Competing Interests: Conflict-of-interest statement: All authors have no financial relationships to disclose., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) differ in treatment and prognosis, warranting an effective differential diagnosis between them. The LR-M category in the contrast-enhanced ultrasound (CEUS) liver imaging reporting and data system (LI-RADS) was set up for lesions that are malignant but not specific to HCC. However, a substantial number of HCC cases in this category elevated the diagnostic challenge., Aim: To investigate the possibility and efficacy of differentiating ICC from HCC classified in the LR-M category according to the CEUS LI-RADS., Methods: Patients with complete CEUS records together with pathologically confirmed ICC and LR-M HCC (HCC classified in the CEUS LI-RADS LR-M category) between January 2015 and October 2018 were included in this retrospective study. Each ICC was assigned a category as per the CEUS LI-RADS. The enhancement pattern, washout timing, and washout degree between the ICC and LR-M HCC were compared using the χ 2 test. Logistic regression analysis was used for prediction of ICC. Receiver operating characteristic (ROC) curve analysis was used to investigate the possibility of LR-M criteria and serum tumor markers in differentiating ICC from LR-M HCC., Results: A total of 228 nodules (99 ICCs and 129 LR-M HCCs) in 228 patients were included. The mean sizes of ICC and LR-M HCC were 6.3 ± 2.8 cm and 5.5 ± 3.5 cm, respectively ( P = 0.03). Peripheral rim-like arterial phase hyperenhancement (APHE) was detected in 50.5% (50/99) of ICCs vs 16.3% (21/129) of LR-M HCCs ( P < 0.001). Early washout was found in 93.4% (93/99) of ICCs vs 96.1% (124/129) of LR-M HCCs ( P > 0.05). Marked washout was observed in 23.2% (23/99) of ICCs and 7.8% (10/129) of LR-M HCCs (P = 0.002), while this feature did not show up alone either in ICC or LR-M HCC. Homogeneous hyperenhancement was detected in 15.2% (15/99) of ICCs and 37.2% (48/129) of LR-M HCCs ( P < 0.001). The logistic regression showed that rim APHE, carbohydrate antigen 19-9 (CA 19-9), and alpha fetoprotein (AFP) had significant correlations with ICC ( r = 1.251, 3.074, and -2.767, respectively; P < 0.01). Rim APHE presented the best enhancement pattern for diagnosing ICC, with an area under the ROC curve (AUC) of 0.70, sensitivity of 70.4%, and specificity of 68.8%. When rim hyperenhancement was coupled with elevated CA 19-9 and normal AFP, the AUC and sensitivity improved to 0.82 and 100%, respectively, with specificity decreasing to 63.9%., Conclusion: Rim APHE is a key predictor for differentiating ICC from LR-M HCC. Rim APHE plus elevated CA 19-9 and normal AFP is a strong predictor of ICC rather than LR-M HCC. Early washout and marked washout have limited value for the differentiation between the two entities., Competing Interests: Conflict-of-interest statement: The authors have no financial relationships to disclose., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Aim: To optimize the hepatobiliary phase delay time (HBP-DT) of Gd-EOB-DTPA-enhanced magnetic resonance imaging (GED-MRI) for more efficient identification of hepatocellular carcinoma (HCC) occurring in different degrees of cirrhosis assessed by Child-Pugh (CP) score., Methods: The liver parenchyma signal intensity (LPSI), the liver parenchyma (LP)/HCC signal ratios, and the visibility of HCC at HBP-DT of 5, 10, 15, 20, and 25 min ( i.e ., DT-5, DT-10, DT-15, DT-20, and DT-25 ) after injection of Gd-EOB-DTPA were collected and analyzed in 73 patients with cirrhosis of different degrees of severity (including 42 patients suffering from HCC) and 18 healthy adult controls., Results: The LPSI increased with HBP-DT more significantly in the healthy group than in the cirrhosis group ( F = 17.361, P < 0.001). The LP/HCC signal ratios had a significant difference ( F = 12.453, P < 0.001) among various HBP-DT points, as well as between CP-A and CP-B/C subgroups ( F = 9.761, P < 0.001). The constituent ratios of HCC foci identified as obvious hypointensity (+++), moderate hypointensity (++), and mild hypointensity or isointensity (+/-) kept stable from DT-10 to DT-25: 90.6%, 9.4%, and 0.0% in the CP-A subgroup; 50.0%, 50.0%, and 0.0% in the CP-B subgroup; and 0.0%, 0.0%, and 100.0% in the CP-C subgroup, respectively., Conclusion: The severity of liver cirrhosis has significant negative influence on the HCC visualization by GED-MRI. DT-10 is more efficient and practical than other HBP-DT points to identify most of HCC foci emerging in CP-A cirrhosis, as well as in CP-B cirrhosis; but an HBP-DT of 15 min or longer seems more appropriate than DT-10 for visualization of HCC in patients with CP-C cirrhosis., Competing Interests: Conflict-of-interest statement: All the authors listed above have no potential conflicts of interest relevant to this article to report.

Aim: To compare the outcomes of preoperative endoscopic nasobiliary drainage (ENBD) and endoscopic retrograde biliary drainage (ERBD) in patients with malignant distal biliary obstruction prior to pancreaticoduodenectomy (PD)., Methods: Data from 153 consecutive patients who underwent preoperative endoscopic biliary drainage prior to PD between January 2009 and July 2016 were analyzed. We compared the clinical data, procedure-related complications of endoscopic biliary drainage (EBD) and postoperative complications of PD between the ENBD and ERBD groups. Univariate and multivariate analyses with odds ratios (ORs) and 95% confidence intervals (95%CIs) were used to identify the risk factors for deep abdominal infection after PD., Results: One hundred and two (66.7%) patients underwent ENBD, and 51 (33.3%) patients underwent ERBD. Endoscopic sphincterotomy was less frequently performed in the ENBD group than in the ERBD group ( P = 0.039); the EBD duration in the ENBD group was shorter than that in the ERBD group ( P = 0.036). After EBD, the levels of total bilirubin (TB) and alanine aminotransferase (ALT) were obviously decreased in both groups, and the decreases of TB and ALT in the ERBD group were greater than those in the ENBD group ( P = 0.004 and P = 0.000, respectively). However, the rate of EBD procedure-related cholangitis was significantly higher in the ERBD group than in the ENBD group ( P = 0.007). The postoperative complications of PD as graded by the Clavien-Dindo classification system were not significantly different between the two groups ( P = 0.864). However, the incidence of deep abdominal infection after PD was significantly lower in the ENBD group than in the ERBD group ( P = 0.019). Male gender (OR = 3.92; 95%CI: 1.63-9.47; P = 0.002), soft pancreas texture (OR = 3.60; 95%CI: 1.37-9.49; P = 0.009), length of biliary stricture (≥ 1.5 cm) (OR = 5.20; 95%CI: 2.23-12.16; P = 0.000) and ERBD method (OR = 4.08; 95%CI: 1.69-9.87; P = 0.002) were independent risk factors for deep abdominal infection after PD., Conclusion: ENBD is an optimal method for patients with malignant distal biliary obstruction prior to PD. ERBD is superior to ENBD in terms of patient tolerance and the effect of biliary drainage but is associated with an increased risk of EBD procedure-related cholangitis and deep abdominal infection after PD., Competing Interests: Conflict-of-interest statement: The authors declare that there is no conflict of interest related to this report.

Hepatic epithelioid hemangioendothelioma (HEH) is a rare tumor of vascular endothelial origin. Spontaneous rupture of HEH is a life-threatening complication and is extremely rare. HEH has variable malignant potential, and the clinical diagnosis remains challenging. Here we report a case of HEH with spontaneous rupture. A 44-year-old man presented with constant cutting pains over the right upper abdomen after eating. He had hemoptysis 11 d previously. Diagnostic abdominal puncture demonstrated active bleeding. Chest and abdominal computer tomography scan showed multiple ground-glass nodules over the lungs, multiple low-density intrahepatic nodules and massive hemorrhage. Transcatheter arterial embolization and exploratory laparotomy were performed and subsequent immunohistochemical examination confirmed a diagnosis of HEH.

Primary splenic angiosarcoma (PSA) is an unusual and highly malignant vascular tumour with a high rate of metastatic. Moreover, the research on prognosis of the disease is poor. The epidemiology, etiology, clinical diagnosis and treatment of the disease remain challenging, because case reports of the disease are few in number. In accordance with other malignant tumors, PSA is very aggressive, and the majority of patients in which this disease is found are at an advanced stage. Almost all patients die within 12 mo of diagnosis irrespective of treatment. We report here a woman who had complained of upper bellyache and anorexia for 10 d. Magnetic resonance imaging showed enlargement of the spleen with multiple heterogeneous masses in the lower pole of the spleen. A hand-assisted laparoscopic splenectomy was performed which allowed histopathologic diagnosis. The patient was diagnosed with PSA and liver metastasis, and succumbed to the disease 35 d after surgery. The literature was finished combined with the clinical features, diagnosis and management of PSA.

Aim: To examine transforming growth factor-β1 (TGF-β1) promoter methylation in gastric cancer and to determine if Helicobacter pylori (H. pylori) or interleukin (IL)-1β could induce TGF-β1 hypermethylation in vitro., Methods: We examined the frequency and extent of TGF-β1 promoter methylation using methylation-specific PCR in the gastric tissues from 47 gastric cancer patients and 39 non-gastric cancer subjects. H. pylori infection was confirmed by a positive result from either a serological test, histological analysis or C¹³ urea breath test. GES-1 and MKN-45 cells co-cultured with H. pylori or treated with IL-1β for 12, 24 and 48 h in vitro tested the effects of H. pylori or IL-1β on TGF-β1., Results: Twenty-four/forty-seven (51%) cases of gastric cancer (GC) tissues showed TGF-β1 promoter methylation, 15/47 (31.9%) cases of matched non-cancerous gastric mucosa tissues from the GC patients, and 11/39 (28%) case of the normal gastric mucosa tissues from non-GC subjects showed TGF-β1 promoter methylation (51% vs 28%, P < 0.05). Significantly higher levels of methylation of TGF-β1 were found in the tumor tissues than in non-tumor tissues from GC patients (0.24 ± 0.06 vs 0.17 ± 0.04, P < 0.05) and normal gastric tissues from non-GC subjects (0.24 ± 0.06 vs 0.15 ± 0.03, P < 0.05). TGF-β1 methylation was found in 48.3% of H. pylori-positive gastric mucosal tissues whereas only 23.1% of H. pylori-negative gastric mucosal tissues showed TGF-β1 methylation (48.3% vs 23.1%, P < 0.05). IL-1β appeared to induce a dose-dependent methylation of TGF-β1 and the strongest methylation was observed in GES-1 cells treated with 2.5 ng/mL of IL-1β for 48 h. Further studies showed that pre-treatment of GES-1 cells with 20 ng/mL IL-1RA for 1 h could partially abolish the effect of IL-1β on TGF-β1 methylation. Infection of GES-1 cells by H. pylori was not found to induce significant TGF-β1 promoter methylation., Conclusion: Our data revealed that TGF-β1 promoter is methylated in GC patients. IL-1β may be an important mediator for H. pylori induced gene methylation during GC development.

Aim: To evaluate a novel biosensor-based microarray (BBM) assay for detecting rs12979860 and rs8099917 genotypes., Methods: Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed, constructed and arrayed on an optical biosensor to develop a microarray assay. Two sets of primers were used in a one tube polymerase chain reaction (PCR) system to amplify two target fragments simultaneously. The biosensor microarray contained probes that had been sequenced to confirm that they included the rs8099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards. In addition to rehybridization of four probes of known sequence, a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested. The target fragments of all 40 samples were amplified in a 50 μL PCR system. Ten μL of each amplicon was tested by BBM assay, and another 40 μL was used for sequencing. The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient. The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 10(3)-10(4) white cells/μL., Results: As shown by polyacrylamide gel electrophoresis, two target segments of the interleukin 28B-associated polymorphisms (SNPs) were successfully amplified in the one-tube PCR system. The lengths of the two amplified fragments were consistent with the known length of the target sequences, 137 and 159 bps. After hybridization of the PCR amplicons with the probes located on the BBM array, the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye. The signals were distinct from each other, could be interpreted visually, and accurately recorded using an ordinary digital camera. To evaluate the specificity of the assay, both the plasmids and clinical samples were applied to the microarray. First, 30 PCR amplicons of the various SNP alleles were hybridized on the BBM microarray. Full agreement between plasmids and the BBM assay was observed, with 30/30 correct matches (100%). The kappa value for the BBM assay with plasmids was 1.00 (P < 0.05). For the 40 clinical blood samples, the BBM assay hybridization and direct sequencing results were compared for each amplicon. For patient blood samples, agreement was 28/28 for rs8099917T/T, 9/11 for rs8099917T/G, 1/1 for rs8099917G/G, 24/24 for rs12979860C/C, 11/14 for rs12979860C/T, and 2/2 for rs12979860T/T. Only five clinical samples of amplicon assay and direct sequencing results were discordant and heterozygotes: 2/11 rs8099917T/G and 3/14 rs12979860C/T. The agreement of outcomes between BBM assay and direct sequencing for the detection of rs8099917 and rs12979860 was 95% and 92.5%, respectively; and the corresponding kappa values were 0.88 and 0.85 (A kappa value > 0.75 was defined as substantial agreement). The BBM assay and sequencing had similar specificities for detection and identification of the two SNPs and their alleles. The sensitivity evaluation showed that the BBM assay could detect and identify SNP sequences present in blood samples containing as few as 10(2) white blood cells/μL., Conclusion: This biosensor microarray assay was highly specific, sensitive, rapid and easy to perform. It is compatible with clinical practice for detection of rs8099917 and rs12979860.

Aim: To explore the DNA image cytometry (DNA-ICM) technique as a primary screening method for esophageal squamous precancerous lesions., Methods: This study was designed as a population-based screening study. A total of 582 local residents aged 40 years-69 years were recruited from Linzhou in Henan and Feicheng in Shandong. However, only 452 subjects had results of liquid-based cytology, DNA-ICM and pathology. The sensitivity and specificity of DNA-ICM were calculated and compared with liquid-based cytology in moderate dysplasia or worse., Results: Sensitivities of DNA-ICM ranging from at least 1 to 4 aneuploid cells were 90.91%, 86.36%, 79.55% and 77.27%, respectively, which were better than that of liquid-based cytology (75%). Specificities of DNA-ICM were 70.83%, 84.07%, 92.65% and 96.81%, but the specificity of liquid-based cytology was 91.91%. The sensitivity and specificity of a combination of liquid-based cytology and DNA-ICM were 84.09% and 85.78%, respectively., Conclusion: It is possible to use DNA-ICM technique as a primary screening method for esophageal squamous precancerous lesions.

Aim: To assess the application of the Kasai procedure in the surgical management of hilar bile duct strictures., Methods: Ten consecutive patients between 2005 and 2011 with hilar bile duct strictures who underwent the Kasai procedure were retrospectively analyzed. Kasai portoenterostomy with the placement of biliary stents was performed in all patients. Clinical characteristics, postoperative complications, and long-term outcomes were analyzed. All patients were followed up for 2-60 mo postoperatively., Results: Patients were classified according to the Bismuth classification of biliary strictures. There were two Bismuth III and eight Bismuth IV lesions. Six lesions were benign and four were malignant. Of the benign lesions, three were due to post-cholecystectomy injury, one to trauma, one to inflammation, and one to inflammatory pseudotumor. Of the malignant lesions, four were due to hilar cholangiocarcinoma. All patients underwent Kasai portoenterostomy with the placement of biliary stents. There were no perioperative deaths. One patient experienced anastomotic leak and was managed conservatively. No other complications occurred perioperatively. During the follow-up period, all patients reported a good quality of life., Conclusion: The Kasai procedure combined with biliary stents may be appropriate for patients with hilar biliary stricture that cannot be managed by standard surgical methods.

Aim: to determine whether mitochondrial dysfunction resulting from high-fat diet is related to impairment of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt, also known as PKB) pathway., Methods: rat models of nonalcoholic fatty liver were established by high-fat diet feeding. The expression of total and phosphorylated P13K and Akt proteins in hepatocytes was determined by Western blotting. Degree of fat accumulation in liver was measured by hepatic triglyceride. Mitochondrial number and size were determined using quantitative morphometric analysis under transmission electron microscope. The permeability of the outer mitochondrial membrane was assessed by determining the potential gradient across this membrane., Results: after Wistar rats were fed with high-fat diet for 16 wk, their hepatocytes displayed an accumulation of fat (103.1 ± 12.6 vs 421.5 ± 19.7, P < 0.01), deformed mitochondria (9.0% ± 4.3% vs 83.0% ± 10.9%, P < 0.05), and a reduction in the mitochondrial membrane potential (389.385% ± 18.612% vs 249.121% ± 13.526%, P < 0.05). In addition, the expression of the phosphorylated P13K and Akt proteins in hepatocytes was reduced, as was the expression of the anti-apoptotic protein Bcl-2, while expression of the pro-apoptotic protein caspase-3 was increased. When animals were treated with pharmacological inhibitors of P13K or Akt, instead of high-fat diet, a similar pattern of hepatocellular fat accumulation, mitochondrial impairment, and change in the levels of PI3K, Akt, Bcl-2 was observed., Conclusion: high-fat diet appears to inhibit the PI3K/Akt signaling pathway, which may lead to hepatocellular injury through activation of the mitochondrial membrane pathway of apoptosis.

Aim: To investigate the possible correlation between osteoglycin expression and gelatinase activity of mouse hepatocarcinoma Hca-F cells., Methods: A eukaryotic expression plasmid pIRESpuro3 osteoglycin(+) was constructed and transfected into Hca-F cells to investigate the possible correlation between osteoglycin expression and gelatinase activity of Hca-F cells cultured with extract of lymph node, liver, spleen or in DMEM medium. The activity of gelatinases was examined through zymographic analysis., Results: High expression of osteoglycin attenuated the gelatinase activity of Hca-F cells cultured with extract of lymph node, and at the same time, decreased the metastatic potential of Hca-F cells to peripheral lymph nodes in vivo., Conclusion: High expression of osteoglycin decreases the gelatinase activity of Hca-F cells cultured with extract of lymph node; regulation of gelatinase activity might be one of mechanisms that osteoglycin contributes to lymphatic metastasis suppression.

Aim: To determine the role of Sonic hedgehog (Shh) pathway in colorectal adenocarcinomas through analysis of the expression of Shh pathway-related molecules, Shh, Ptch1, hedgehog-interacting protein (Hip), Gli1, Gli3 and PDGFRalpha., Methods: Expression of Shh in 25 colorectal adeno-carcinomas was detected by RT-PCR, in situ hybridization and immunohistochemistry. Expression of Ptch1 was observed by in situ hybridization and immunohistochemistry. Expression of Hip, Gli1, Gli3 and PDGFRalpha was analyzed by in situ hybridization., Results: Expression of cytokeratin AE1/AE3 was observed in the cytoplasm of colorectal crypts. Members of the Hh signaling pathway were expressed in colorectal epithelium. Shh was expressed in cytoplasm of dysplastic epithelial cells, while expression of Ptch1, Hip and Gli1 were mainly detected in the malignant crypts of adenocarcinomas. In contrast, PDGFRalpha was expressed highly in aberrant crypts and moderately in the stroma. Expression of Gli3 could not be detected in colorectal adenocarcinomas., Conclusion: These data suggest that Shh-Ptch1-Gli1 signaling pathway may play a role in the progression of colorectal tumor.

Aim: To identify genes differentially expressed in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis., Methods: A subtracted cDNA library of mouse hepatocarcinoma cell line with low potential of lympho-genous metastasis Hca-P and its synogenetic cell line Hca-F with high metastatic potential was constructed by suppression subtracted hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed., Results: Fifteen differentially expressed cDNA fragments of Hca-P were obtained which revealed 8 known genes, 4 expressed sequence tags (ESTs) and 3 cDNAs showed no homology., Conclusion: Tumor metastasis is an incident involving multiple genes. SSH is a useful technique to detect differentially expressed genes and an effective method to clone novel genes.

Aim: To study the expression of Sonic hedgehog pathway-related molecules, Sonic hedgehog (Shh) and Gli1 in gastric carcinoma., Methods: Expression of Shh in 56 gastric specimens including non-cancerous gastric tissues, gastric adenocarcinoma, gastric squamous cell carcinoma was detected by RT-PCR, in situ hybridization and immunohistochemistry. Expression of Gli1 was observed by in situ hybridization., Results: The positive rate of Shh and Gli1 expression was 0.0%, 0.0% in non-cancerous gastric tissues while it was 66.7%, 57.8% respectively in gastric adenocarcinoma, and 100%, 100% respectively in gastric squamous cell carcinoma. There was a significant difference between the non-cancerous gastric tissues and gastric carcinoma (P<0.05). Elevated expression of Shh and Gli1 in gastric tubular adenocarcinoma was associated with poorly differentiated tumors while the expression was absent in gastric mucinous adenocarcinoma., Conclusion: The elevated expression of Shh and Gli1 in gastric adenocarcinoma and gastric squamous cell carcinoma shows the involvement of activated Shh signaling in the cellular proliferation of gastric carcinogenesis. It suggests Shh signaling gene may be a new and good target gene for gastric tumor diagnosis and therapy.

Aim: To construct the eukaryotic expression plasmid containing HCV NS3 segment and to analyze the expression of NS3 protein in normal human hepatocyte HL-7702., Methods: We amplified HCV NS3 fragment from plasmid pBRTM/HCV 1-3011 containing the whole length of HCV genome, recombined it with expression vector pcDNA3.1(-) to form the eukaryotic expression vector pcDNA3.1(-)/NS3, and transfected human HL-7702 hepatocytes with the recombined plasmid by cationic polymers. The expressed HCV NS3 protein was detected and analyzed by immunohistochemical method and Western blot., Results: The amplified NS3 fragments had correct molecule weight and sequence. The successfully constructed eukaryotic expression plasmids were transfected to HL-7702 cells. The expressed NS3 proteins had correct molecular weight 70000., Conclusion: Eukaryotic expression vector pcDNA3.1 (-)/NS3 containing NS3 segment of HCV can be constructed, the sequence of NS3 fragments is consistent with the template. Normal human HL-7702 hepatocytes can efficiently express specific HCV NS3 protein in vitro.

Aim: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis., Methods: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed., Results: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes., Conclusion: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.

Aim: In order to obtain lymphogenous metastasis-associated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential., Methods: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e. biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip(r) MOE430A (containing 22 690 transcripts, including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics., Results: Out of the 14 500 known genes investigated, 110 (0.8%) were up regulated at least 2(3) fold. Among the total 4 371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 2(3) fold. According to the Gene Ontology and TreeView analysis, the 110 genes were further classified into two groups: differential biological process profile and molecular function profile., Conclusion: Using high-throughput gene chip method, a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription, chaperone activity, motor activity, protein kinase activity, receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation. Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments. ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.

Aim: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential., Methods: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5alpha competent cells. Individual clones were randomly selected and used for PCR amplification. Vector DNA from positive clones was isolated for sequencing., Results: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13, ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes., Conclusion: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.

Aim: To study and compare the difference of activation-induced cell death (AICD) in peripheral blood T-lymphocytes(PBL-Ts) from patients with chronic hepatitis B (CHB) and the normal people in vitro, and to explore the role of AICD in chronic hepatitis B virus (HBV) infection and the pathogenesis of CHB., Methods: Twenty-five patients and fourteen healthy people were selected for isolation of PBL-Ts. During cultivation, anti-CD3 mAb, PMA and ionomycin were used for AICD of PBL-Ts. AICD ratio of PBL-Ts was detected with TdT-mediated dUTP nick end labeling and assessed by flow cytometry., Results: When induced with anti-CD3, PMA and ionomycin in vitro, AICD ratio of PBL-Ts from CHB patients was significantly higher than that from healthy control (17.24+/-1.21 vs. 6.63+/-1.00, P<0.01) and that from CHB patients without induction (17.24+/-1.21 vs. 9.88+/-1.36, P<0.01). There was a similar AICD ratio of PBL-Ts between induction group and without induction group, but no difference was found before and after induction in healthy control. The density of INF-gamma in culture media of induction groups of CHB was lower than that of other groups (P<0.01). There was no difference between these groups in density of IL-10 (P>0.05)., Conclusion: When induced during cultivation in vitro, PBL-Ts from CHB have AICD very commonly. This phenomenon has a potentially important relation with pathogenesis of CHB and chronicity of HBV infection.