[Study on the state of macrophage infiltration in the progression of non-alcoholic fatty liver disease induced by high-fat diet in mice].

Objective: Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease. Macrophages are an important cell-mediated immune response in the pathogenesis of NAFLD. However, the dynamic changes of intrahepatic infiltration during the pathogenesis of NAFLD remains unclear. Therefore, this study intends to investigate the changes and their correlations with morphological indicators, hepatic histopathological index, and intrahepatic macrophage infiltration in the progression of NAFLD induced by high-fat diet in mice. Methods: C57BL/6J mice were fed with 42% high-fat diet, and the morphological data and liver tissue were obtained at 1, 2, 4, 8 and 12 months, respectively. Hepatic histopathological characteristics were evaluated by HE stain. Immunohistochemical staining was used to detect the number of F4/80 positive cells in liver tissue at different stages to evaluate the degree of intrahepatic macrophage infiltration. Results: (1) The body weight, liver weight, and liver weight/body weight of mice fed with high-fat diet had gradually increased. (2) HE staining results showed that mice fed with high-fat diet had mainly developed simple steatosis within 1 to 2 months. In addition, a balloon-like hepatocyte degeneration and intralobular inflammation had begun to appear at 4 months, indicating that non-alcoholic steatohepatitis had started, and can be seen very clearly at 8 to 12 months. (3) Immunohistochemical staining results suggested that in the simple steatosis stage of NAFLD, the intrahepatic macrophage infiltration was not significant. However, after NAFLD activity score > 3, a large amount of infiltration had appeared and cluster-like changes in the later stage. (4) Correlation analysis results indicated that the degree of macrophage infiltration was not related to the mice morphological indicators (body weight, liver weight and liver weight/body weight) and pathological indicators (percentage of hepatic steatotic change, and degree of hepatocyte ballooning and intralobular inflammation), but was significantly related to the NAFLD activity score. Conclusion: High-fat diet can successfully induce NAFLD in mice and progress to the stage of non-alcoholic steatohepatitis. At the same time, high-fat diet can induce macrophage infiltration in liver tissue of mice and the changing trend of infiltration is related to NAFLD activity score.

[FibroTouch diagnostic efficiency and influencing factors for liver fibrosis in autoimmune liver diseases].

FibroTouch (FT) has been used widely in clinic. Studies of the FT diagnostic efficiency and influencing factors of liver stiffness measurement (LSM) of liver fibrosis in autoimmune liver diseases (AILD) have shown that FT has a good diagnostic efficiency and accuracy, especially in AIH. However, for patients with primary biliary cholangitis and overlap syndrome of autoimmune hepatitis, FT results should be vigilant. In addition, the diagnostic efficiency of FT. Notably, when using FT to diagnose AILD, the value of elastic stiffness depends on five influencing factors, such as, age, total bile acid, international standardized ratio, FIB-4 index, and prothrombin time.

[The influence and mechanisms of lysine-specific demethylase 1 (LSD1) on invasion and metastasis of colon cancer cells].

Objective: To investigate the effect and molecular mechanisms of LSD1 on proliferation and metastasis of colon cancer. Methods: The influence of down-regulated LSD1 expression on proliferation, invasion and apoptosis of colon cancer cells were detected by transwell invasion assay, cell proliferation assay and apoptosis assay, respectively. Results: Three independent siRNAs targeting LSD1 (siRNA-1554, siRNA-705, and siRNA-1973) were transfected to SW620 cells to detect gene-silencing efficiency, and the result showed that the knockdown effect of siRNA-705 were better than the other two siRNAs at both mRNA and protein levels. Using siRNA-705 and pargyline (2.5 mmol/L), we performed transwell invasion assay, cell proliferation assay and apoptosis assay in SW620 cell lines, and found the significant suppression of invasion and growth. Cell apoptosis were induced by siRNA and pargyline (P<0.05). Interestingly, up-regulation of E-cadherin and down-regulation of N-cadherin were observed after treated with siRNA-705 and pargyline for 72 hours. Conclusion: Inhibition of LSD1 could impair proliferation and invasiveness, and induce apoptosis of colon cancer cells in vitro. It leads to up-regulation of E-cadherin and down-regulation of N-cadherin. All of above may play important roles in invasion and metastasis of colon cancer.

Integrin alphanvbeta6 acts as a prognostic indicator in gastric carcinoma.

AIMS: To investigate the relationships between integrin alphanubeta6 expression and the clinical-pathological features of gastric carcinoma and whether integrin alphanubeta6 can act as a prognostic indicator in gastric carcinoma. MATERIALS AND METHODS: We generated the microarray of 300 human gastric carcinoma specimens, and used the method of immunohistochemistry to investigate the expression of alphanubeta6 in them and the relationships between the expression of alphanubeta6 and the clinical-pathological features of the tumours. Meanwhile, we retrospectively analysed the relationship between alphanubeta6 expression and the survival times of the patients. RESULTS: The expression of alphanubeta6 was detected in 36.7% of gastric carcinomas, and the expression was associated with Lauren type, differentiation, N stage and TNM stage of the tumours (the P values were 0.004, 0.035, 0.024 and 0.001, respectively). The Kaplan-Meier plot showed that patients who were alphanubeta6 negative had much longer survival times than those who were alphanubeta6 positive (P<0.0001). The survival estimates showed a striking difference in median survival between the negative and positive alphanubeta6 expression patients, especially in early stage tumours. Univariate analysis indicated that significant factors for prognosis included alphanubeta6 expression, differentiation, TNM stage, T stage, N stage, M stage and R classification (R0: potentially curative resection; R1: had residual microscopic disease after resection; R2: had residual macroscopic disease after resection), whereas in multivariate analysis using the Cox regression model, only alphanubeta6 expression, M stage, TNM stage and R classification retained significance for prognosis. CONCLUSIONS: Positive alphanubeta6 expression in gastric carcinoma is linked to significantly reduced survival times and, even more important, is that its value as a prognostic marker is significant for early stage tumours.

cDNA cloning and high-level expression of a thrombin-like enzyme from Agkistrodon acutus venom.

Agkistrodon acutus (Guenther), a poisonous snake species of the family of Crotalidae, is mainly found south of the Yellow River in China. The main symptom of this poison is massive hemorrhage, in which thrombin-like enzymes (TLEs) in the venom play an important role. TLEs are abundant, especially in the venom of A. acutus. We isolated the total RNA from the venom gland tissue of A. acutus and amplified the cDNAs of the TLEs using reverse transcription-polymerase chain reaction (RT-PCR). The cDNAs were cloned into vector pThioHis B and were expressed as fusion proteins in the form of inclusion bodies, which accounted for nearly 50% of the total cell proteins. The inclusion bodies were washed, dissolved, refolded and purified by affinity chromatography. The purity was higher than 97%, as indicated by capillary zone electrophoresis. The renatured recombinant enzyme exhibited arginine esterase activity, as tested by the BAEE method, and also showed a fibrinogen cleavage effect, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This method provides a fast and convenient system for studying the structure-function relationships in TLE isoenzymes, and also a practical way for mass production of TLEs in the pharmaceutical industry.

[Detection of nucleotide mutation by direct sequencing method using non-radio isotopic marker].

We report a protocol which can analyze DNA by the dideoxy method. First, we prepared DNA from paraffin specimen of colon cancer and normal tissue by the method using proteinase and phenol. Polymerase chain reaction (PCR) was performed as follows. The primers used were oligonucleotides corresponding to the sequence of exon 5 on p53. An initial denaturing step was carried out at 94 degrees C for 2 min. Products were amplified for 40 cycles at 94 degrees C for 1 min, 60 degrees C for 1 min, and 72 degrees C for 1 min. Specific PCR products derived from p53 gene were purified. Protocol for the PCR-sequencing reaction: The reaction mixture was divided into four 4 microliters fraction. Each fraction was mixed with 2 microliters of NTP solution including non-RI dideoxynucleotides (TOYOBO). PCR was carried out as follows: an initial denaturing step at 94 degrees C for 1 min, then 30 cycles at 94 degrees C for 1 min, 60 degrees C for 1 min, and 72 degrees C for 1 min. Prior to loading in a denaturing 8% polyacrylamide-6M Urea gel, the samples were heated to 94 degrees C for 2 min then quickly chilled in ice-water. Electrophoresis was carried out at 1000V for 3hr and transcribed to a nylon membrane. The ladders of DNA were obtained by Non-RI Detection Kit (TOYOBO). We determined the sequence of 167 nucleotides. Results indicated that the point mutations in DNA could be easily detected.

Fourier-phase method for the location of moving objects.

A novel method is presented that permits one to locate a moving object. According to this method, the change in location of a moving object can be detected from its Fourier spectrum. This method has the advantages of a high-precision locating mechanism and the capacity to permit one to ignore the change in the orientation and the size of the moving object. The principles are introduced, a computer simulation and experimental demonstration are given, and the practicality of this method is discussed.

[Endoscopic bipolar circumactive electrocoagulation in acute upper gastrointestinal bleeding].

Bipolar circum-active probe (BICAP) is a new endoscopically deliverable bipolar electrocoagulator. 57 patients with acute upper gastrointestinal bleeding have been treated by the endoscopic BICAP and the value of the technique in the control of acute bleeding has been assessed. 57 patients underwent 47 qg probe applications with a mean value of 8.3 for each case. Immediate hemostasis was achieved in out of 57 cases (93%). Rebleeding after hemostasis by BICAP was noted in 4 out of 53 cases (7.5%). There were no complications related to the procedure. It is an important technique for the control of acute upper gastrointestinal bleeding.