In order to investigate the expression of Notch-1 in rats with acute pancreatitis (AP) and its relation with apoptosis of pancreatic cells, mild AP (MAP) and severe AP (SAP) models were established by retrograde injection of different concentrations of sodium taurocholae into pancreatic duct. The apoptosis index and the expression of Notch-1 protein and mRNA were detected by using TUNEL, Western blot and real-time PCR in MAP and SAP at different time intervals. The results showed that in MAP group the apoptosis index was significantly elevated during 4 to 24 h after induction of pancreatitis, but there was no significant difference among different time intervals. In SAP group, the apoptosis index reached the peak at 4th h after induction of pancreatitis, then gradually declined. There was significant difference in apoptosis index between MAP and SAP groups. Starting from 4th after induction of pancreatitis, the expression of Notch-1 in both MAP and SAP group was increased at different time intervals, but that in SAP group was significantly higher than in MAP group (P<0.05). The expression of Notch in MAP and SAP groups reached the peak at 12th and 8th h respectively after induction of pancreatitis. It was concluded that there was significant difference in apoptosis index and the Notch-1 expression in different types of AP. The overexpression of Notch-1 could aggravate AP by inhibiting the apoptosis of pancreatic cells.
To clone and sequence the human tissue kallikrein gene of Chinese, and to construct eukaryotic expression recombinant of KK, total RNA was extracted from human pancreas and human tissue kallikrein gene cDNA was amplified by PCR after reverse-transcription by using Oligo(dT) primer. The original kallikrein cDNA was recovered and filled with Klenow enzyme and inserted into KS plasmid. After restriction endonuclease digestion, KK cDNA was sequenced by ABI377 analyzer. Then the KK gene was amplified from pBluescript KSKK and inserted into pcDNA3. A sequence comparison showed that the cloned kallikrein gene was only one nucleotide different from that reported in the Genbank. The coding amino acid was Asp in the Genbank gene, while the coding amino acid of Chinese kallikrein gene was Asn. The KK cDNA fragment was inserted into the eukaryotic expression vector pcDNA3. The cloned kallikrein gene and the pcDNA3KK can be used for further study in gene therapy.. .
The distribution of hepatitis B virus genotype in Hubei province and its clinical significance were investigated. HBV genotypes of 276 patients were detected by PCR-microplate sandwich hybrization-ELISA technique. The level of HBV DNA was detected by using PCR-fluorescence quantification test. Among 276 patients, there were 78 cases of HBV asymptomatic carriers, 110 cases of chronic hepatitis B (CHB), 62 cases of severe hepatitis (SH) or liver cirrhosis (LC) and 26 cases of hepatocellular carcinoma (HCC). The genotypes of HBV included C, B, mixtures (B+C, B+D, C+D) and D, accounting for 55.8%, 25.4%, 16.7% and 2.1% respectively. The average level of HBV DNA in genotypes C, B, mixtures and D was 1.20×106, 7.81×104, 3.26×105 and 5.01×104 copies/mL respectively. The ratio of SH, LC and HCC in genotype B, C and mixtures was 20%, 30% and 48% respectively. Statistical analysis revealed the percentage of genotype mixtures infection was significantly higher than that of genotype B infection. There was no significant difference in the percentage between genotype B and genotype C or between genotype C and mixtures. The distribution of genotype B, C and mixtures in SH, LC and HCC was significantly different. The frequency of HCC was zero in patients with co-infection. Genotype D was only related with SH and LC. The increased ALT could be converted to categorical grades of severity. From mild, moderate to severity, the prevalence of genotype C showed an opposite trend, although no statistically significant difference was observed. The HBeAg positive rate was higher in patients with genotype C infection than in those with genotype B, especially in the patients whose ages were from 31 to 40 years old. Compared with genotype B, genotype C showed a higher HBeAg positive rate in patients with SH and LC. The percentage of SH, LC and HCC was higher in patients with genotype C and mixtures infection. On the contrary, the percentage of genotype B was lower. The HBeAg positive rate in patients with genotype C infection was higher than those with genotype B infection. Genotype C and mixtures may be associated with development of severe liver disease.
To investigate the role of NF-κB in TNF-α induced apoptosis in HSC-T6, a mutant IκBα was transfected into HSC-T6 cells by lipofectin transfection technique and its transient effect was examined 48 h after the transfection. The activation of NF-κB was detected by immune fluorescence cytochemistry and Western blotting with anti-p65 antibody. The apoptosis and the rate of inhibition by TNF-α in both transfected and untransfected HSC-T6 cells were measured respectively by FAC-Scan side scatter analysis and MTT methods. Our results showed that TNF-α could activate NF-κB in untransfected cells but not in transfected HSC-T6 cells. The percentage of apoptosis in transfected cells were significantly higher than that in the untransfected ones (P<0.01) and it was also true of the inhibition rate (P<0.01). It is concluded that the resistance of HSC-T6 towards apoptosis induced by TNF-α can be mediated by NF-κB activation. The inhibition of NF-κB activation by mutant IκBα can attenuate the resistance of HSC-T6 cells and increase its sensitivity to TNF-α.
Gold(Au) nanoparticle HBV DNA or HCV cDNA gene probes were prepared and were used to detect HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection directly by transmission electron microscopy (TEM). PCR identifying HBV and HCV in serum of patients with HBV and HCV coinfection was established. Alkanethiol-modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA or HCV cDNA gene probes through covalent binding of Au-S. HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfectionw as added to the detection system composed of nanoparticle HBV DNA and(or) HCV cDNA gene probes. The results showed that HBV DNA and HCV RNA could be specifically amplified by PCR. The zones of DNA amplification appeared in 431 bp and 323 bp respectively. When HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection were added to the detection system, TEM displayed the nanoparticles self-assembled into large network aggregates. It was concluded that the detection of HBV and HCV coinfection by TEM was convenient and efficient with high specificity and sensitivity.
In order to detect the expression of Oct4 in bladder cancer tissue and cell line BIU-87, immunohistochemistry was used in 49 bladder cancer biopsy samples and immunofluorescence and reverse transcription-PCR were performed on bladder cancer cell line BIU-87. Forty of 49 bladder cancer samples showed the expression of Oct4 in about 0.6% cancer cells. The positive rate and density of Oct4 expression had no obvious relationship with the grade, recurrence or metastasis of bladder cancer (P>0.05). A few Oct4 positive cells were found in bladder cancer cell line BIU-87, which was also confirmed by RT-PCR. This study indicated the existence of few Oct4 positive cells in bladder cancer, which may be the bladder cancer stem cells. This study may provide the foundation for isolation and identification of bladder cancer stem cells.