Cell Differentiation ; Cell Line, Tumor ; metabolism ; Cell Proliferation ; Humans ; Trans-Activators ; biosynthesis ; genetics ; Transfection

To investigate the protein and mRNA expression of pancreas/duodenal homeobox-1 (PDX-1), a transcription factor as a marker for pancreatic stem cells, in pancreatic ductal cells of rats after partial (90%) pancreatectomy and evaluated the significance of the PDX-1 expression. Western blot and Reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the expression of PDX-1 protein and mRNA respectively. PDX-1 protein was only faintly detected in pancreatic ductal cells on the day 1 after partial pancreatectomy. On the day 2 and 3 after operation in operation group, a 2-3 fold increased PDX-1 protein was observed, corresponding to the characteristic 42-kD protein in Western blot. There was significant difference between operation group and sham-operation group (P<0.05). PDX-1 protein expression on the day 5 and 7 after operation had already been no difference from control group (P>0.05). RT-PCR revealed the PDX-1 mRNA expression showed no significant difference between operation group at various time points and sham-operation group (P> 0.05). These results indicate that there was overexpression of PDX-1 in the cells of pancreatic epithelium during the regeneration of remnant pancreas after partial pancreatectomy in adult rats, suggesting the pancreatic stem cells in pancreatic ductal epithelial cells are involved in the regeneration of remnant pancreas and the expression of PDX-1 in ductal cells was regulated posttranscription.
Epithelial Cells/metabolism ; Homeodomain Proteins/*biosynthesis ; Homeodomain Proteins/genetics ; Pancreatectomy/methods ; Pancreatic Ducts/cytology ; Pancreatic Ducts/*metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Rats, Sprague-Dawley ; Trans-Activators/*biosynthesis ; Trans-Activators/genetics

OBJECTIVETo construct a retroviral vector carrying HBX gene and investigate its expression in LO2 human hepatocytes.

METHODSHBX gene was amplified by PCR and subcloned into the retroviral vector pSEB-Flag to construct a retroviral plasmid (pSEB-Flag-HBX) expressing HBX. The HBX gene insert was confirmed by restriction enzyme digestion, PCR and DNA sequencing. The recombinant retroviruses carrying HBX gene were generated in 293T cells co-transfected with pSEB-Flag-HBX and the packaging plasmids pAmpho, and used to infect LO2 human hepatocyte. After selection with blasticidin, the mRNA and protein expressions of HBx were determined by the reverse transcription-PCR and Western blotting, respectively.

RESULTSThe retroviral plasmid (pSEB-Flag-HBX) carrying HBX was constructed successfully. The recombinant retrovirus efficiently delivered HBX gene into LO2 human hepatocyte, resulting in stable expression of HBX mRNA and HBx protein as shown by RT-PCR and Western blotting, respectively.

CONCLUSIONThe recombinant retrovirus pSEB-Flag-HBX has been successfully constructed, which is capable of delivering the target gene HBX into LO2 human hepatocytes and results in stable expression of HBx to serve as an ideal model to study the effect of HBx on the development of hepatocellular carcinoma.

Cell Line ; Gene Expression ; Genetic Vectors ; Hepatocytes ; cytology ; Humans ; Plasmids ; RNA, Messenger ; genetics ; Retroviridae ; genetics ; Trans-Activators ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate the expression of Shh and its receptors Ptc1 and Ptc2 mRNA in the cap stage of mouse molar and discuss its role in early tooth morphogenesis.

METHODSThe embryonic mouse heads of early tooth development (E10.5 - E15.5) were obtained and 5 micro m serial sections were made. Immunohistochemical staining of PCNA was carried out by SP method. The expression pattern of Shh, Ptc1, and Ptc2 mRNA was analysed by in situ hybridization.

RESULTSE14.5, outer dental epithelium, inner dental epithelium, stellate reticulum and underlying dental mesenchyme were PCNA positive. Most of the enamel knot cells were PCNA negative. A few of the enamel knot cells were PCNA positive. Shh, Ptc1, and Ptc2 mRNA were strongly expressed in outer dental epithelium, inner dental epithelium, stellate reticulum and the enamel knot.

CONCLUSIONIn the cap stage, Shh as a paracrine and autocrine signaling molecule might stimulate epithelium and mesenchyme proliferation.

Animals ; Hedgehog Proteins ; Mice ; Molar ; metabolism ; Patched Receptors ; Patched-1 Receptor ; RNA, Messenger ; biosynthesis ; Receptors, Cell Surface ; biosynthesis ; genetics ; Tooth Germ ; growth & development ; metabolism ; Trans-Activators ; biosynthesis

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cyclin D1 ; biosynthesis ; Hepatitis B virus ; Humans ; Liver ; pathology ; Liver Cirrhosis ; metabolism ; pathology ; NF-kappa B ; biosynthesis ; Trans-Activators ; biosynthesis

To investigate the protein and mRNA expression of pancreas/duodenal homeobox-1 (PDX-1), a transcription factor as a marker for pancreatic stem cells, in pancreatic ductal cells of rats after partial (90%) pancreatectomy and evaluated the significance of the PDX-1 expression. Western blot and Reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the expression of PDX-1 protein and mRNA respectively. PDX-1 protein was only faintly detected in pancreatic ductal cells on the day 1 after partial pancreatectomy. On the day 2 and 3 after operation in operation group, a 2-3 fold increased PDX-1 protein was observed, corresponding to the characteristic 42-kD protein in Western blot. There was significant difference between operation group and sham-operation group (P<0.05). PDX-1 protein expression on the day 5 and 7 after operation had already been no difference from control group (P>0.05). RT-PCR revealed the PDX-1 mRNA expression showed no significant difference between operation group at various time points and sham-operation group (P> 0.05). These results indicate that there was overexpression of PDX-1 in the cells of pancreatic epithelium during the regeneration of remnant pancreas after partial pancreatectomy in adult rats, suggesting the pancreatic stem cells in pancreatic ductal epithelial cells are involved in the regeneration of remnant pancreas and the expression of PDX-1 in ductal cells was regulated posttranscription.
Animals ; Epithelial Cells ; metabolism ; Homeodomain Proteins ; biosynthesis ; genetics ; Pancreatectomy ; methods ; Pancreatic Ducts ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Trans-Activators ; biosynthesis ; genetics

The HBV X gene was amplified by PCR according to the pecob6 containing the whole fragment of adw subtype of HBV, then the fragment was inserted into the multiple clone site of pAdTrack-CMV. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 and the recombinant adenoviral plasmid pAd-X was generated. Then plasmid pAd-X was digested with Pac I and transfected into 293 cells for packaging and amplifying. Infection titer and rate were monitored by green fluorescent protein (GFP) expression. With restriction endonuclease analysis and PCR methods, it has been confirmed that HBV X gene was cloned into the adenovirus vector successfully. The expression of X protein in HepG2 cells was detected by Western-blot. The recombined adenovirus Ad-X was constructed successfully, which would contribute to the advanced functional study of HBV X protein.
Adenoviridae ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; metabolism ; Hep G2 Cells ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; Trans-Activators ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate whether the sonic hedgehog signaling pathway is involved in the development of human fetal prostate, and to evaluate the changing staining patterns of its molecules, sonic hedgehog (SHH), patchedl (PTC1), smoothened (SMO), and GLI1, in the human fetal prostate at various gestation stages.

METHODSFifteen human fetal prostate specimens at various developmental stages (10 - 39 weeks) were included in this study. SHH, PTC1, SMO and GLI1 were detected in all the specimens by immunohistochemical technique. All the slides were observed and assessed under the light microscope.

RESULTSSHH, PTC1, SMO and GLI1 could be detected in human fetal prostate tissues, and their expression formed two surges, the former at week 16, and the latter at week 28. The staining of SHH and SMO was distributed only in the ductal epithelium but not in the stroma. The expression of PTC1 and GLI1 could be found mainly in the epithelium, with minimal staining in the stroma.

CONCLUSIONThe sonic hedgehog signaling pathway is involved in the development of the human fetal prostate. The high expression of its molecules at early gestation stages might be associated with the induction of prostatic buds, while their abundant expression at later gestation stages might be related to the prostate ductal branching, growth, differentiation and morphogenesis.

Gene Expression Regulation, Developmental ; physiology ; Hedgehog Proteins ; biosynthesis ; Humans ; Male ; Oncogene Proteins ; biosynthesis ; Patched Receptors ; Prostate ; embryology ; metabolism ; Receptors, Cell Surface ; biosynthesis ; Receptors, G-Protein-Coupled ; biosynthesis ; Signal Transduction ; physiology ; Smoothened Receptor ; Trans-Activators ; biosynthesis ; Zinc Finger Protein GLI1

OBJECTIVEResearch was done on the possible roles of beta-catenin, p53 and proliferating cell nuclear antigen (PCNA) in the carcinogenesis of colorectal adenoma (CRA).

METHODSbeta-catenin and p53 and PCNA expressions were studied with immunohistochemical stain in 77 specimens of CRA together with mild epithelial dysplasia (CRA-MD), CRA with moderate/severe epithelial dysplasia (CRA-D/SD) and CRA with cancerous changes (CRA-C).

RESULTSThe percentage of abnormal expression of beta-catenin increased during the transition from CRA-MD to CRA-D/SD to CRA-C (P < 0.01). The nuclear expressions of beta-catenin in CRA-D/SD and CRA-C were all significantly higher than that in CRA-MD. Expression of p53 and PCNA were increased from CRA-MD to CRA-D/SD to CRA-C, with the positive rates in these three groups of 10.3%, 43.8%, 75.0% for p53 and 17.2%, 62.5%, 87.5% for PCNA, respectively. 69.7% of cases with positive nuclear beta-catenin expression showed strong PCNA positivity which was much higher than the 36.4% of cases without nuclear beta-catenin expression (P < 0.05). The percentage of strong PCNA expression in the p53 positive cases was significant higher than that in cases with negative p53 expression (72.4% vs 37.5%, P < 0.05). Nuclear beta-catenin and p53 co-expression rates in CRA-C reached 50%.

CONCLUSIONbeta-catenin, p53 and PCNA may play important roles in the carcinogenesis of colorectal adenoma.

Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; biosynthesis ; Colorectal Neoplasms ; diagnosis ; metabolism ; Cytoskeletal Proteins ; biosynthesis ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Prognosis ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Trans-Activators ; biosynthesis ; Tumor Suppressor Protein p53 ; biosynthesis ; beta Catenin

The MHC class II transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC class II restricted antigen presentation. Previously we suggested another role of CIITA in Th1/Th2 balance by demonstrating that forced expression of CIITA in murine T cells repressed Th1 immunity both in vitro and in vivo. However, the results were contradictory to the report that CIITA functioned to suppress the production of Th2 cytokine by CD4+T cells in CIITA deficient mice. In this study, we investigated the influence of constitutive expression of CIITA in T cells on Th2 immune response in vivo using murine experimental colitis model. In the dextran sodium sulfate-induced acute colitis, a disease involving innate immunity, CIITA transgenic mice and wild type control mice showed similar progression of the disease. However, the development of oxazolone-induced colitis, a colitis mediated by predominantly Th2 immune response, was aggravated in CIITA-transgenic mice. And, CD4+T cells from the mesenteric lymph node of CIITA-transgenic mice treated with oxazolone exhibited a high level of IL-4 secretion. Together, these data demonstrate that constitutive expression of CIITA in T cells skews immune response to Th2, resulting in aggravation of Th2-mediated colitis in vivo.
Trans-Activators/*physiology ; Th2 Cells/*immunology ; T-Lymphocytes/*metabolism ; Oxazolone/pharmacology ; Nuclear Proteins/*physiology ; Mice, Transgenic ; Mice, Inbred C57BL ; Mice ; Interleukin-4/biosynthesis ; Colitis/*etiology ; Animals