Carcinoma ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Male ; Pancreatic Neoplasms ; genetics ; Spectral Karyotyping ; methods

OBJECTIVE: To investigate effects of antioxidant stress protein heme oxygenase-1 (HO-1) on lipopolysaccharide (LPS)-induced endoplasmic reticulum stress (ERS) of rat hepatocytes. METHODS: The BRL cells (rat hepatocyte cell line) were cultured. The hepatocytes were treated with LPS, LPS+HO-1 siRNA, HO-1 siRNA and PBS solution, respectively. The cell viability was measured by trypan blue exclusion test. The apoptosis cells were detected by the fluorescent dye Hoechst 33258. Expressions of GRP78, CHOP, caspase-12 and HO-1 were detected by Western blotting. RESULTS: LPS caused an increase of HO-1 protein expression of rat hepatocytes in a dose-dependent and time-dependent manner, a up-regulation of GRP78, CHOP and caspase-12, a decrease in cell viability, and an increase in apoptosis rate of hepatocytes. Pretreatment of HO-1 siRNA inhibited the up-regulation of LPS-induced HO-1, however, aggravated ERS and cellular injury. CONCLUSION HO-1 inhibites ERS-mediated cellular injury of rat hepatocytes induced by LPS.
Animals ; Apoptosis/physiology* ; Endoplasmic Reticulum/metabolism* ; Endoplasmic Reticulum Stress/physiology* ; Heme Oxygenase-1/pharmacology* ; Hepatocytes/metabolism* ; Lipopolysaccharides/pharmacology* ; Rats

Toll like receptor ( TLRs) is a highly conserved transmembrane protein expressed in epithelial cells and immune cells,which is a kind of important receptor to identify the damage associated molecular patterns (DAMPs). TLRs and DAMP produced by tumor cells after chemotherapy combine to produce the corresponding biological effects via MyD88 dependent and independent path-ways. At present,the research application of TLRs identifying DAMP clinically is mainly focused on promoting the release of the DAMP and enhancing the expression of TLRs to strengthen the immune effect of chemotherapy drugs.

Objective To evaluate the effects of amino acids (AA) on the development of in vitro cultured preimplantation embryos of Kunming mice, and define the optimal AA concentration for embryo culture. Methods Totally 630 zygotes were collected from the oviducts of superovulated female Kunming mice, which were cultured in protein-free potassium simplex optimized medium (mKSOM) supplemented with Eagle's essential amino acids and Eagle's non-essential amino acids of different concentrations (mKSOM, mKSOM+1/16AA, mKSOM+1/8AA, mKSOM+1/4AA, mKSOM+1/2AA, mKSOM+AA, and mKSOM+2AA). Results The embryos cultured with the amino acids showed higher development rate to both 8-cell embryo stage and blastocyst stage than those cultured without amino acids. The correlation of amino acid concentration with 8-cell and blastocyst development rates conformed to the cubic model, with the highest development rate to both of the two stages observed at half of the amino acid concentration. Conclusion Amino acids can promote the development of preimplantation Kunming mouse embryos, but excessively high concentration of amino acids impair embryo development possibly because of metabolic and osmotic pressure changes of the embryos as well as toxicity of ammonium resulting from the metabolism of amino acids.

Objective To evaluate the effects of amino acids (AA) on the development of in vitro cultured preimplantation embryos of Kunming mice, and define the optimal AA concentration for embryo culture. Methods Totally 630 zygotes were collected from the oviducts of superovulated female Kunming mice, which were cultured in protein-free potassium simplex optimized medium (mKSOM) supplemented with Eagle's essential amino acids and Eagle's non-essential amino acids of different concentrations (mKSOM, mKSOM+1/16AA, mKSOM+1/8AA, mKSOM+1/4AA, mKSOM+1/2AA, mKSOM+AA, and mKSOM+2AA). Results The embryos cultured with the amino acids showed higher development rate to both 8-cell embryo stage and blastocyst stage than those cultured without amino acids. The correlation of amino acid concentration with 8-cell and blastocyst development rates conformed to the cubic model, with the highest development rate to both of the two stages observed at half of the amino acid concentration. Conclusion Amino acids can promote the development of preimplantation Kunming mouse embryos, but excessively high concentration of amino acids impair embryo development possibly because of metabolic and osmotic pressure changes of the embryos as well as toxicity of ammonium resulting from the metabolism of amino acids.

Objective To analyze the clinical diagnosis and treatment data of 20 children with hypophosphatemic rickets (HR) in order to improve the clinical diagnosis and treatment of HR.Methods The retrospective analysis of clinical data of 20 cases with HR who were hospitalized at Children's Hospital of Nanjing Medical University from May,2010 to April,2016 was performed to summarize the clinical characteristics.All patients were analyzed for the phosphate regulating gene with homologies to endopeptidase on the X chromosome(PHEX) gene by direct sequencing.If no mutations were detected,multiplex ligation-dependent probe amplification analysis was performed.Results All of the 20 cases with HR showed different degrees of growth retardation and typical X-ray rickets.After treatment,the clinical features were improved.Height standard deviation score (HSDS) was improved significantly with longer treatment time,and the difference was statistically significant(P =0.027).There was a correlation between the blood phosphorus fluctuation and secondary hyperparathyroidism(P ＜ 0.05).Nineteen cases had PHEX gene mutations.Truncating mutations was the most frequent mutation type,and 4 new mutations were found.Conclusions Clinical characteristics,laboratory test results and X-ray examination are important clinical index for the diagnosis of HR,and PHEX gene test can be used as an important auxiliary diagnostic tool.Early diagnosis and treatment can significantly improve the clinical manifestations of the patients.

OBJECTIVETo investigate effect of oxidized low-density lipoprotein (ox-LDL) on memory CD8T cell subpopulation differentiation in mice with autoimmune diabetes.

METHODSCultured splenic CD8T cells from pre-diabetic NOD mice isolated with magnetic beads were treated with 30 µg/mL ox-LDL and 10 U/mL interleukin-2 (IL-2) for 24 h and the control cells were treated with IL-2 only. Flow cytometry was used to determine the percentage of splenic CD8IFN-γT cells, expressions of CD8, CD44 and CD62L on the T cells, and the activation of T cell factor-1 (TCF-1) and STAT-3. The CD127memory T cells were purified and transplanted into the pre-diabetic NOD mice via the tail vein, and the blood glucose was recorded weekly and survival time of the mice was monitored.

RESULTSTreatment with ox-LDL significantly reduced islet β cell-specific cytotoxic CD8T cells as compared with the control group [(0.7∓0.03)% vs (2.7∓0.14)%, P<0.01]. The percentage of effector memory CD8T cells (Tem) in the total memory CD8T cells was reduced [(10.3∓0.71)% vs (30.3∓1.36)%, P<0.01] and that of stem cell-like memory T cells was significantly increased [(72.3∓3.8)% vs (55.1∓2.61)%, P<0.05] following ox-LDL treatment, which also resulted in significantly decreased activation of TCF-1 [(14.5∓0.82)% vs (34.2∓1.23)%, P<0.01] and pSTAT-3 [(3.3∓0.12)% vs (22.1∓1.1)%, P<0.01]. Transplantation of ox-LDL-treated memory T cells in pre-diabetic NOD mice obviously inhibited the increase of blood glucose and prolonged the survival time of the mice (P<0.05).

CONCLUSIONOx-LDL decreases the activation of transcriptional factors TCF-1 and phosphorylation of STAT-3, inhibits the formation of effector memory CD8T cells with long-term cytotoxicity, but promote the generation of stem cell-like memory CD8T cells, which result in suppression of islet β cell-specific effector cytotoxic CD8T cell differentiation to lessen autoimmune injury to the islet β cells.

OBJECTIVETo investigate the changes of topoisomerase IIalpha (TOP2A) and HER2/neu genes in pancreatic ductal adenocarcinomas of Chinese patients, and to determine their roles during carcinogenesis and tumor progression.

METHODSExpressions of TOP2A and HER2/neu proteins were detected by using immunohistochemistry, while gene amplifications of TOP2A and HER2/neu were assessed by using multi-color fluorescence in situ hybridization (FISH). All the samples were of paraffin embedded and 10% formalin fixed tissue, including 26 cases of pancreatic ductal adenocarcinomas with adjacent non-neoplastic pancreatic tissues, 10 cases of chronic panreatitis, and 10 cases of normal pancreas. The correlation between TOP2A and HER2/neu gene status was analyzed.

RESULTSBy immunohistochemistry, the nuclear positive index of TOP2A in pancreatic ductal adenocarcinomas varied from 0.5% to 70%, and the positive rate of HER2/neu in pancreatic ductal adenocarcinomas was 46.2% (12/26). By FISH, 9/10 TOP2A amplified adenocarcinomas showed TOP2A and HER2/neu gene coamplification, while one case with HER2/neu gene amplification adenocarcinoma showed no TOP2A amplification. No expression of TOP2A, HER2/neu proteins and no amplification of TOP2A and HER2/neu gene were detected in adjacent non-neoplastic pancreatic tissues, chronic pancreatitis tissues and normal pancreas. No relationship was found between protein expression and gene amplification of TOP2A and HER2/neu (P > 0.05). TOP2A gene amplification was significantly correlated with HER2/neu gene amplification (P < 0.01).

CONCLUSIONSProtein expression of TOP2A and HER2/neu are not associated with the gene amplification. There is a significant correlation between TOP2A amplification and HER2/neu gene amplification. Co-amplification of TOP2A and HER2/neu may play an important role in the carcinogenesis and progression of pancreatic carcinoma. Evaluation of the status of TOP2A and HER2/neu may be helpful to achieve target therapy of pancreatic carcinoma.

Adenocarcinoma ; genetics ; metabolism ; pathology ; secondary ; Adult ; Aged ; Antigens, Neoplasm ; genetics ; metabolism ; Carcinoma, Pancreatic Ductal ; genetics ; metabolism ; pathology ; secondary ; DNA Topoisomerases, Type II ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Gene Amplification ; Gene Expression Regulation, Neoplastic ; Genes, erbB-2 ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Liver Neoplasms ; metabolism ; secondary ; Lymphatic Metastasis ; Male ; Middle Aged ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; Poly-ADP-Ribose Binding Proteins ; Receptor, ErbB-2 ; genetics ; metabolism

OBJECTIVETo investigate the roles of K-ras gene in the tumorigenesis of ovarian serous borderline and malignant tumors.

METHODSFifty one tissue samples of ovarian serous tumors, including 18 conventional serous borderline tumors, 11 micropapillary serous borderline tumors, 12 invasive micropapillary serous carcinomas, and 10 conventional serous carcinomas were investigated for the presence of K-ras mutation. DNA was extracted after microdissection of the tumor tissue, the exon 1 of K-ras gene was amplified by PCR, and the presence of mutation at the codons 12 and 13 was evaluated by direct sequencing analysis.

RESULTSGGT to GTT mutation at codon 12 of the K-ras gene was found in one conventional serous borderline tumors, resulting in valine to glycine substitution. All other 50 cases showed no K-ras mutation. All tumors had a wild-type codon 13.

CONCLUSIONSMutations of K-ras at codons 12 and 13 in ovarian serous tumors are very rare in this series of patients, suggesting a difference present between the Chinese and Caucasian populations. K-ras mutations may play a less important role in the tumorigenesis of ovarian serous tumor of the Chinese patients.

Adolescent ; Adult ; Aged ; Amino Acid Substitution ; Codon ; Cystadenocarcinoma, Serous ; genetics ; Cystadenoma, Serous ; genetics ; Female ; Genes, ras ; genetics ; Humans ; Middle Aged ; Ovarian Neoplasms ; genetics ; Young Adult

OBJECTIVETo investigate the genetic status of 13q and its role in the oncogenesis and progress of soft tissue tumors.

METHODSForty-one soft tissue tumors, including 9 benign tumors, 9 tumors of malignant potential and 23 sarcomas, were studied by fluorescence in-situ hybridization (FISH) using dual color probes. The probes were generated from BAC clones RP11-685I15, RP11-352N7 and RP11-505F3, corresponding to Rb, RFP2, KCNRG and KLF5 genes respectively.

RESULTSLoss of heterozygosity (LOH) of RP11-685I15 were found in 8/41 cases, LOH of RP11-352N7 was seen in 4/41 cases and LOH of RP11-505F3 was present in 3/41 cases. LOH of all 3 loci were detected in 2 cases. LOH of RP11-61K9, an internal control locus, was detected in 2 cases. One case of malignant peripheral nerve sheath tumor showed amplification at all 3 loci. Amplification of RP11-505F3 was seen in another 2 cases.

CONCLUSIONSA significant percentage of soft tissue tumors exhibited chromosomal instability, reflected by an increase of LOH at tumor-suppressing gene loci. The incidence of 13q abnormality was different in various types of soft tissue tumors, indicating that alterations of Rb, RFP2, KCNRG and KLF5 tumor suppressing genes may play diverse roles in different types of soft tissue tumor.

Adolescent ; Adult ; Aged ; Chromosomal Instability ; Chromosomes, Human, Pair 13 ; Female ; Gastrointestinal Stromal Tumors ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Loss of Heterozygosity ; Male ; Middle Aged ; Retroperitoneal Neoplasms ; genetics ; Soft Tissue Neoplasms ; genetics ; Young Adult