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.

BACKGROUNDThe blood vessels of a transplanted organ are the interface between donor and recipient. The endothelium in the blood vessels is thought to be the major target for graft rejection. Endothelial cells of a transplanted organ can be of recipient origin after transplantation. In this study, we tested whether endothelial chimerism correlated with the graft rejection and cold ischemia.

METHODSWe studied the biopsy samples from 34 renal transplants of female recipients who received the kidney from a male donor for the presence of endothelial cells of recipient origin. We examined the tissue sections of renal biopsy samples by fluorescence in situ hybridization (FISH) for the presence of endothelial cells containing two X chromosomes using a biotinylated Y chromosome probe and digoxigenin labelled X chromosome probe, and then analyzed the relationship between the endothelial cell chimerism and the rejection and cold ischemia.

RESULTSEndothelial chimerism was common and irrespective of rejections (P > 0.05). The cold ischemic time of chimerism group was longer than no chimerism group ((14.83 +/- 4.03) hours vs (11.27 +/- 3.87) hours, P < 0.05).

CONCLUSIONSThere is no correlation between the percentage of recipient endothelial cells in vascular endothelial cells and the type of graft rejection. The endothelium damaged by ischemic injury might be repaired by the endothelial cells from the recipient.

Animals ; Biopsy ; Endothelial Cells ; pathology ; Female ; Graft Rejection ; Humans ; In Situ Hybridization, Fluorescence ; Kidney ; pathology ; Kidney Transplantation ; Male ; Mice ; Time Factors ; Transplantation Chimera ; Transplantation, Homologous

OBJECTIVEto investigate the chromosomal characteristics of pancreatic ductal adenocarcinomas by spectral karyotyping.

METHODScytogenetic aberrations of pancreatic cancer cell line P2 established from a Chinese patient was investigated by spectral karyotyping (SKY). Chromosomal alterations were further evaluated in 10 cases of pancreatic cancer and 10 cases of chronic pancreatitis by two color fluorescence in situ hybridization (FISH) by using EGFR/CEP7 probe and paraffin embedded tissue samples.

RESULTShypotriploid and 26 chromosomal aberrations were revealed in cell line P2. Recurrent chromosomal numerical alterations included loss of chromosome 4, 9, 18, 19, 22, Y, 10p, 15p, 8p, 6q and 12p, with gain of chromosome 7 and 12q. Frequent chromosomal structural abnormalities included der(9;15)(q10;q10), der(10)(3;10)(?;q26) and der(12)(8;12)(?;p13). Four of 10 cases showed EGFR copy number changes by FISH.

CONCLUSIONShighly complex chromosomal rearrangements occur in pancreatic cancers. Additional studies of more cases are needed, including FISH analysis of EGFR copy number changes, to reach a conclusion.

Adenocarcinoma ; genetics ; Aged ; Cell Line, Tumor ; Chromosome Aberrations ; Chromosome Deletion ; Chromosome Duplication ; Female ; Gene Dosage ; Genes, erbB-1 ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; methods ; Male ; Middle Aged ; Pancreatic Neoplasms ; genetics

OBJECTIVETo explore epidermal growth factor receptor (EGFR) and HER2 gene status, to assess the correlation between EGFR and HER2 gene status, and to investigate the role of copy number increase and amplification of EGFR gene and HER2 gene in the tumorigenesis and disease progression of non-small-cell lung cancer.

METHODSUsing Path Vysion kit and LSI EGFR SpectrumOrange/CEP7 Spectrum Green probes, EGFR gene and HER2 gene status were evaluated by fluorescence insitu hybridization (FISH) using formalin-fixed, paraffin-embedded samples from 31 patients with non-small-cell lung cancer, including 20 adenocarcinomas, 2 squamous cell carcinomas, 2 large cell carcinoma, 4 bronchoalveolar carcinomas and 3 adenosquamous carcinomas. The correlation between EGFR and HER2 gene status was analyzed.

RESULTSSix of thirty-one carcinomas showed EGFR gene amplification. Of 25 cases without EGFR gene non-amplification, four tetrasomy and 5 polysomy were detected. Overall, 15 out of 31 carcinomas demonstrated either EGFR gene copy number increase or gene amplification (15/31). HER2 gene amplification was seen in 2 of the 31 cases. Four trisomy, one tetrasomy and nine polysomy cases were found in 29 tumors that had no HER2 gene amplification. Overall, 16 of 31 cases showed either HER2 gene copy number increase and/or amplification (16/31). Synchronous EGFR and HER2 gene numerical changes, i.e. gene copy number increase and gene amplification, were found in 12 of 31 cases (12/31), and almost all such patients had either clinical stage III or IV tumor. EGFR gene numerical changes significantly correlated with HER2 gene abnormality (chi(2)(Adj) = 7.3045, P = 0.0069).

CONCLUSIONSEGFR or HER2 copy number increase is much more frequent than gene amplification in no-small-cell lung cancer. Our data based on gene alterations indicate, for the first time, that there is a significant correlation between EGFR alterations and HER2 abnormalities. Both genes are involved in the tumorigenesis and development of lung cancer. EGFR/HER2 dimer is one of the predominant heterodimerization types in lung cancer. The interactions between EGFR and HER2 may play a rule in the progression of non-small-cell lung cancer.

Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Chromosomes, Human, Pair 17 ; Chromosomes, Human, Pair 7 ; Gene Amplification ; Gene Dosage ; Gene Expression Regulation, Neoplastic ; Genes, erbB-1 ; genetics ; Genes, erbB-2 ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; genetics ; pathology ; Polyploidy

OBJECTIVETo investigate mutations of EGFR gene in non-small cell lung cancers (NSCLC) using scorpions amplification refractory mutation system (Scorpions ARMS) is in comparing the detection sensitivity with that by PCR-direct sequencing method, and in addition to study the correlation between the mutations and the clinicopathological characteristics of the patients.

METHODSTumor cells were collected by microdissection from paraffin embedded tumor specimens and adjacent normal lung tissues of 82 NSCLC patients. The genomic DNA was extracted. Mutations of EGFR gene (exons 18, 19, 20 and 21) were detected by PCR-direct sequencing and Scorpions ARMS methods respectively.

RESULTSSomatic mutations were identified involving the tyrosine kinase domain of the EGFR gene in 42 of 82 cases with a mutation detection rate of 51.2% by Scorpions ARMS assay. In-frame deletions of exon 19 occurred in 20 patients and point mutation occurred at codon 858, 861 (exon 21) in 18 and 1 patients respectively. Two patients had insertions mutations and 1 patient had point mutation occurring at codon 768 (exon 20). Among the 58 informative cases analyzed by PCR-direct sequencing, 25 mutations (detection rate of 30.5%) were identified. In-frame deletions of exon 19 occurred in 13 patients and point mutation occurred at codon 858, 861 (exon 21) in 10 and 1 patients respectively. In addition, 1 patient had point mutation at codon 768 (exon 20). Overall, Scorpions ARMS assay was more sensitive in detecting mutations of EGFR than PCR-direct sequencing.

CONCLUSIONSA higher incidence of somatic mutations of EGFR gene was detected in NSCLC of Chinese patients. Mutations were more common in female, non-smoking patients with adenocarcinoma and bronchioloalveolar carcinoma histology. Scorpions ARMS method is quicker, more sensitive and accurate in detecting the EGFR gene mutations and should provide important therapeutic and prognostic information to the clinicians.

Adenocarcinoma ; genetics ; Carcinoma, Non-Small-Cell Lung ; genetics ; Codon ; genetics ; therapeutic use ; Exons ; genetics ; Female ; Gene Amplification ; Genes, erbB-1 ; genetics ; Humans ; Lung Neoplasms ; genetics ; Mutation ; Point Mutation ; Polymerase Chain Reaction ; methods ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Scorpion Venoms ; chemistry ; Sensitivity and Specificity ; Sequence Deletion