UNLABELLEDThis study is conducted to explore an effective culture method for supporting the embryo development. The cattle fetal ear fibroblasts and the goat fetal ear fibroblasts are transplanted into the enucleated cattle oocytes separately by oocyte intraplasmic nuclear injection method to construct bovine cloned embryos and goat-bovine cloned embryos. The embryos are first cultivated in modified charles rosenkrans 2 amino acid medium (mCR2aa) and modified synthetic oviduct fluid medium (mSOF) separately. Then BSA (8 mg/mL) or FBS (10%) can be added to mSOF according to the different culture period. The supplements and orders, added during the first three days and after three days are as follow: BSA and BSA, BSA and FBS, FBS and BSA, FBS and FBS. On the basis of the cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, the best culture way can be screened out.

RESULTFirst, cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, cultivated in mSOF solution are all higher than those cultivated in mCR2aa( P < 0.05). Second, the cleavage rate and 8/16-cell rate, adding BSA and FBS into mSOF, are in turn 79.8% +/- 7.1%, 49.7% +/- 3.5%, 21.5% +/- 1.8%, and 115.2 +/- 4.3 in bovine cloned embryo, and 40.1% +/- 6.3%, 29.2% +/- 2.0%, 13.4% +/- 2.1% and 100.1 +/- 3.0 in goat-bovine cloned embryo, which are significant higher than other culture groups (P < 0.05).

CONCLUSIONThe goat-bovine cloned embryo can be cultivated by the optimized culture measure of bovine cloned embryo. The best culture ways of bovine cloned embryo and goat-bovine cloned embryo are all to use mSOF supplemented BSA in the first three days and then use mSOF supplemented FBS in the next five days.

Animals ; Cattle ; embryology ; physiology ; Cells, Cultured ; Cloning, Organism ; veterinary ; Ear, External ; cytology ; Embryo Culture Techniques ; methods ; veterinary ; Embryonic Development ; Fibroblasts ; cytology ; transplantation ; Goats ; embryology ; physiology ; Nuclear Transfer Techniques ; Oocytes ; cytology

Cysts ; pathology ; Humans ; Iliac Vein ; pathology ; Male ; Middle Aged

OBJECTIVETo map the frequency and types of EGFR gene mutations present in lung cancer tissues. To evaluate the clinical applicability of a novel real-time double-loop probe PCR of which the ADx-EGFR kit is based, and to compare its performance with traditional Sanger DNA sequencing in the detection of somatic mutations of tumor genes.

METHODSA total of 208 formalin-fixed paraffin-embedded (FFPE) tumor samples were tested. Genomic DNA of the tissue samples was extracted and purified, and subjected to both traditional PCR amplification, Sanger sequencing of EGFR gene in exon 18, 19, 20, 21, and ADx's EGFR mutation detection kit. The mutation rates for EGFR gene in exon 18, 19, 20, 21, as well as the frequency of each mutation detected by the two methods, were analyzed.

RESULTSThe traditional Sanger DNA sequencing technique was successfully performed in 196 out of 208 (94.2%) lung cancer samples, and 22 samples (11.2%) showed EGFR gene mutations. ADx-EGFR kit was successfully used in the lung cancers of all of the 208 cases (100.0%), and 40 samples (19.2%) showed mutations. In the lung cancer samples analyzed, mutations were mainly detected in the exon 19 and exon 21 L858R point mutation, i.e. 4.8% (10/208) and 11.6% (23/208) of total mutations, respectively, and the remaining mutations were rare.

CONCLUSIONSThe success rate of ADx-EGFR real-time PCR for formalin-fixed and paraffin-embedded tissues samples is significantly higher than that of Sanger sequencing (P < 0.01). There are significant differences between the two methods. ADx-EGFR real-time PCR shows a much higher successful detection rate and mutation rate of lung cancer tissues compared with that of Sanger sequencing. As a result, the real-time PCR with ADx-EGFR kit is proved to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is an effective and reliable tool for clinical screening of somatic gene mutations in tumors.

DNA Mutational Analysis ; methods ; Exons ; Genes, erbB-1 ; Humans ; Lung Neoplasms ; genetics ; Paraffin Embedding ; Point Mutation ; Real-Time Polymerase Chain Reaction ; methods

Castleman's disease is a slowly progressive and rare lymphoproliferative disorder. Here, we report a 55-year-old woman with superior mediastinal Castleman's disease being misdiagnosed for a long term. We found a 4.3 cm mass localized in the superior mediastinum accompanied with severe clinical symptoms. The patient underwent an exploratory laparotomy, but the mass failed to be totally excised. Pathologic examination revealed a mediastinal mass of Castleman's disease. After radiotherapy of 30 Gy by 15 fractions, the patient no longer presented previous symptoms. At 3 months after radiotherapy of 60 Gy by 30 fractions, Computed tomography of the chest showed significantly smaller mass, indicating partial remission. Upon a 10-month follow-up, the patient was alive and free of symptoms.
Antigens, CD20 ; metabolism ; Castleman Disease ; diagnosis ; immunology ; pathology ; radiotherapy ; surgery ; Female ; Follow-Up Studies ; Humans ; Mediastinal Diseases ; diagnosis ; immunology ; pathology ; radiotherapy ; surgery ; Mediastinum ; diagnostic imaging ; pathology ; Middle Aged ; Multimodal Imaging ; Positron-Emission Tomography ; Radiotherapy, Intensity-Modulated ; Tomography, X-Ray Computed

OBJECTIVETo understand the distribution of hepatitis B virus genotype in Guangxi and its clinical significance.

METHODSNested polymerase chain reaction (nPCR) was used for amplification of HBV DNA in sera of asymptomatic carrier (ASC) of hepatitis B virus (HBV) and patients with different liver diseases from southern and northern Guangxi. Specimens from 161 subjects were positive for HBV DNA and HBV genotype was determined by using restriction fragment length polymorphism analysis, direct sequencing or cloning sequencing.

RESULTSThe prevalence of genotype A was 3.7% in all samples and that of genotype B, C and D was 21.7%, 72.7% and 1.2%, respectively. No other genotypes (such as genotype E, F, G, H) were found. The prevalence of genotype C showed an increasing trend in ASC, chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) group; in contrast, the prevalence of genotype B showed an opposite trend, although no statistically significant difference was observed, except between ASC and HCC (P=0.05). The HBeAg positive rate was higher, and the anti-HBe positive rate was lower in patients with chronic genotype C infection than in those with genotype B (P<0.05 for both). Liver function test (ALT) abnormality was more severe in genotype C group than in genotypes A and B groups having acute or chronic infection (P<0.01 for all comparisons). The prevalence of genotype C in southern Guangxi was higher than that in northern Guangxi. In contrast, the prevalence of genotype B in southern Guangxi was lower than that in northern Guangxi.

CONCLUSIONS1. The predominant HBV genotypes in Guangxi were genotypes B and C. The major genotype in southern Guangxi was genotype C; while that in northern Guangxi was genotype B, which implied that the distribution of HBV genotype C was consistent with the incidence of HCC in Guangxi. 2. Genotype C maybe associated with development of severe liver diseases including HCC. 3. Genotype A,D and B+C were mostly found in acute, hepatitis and chronic hepatitis group.

Carcinoma, Hepatocellular ; virology ; Carrier State ; virology ; DNA, Viral ; blood ; genetics ; Female ; Genotype ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Liver Cirrhosis ; virology ; Liver Neoplasms ; virology ; Male ; Point Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

OBJECTIVETo study the effects of RhoA down-regulation by RNA interference on the invasion of tongue carcinoma Tca8113 and SCC-4.

METHODSDetermination of the human RhoA sequence as well as the design and constructionof a short specific small interfering RNAs (siRNA) were performed. The siRNA of RhoA gene was transfected into humantongue squamous cell carcinoma Tca8113 and SCC-4 cells line by Lipofectamine 2000. Quantitative real-time polymerasechain reaction was used to examine the mRNA expressionlevels of RhoA. Protein expressions of mRNA, galectin-3,and matrix metalloproteinase (MMP)-9 were evaluated byWestern blot. Transwell invasion assay was performed toassess the invasion ability of tongue carcinoma.

RESULTSRhoA expressions in Tca8113 and SCC-4 cells were reducedsignificantly after transfection of RhoA-siRNA. Protein levels f galectin-3 and MVP-9 were also down-regulated significantly. Invasion ability was inhibited as well.

CONCLUSIONRhoA-siRNA can effectively inhibit RhoA expression in Tca8113 and SCC-4 cells. The invasion ability of tongue carcinoma cells decreased with down-regulation of the protein expressions of galectin-3 and MMP-9, indicating that RhoA-siRNA can inhibit invasion of tongue carcinoma. Results show that RhoA may play an important role in the processes of invasion and metastasis of tongue carcinoma.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Down-Regulation ; Galectin 3 ; metabolism ; Gene Silencing ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Tongue Neoplasms ; genetics ; metabolism ; pathology ; Transfection

BACKGROUNDExposure of cells to sublethal concentrations of hydrogen peroxide (H2O2) can alleviate subsequent oxidative stress-induced apoptosis. We assessed the effects of H2O2 preconditioning on the therapeutic potential of human umbilical cord Wharton's Jelly mesenchymal stem cells (WJ-MSCs) in a murine model of myocardial infarction.

METHODSWJ-MSCs were incubated in the media for 2 hours with or without 200 µmol/L H2O2. Mice underwent left anterior descending coronary artery ligation, and received injection of phosphate buffered saline, 1×10(6) WJ-MSCs, or 1×10(6) H2O2 preconditioned WJ-MSCs 3 hours later via tail vein. Echocardiography was performed 0, 7, 14 and 28 days after surgery, and the mice were euthanized on day 28 for histological analysis. In vitro cytokine concentrations in the WJ-MSC cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The effect of WJ-MSC cell supernatant on the migration and proliferation of endothelial cells were observed by transwell migration and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assays.

RESULTSEchocardiographic measurements revealed a significant improvement in the left ventricular contractility of the WJ-MSCs-H2O2 group compared to the WJ-MSCs group. Histological analysis revealed increased neovascularization and reduced myocardial fibrosis in the WJ-MSCs-H2O2 group compared to the WJ-MSCs group. Pretreatment of WJ-MSCs with H2O2 increased the secretion of interleukin-6 (IL-6) into the cell culture supernatant by approximately 25-fold. The culture supernatant from WJ-MSCs-H2O2 significantly increased the migration and proliferation of endothelial cells; these effects could be blocked using an anti-IL-6 antibody.

CONCLUSIONSThis study demonstrates that H2O2 preconditioning significantly enhanced the therapeutic potential of WJ-MSCs, possibly by stimulating the production of IL-6 by WJ-MSCs, which may cause migration and proliferation of endothelial cells and increase neovascularization.

Animals ; Cell Movement ; physiology ; Echocardiography ; Enzyme-Linked Immunosorbent Assay ; Humans ; Hydrogen Peroxide ; pharmacology ; Immunohistochemistry ; Interleukin-6 ; metabolism ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Myocardial Infarction ; pathology ; therapy ; Reactive Oxygen Species ; metabolism ; Wharton Jelly ; cytology

OBJECTIVETo investigate the ability of secreting interferon-gamma (IFN-gamma) of the peripheral blood monocular cells (PBMC) stimulated by hepatitis B virus (HBV)-specific cytotoxic T lymphocyte (CTL) epitopes peptides and to analyze the difference of CTL immune response in patients with HBV infection.

METHODSFour HLA-A2-restricted HBV cytotoxic T lymphocyte epitopes [Tp: HBV polymerase 575-583 (FLLSLGIHL), Te1: envelope 28-39 (IPQSLDSWWTSL), Te2: envelope 183-191 (FLLTRILTI) and Tc: core 18-27 (FLPSDFFPSV)] were synthesized. Human leucocyte antigen (HLA)-A2 typing was detected by Flow cytometry. PBMCs which were isolated from patients with chronic hepatitis B(CHB), patients with chronic severe hepatitis B(CSH), subjects with past HBV infection(N1) and healthy blood donors (N2) were stimulated by the four HLA-A2-restricted HBV CTLs epitopes. Enzyme linked immunospot (ELISPOT) assay was used to detect the frequency of secreting IFN-gamma CTL in each group.

RESULTS(1) HLA-A2 typing: 20 of 44 patients with CHB (45.5%) were HLA-A2 positive, 10/18 (55.6%) in CSH and 6/10 (60%) in group N1 were HLA-A2 positive.10 healthy blood donors' HLA-typing was detected in the early study.(2) ELISPOT results: (1) The total responses to the four epitopes in CHB, CSH, N1 and N2 groups were 50% (10/20), 10% (1/10), 83.3% (5/6) and 10% (1/10), respectively. The response in N1 group was significantly higher than that in CSH group (chi(2) = 9.000, P = 0.008) and N2 group (chi(2) = 9.000, P = 0.008). (2) The CTL average magnitude response to Tp epitope, Te1 epitope, Te2 epitope and Tc epitope was also significantly higher in past HBV infection group (77 SFC/10(6) PBMC, 59 SFC/10(6) PBMC, 100 SFC/10(6) PBMC and 57 SFC/10(6) PBMC, respectively) than that of CSH group (10 SFC/10(6) PBMC, 0 SFC/10(6) PBMC, 0 SFC/10(6) PBMC and 20 SFC/10(6) PBMC respectively, all P < 0.01) and N2 group (15 SFC/10(6) PBMC, 0 SFC/10(6) PBMC, 22 SFC/10(6) PBMC and 30 SFC/10(6) PBMC respectively, all P < 0.01).

CONCLUSIONThis study indicates that the T cell immune response to HBV-specific epitopes might be detected either in patient with chronic HBV infection or with previous HBV infection. This response should be much higher in patients with past HBV infection, even the virus had been cleared for long time. These results demonstrate that HBV-specific CTL might play an important role in the clearance of the virus.

Adult ; Blood Donors ; Case-Control Studies ; Enzyme-Linked Immunosorbent Assay ; methods ; Epitopes, T-Lymphocyte ; immunology ; Female ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B virus ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Interferon-gamma ; immunology ; secretion ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic ; immunology ; Young Adult

OBJECTIVETo investigate the delayed cardioprotection induced by remifentanil in intact rat ischemia-reperfusion (I/R) models.

METHODSTotally 42 adult male Wistar rats weighing 200-300 g were randomly divided into 7 groups (n = 6 in each group): In Group I, rats were injected with normal saline via tail vein, performed with the regimen of 3 x 5-min intravenous (i.v.) infusion at a rate of 0.1 ml x kg(-1) min(-1) 24 h before I/R; In Group II, rats were treated according to the same experimental protocols as in Group I except receiving additional naloxone (0.1 mg/kg) 10 minutes before normal saline pretreatment; In Groups III, IV, V, and VI, rats were treated with remifentanil via tail vein, performed with the regime of 3 x 5-min i.v. infusion at a rate of 2 microg x kg(-1) x min(-1) 12 h, 24 h, 48 h, and 72 h before I/R; In Group VII, the rats were treated according to the same experimental protocols as in Group IV except that they received additional naloxone (0.1 mg/kg) 10 minutes before remifentanil pretreatment. Heart rate (HR), mean arterial pressure (MAP), and a lead II electrocardiogram were continuously monitored during IR process. To determine plasma concentration of creatine kinase myocardial isoenzyme-MB (CK-MB), arterial blood samples were obtained immediately before ischemia, and at the end of ischemia and reperfusion. After a 120-min reperfusion, heart was removed for the measurement of myocardial infarct size. Infarct size (IS) was expressed as percentage of the area at risk.

RESULTSHR, MAP, and rate-pressure product were not significantly different at each time points among all groups (P > 0.05). Compared with Group I, plasma concentrations of CK-MB at the end of ischemia and reperfusion and myocardial infarct size were significantly lower in Groups IV and V (P < 0.05). Compared with Group IV, plasma concentrations of CK-MB at the end of ischemia and reperfusion were significantly higher and myocardial infarct size was significantly larger in Group VII (P < 0.05).

CONCLUSIONRemifentanil preconditioning induces delayed cardioprotection in intact rat ischemia-reperfusion model, which may be triggered via opioid receptors.

Animals ; Disease Models, Animal ; Ischemic Preconditioning, Myocardial ; Male ; Myocardial Reperfusion Injury ; physiopathology ; prevention & control ; Piperidines ; pharmacology ; Rats ; Rats, Wistar