AIM: To establish a fluorogenic quantitative polymerase chain reaction (FQ-PCR) method for the routine examination of c-erbB-2 gene expression in breast cancer. METHODS: The c-erbB-2 standard gene was obtained by in vitro amplification of cloned c-erbB-2 fragment in plasmid PGEM-T easy vector. FQ-PCR product was detected by using a 7700 ABI PRISM sequence detector system and c-erbB-2 standard curve was obtained to quantity c-erbB-2 in unknown samples. RESULTS: “S” kinetics curve of FQ-PCR amplification was generated by relating the fluorescence signal intensity (△Rn) to the cycle number. The standard curve of c-erbB-2 was constructed by the linear relationship between the cycle threshold (ct) and the log of starting copy number. The high correlation (0.999) revealed the reliability of FQ-PCR. CONCLUSION: The FQ-PCR is a rapid, sensitive, reliable method for quantity of c-erbB-2 gene expression.

Experiments were conducted to investigate the suitability of the multistage in-situ reaction analyzer based on a micro fluidized bed ( MFB-MIRA) for measuring the rapid change of the gas concentration during gas-solid reactions. The results showed that the control performance of capillary temperature had a great impact on the stability of on-line measurement. Based on the observed regular patterns, the capillary temperature control system was equipped with a precision temperature controller. The control precision of capillary temperature reached ± 0 . 2℃, which guaranteed the high stabilities of the sampling flow rate and the chamber vacuum. The measured results of the modified gas monitoring system showed the periodic fluctuations of the on-line measurement were eliminated. The stability of measurement was significantly improved. The fluctuating range and relative standard deviation of the measured response to O2 in air changed from 1. 9% and 0. 5% to 1. 4% and 0. 2%, respectively. A pressure regulating device was also developed to control the absolute pressure at the gas sampling point. The control precision reached ± 0. 02 kPa. The measured results showed that the response of the process mass spectrometer was positively correlated with the absolute pressure at the sampling point, indicating the necessity of the pressure regulating device. The accuracy and repeatability of process mass spectrometer were improved. This study has enhanced the suitability of MFB-MIRA for studying rapid gas-solid reactions and broadened the scope of reliable applications of MFB-MIRA and process mass spectrometer.

Objective To retrospectivel y analyze the abdominal CT findings and pathological results of the chronic schist osomiasis so as to improve the diagnostic accuracy of the disease. M ethods The plain abdominal CT scanning was performed in 103 cases an d enhanced CT scanning in 81 cases. The pathological specimen which was consist ent with the section of CT scan was obtained in each cases. Results On CT scanning, liver cirrhosis was seen in 84 cases, various calci fication in liver in 71 cases, liver cancer in 12 cases, enlargement of sple en in 78 cases, calcification in spleen in 13 cases, wall-thickening in colon i n 27 cases, calcification in colon in 31 cases, and colon cancer in 9 cases. Pa thological examination revealed various fibrosis and formation of pseudolobule. The eggs and calcification could be seen in pseudolobule and septa, colonic sub mucosa, and regional lymph nodes. Fibrous hyperplasia in colonic wall and hyper plasia in mucous membrane were obvious. Fibrous hyperplasia and calcification w ere seen in spleen, but the eggs were not found. Conclusion The liver and colon are the major organs affected by chronic schistosomias is in abdomen, and the CT findings are obvious too. The pathological features o f spleen are accompanied with liver cirrhosis. CT is the important imaging meth od in diagnosing chronic schistosomiasis and pathological changes.

OBJECTIVETo evaluate the effectiveness of locking plate external fixator in treating middle and distal tibial fractures.

METHODSFrom January 2010 to January 2013,18 patients suffered from middle and distal tibial fractures were treated by locking plate external fixator,including 11 males and 7 females, with an average age of 53.5 (ranged from 13 to 80) years old,the course of disease ranged from 2 h to 3 d. According to AO classification, 4 cases were type A,11 cases were type B and 3 were type C. Among them,6 patients were open fracture, including 2 cases with type I, 3 cases with type II and 1 case with type III, according to Gustilo classification), 12 patients were close fracture. Operation time, postoperative complications were observed, and Johner-Wruhs scoring were used to evaluate clinical outcomes.

RESULTSAll patients were followed up from 6 to 15 (meaned 11) months. Two cases occurred skin necrosis (1 case occurred bone exposure), 2 cases occurred delayed union (all were open fracture), and 1 case occurred nail infection. No screw loosening or broken occurred. According to Johner-Wruhs scoring, 10 cases obtained excellent result,6 cases good,and 2 cases fine.

CONCLUSIONLocking plate external fixator for the treatment of middle and distal tibial fractures, which has advantages of lessen damage, shorter operative time, less complications and rapid functional recovery, is one of good choice.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Plates ; utilization ; External Fixators ; utilization ; Female ; Fracture Fixation ; instrumentation ; methods ; Humans ; Male ; Middle Aged ; Tibial Fractures ; surgery ; Treatment Outcome ; Young Adult

Objective To propose a new blood purification modality-hemodialysis with plasma- based dialysate (HD-PBD) plus high volume hemofiltration (HVHF) for patients with liver failure, and to evaluate the effect of this treatment on plasma cytokines.Methods Twelve patients with liver failure were included in this study.All patients received HD-PBD therapy in the first 6 hours,and then were treated with HVHF for 24 hours with the same filter (AV600).The levels of TNF-?,IL-1?, IL-6 and IL-8 in plasma before and after HD-PBD plus HVHF for 6 and 24 hours were examined respectively by ELISA,and changes of clinical parameters were observed at the same time point. Serum bilirubin,total bile acids (TBA),serum ammonia,blood urea nitrogen (BUN) and serum creatinine (Scr) were detected before and after treatment.Arterial blood gas analysis and the concentration of electrolytes were monitored before and after treatment.Results (1)HD-PBD for 6 hours was more effective than HVHF for 24 hours in removal of serum bilirubin and TBA(P＜0.05). (2)Serum ammonia,BUN,Ser,arterial blood HCO_3~-,PCO_2,PO_2 and electrolytes did not show significant difference before and after HD-PBD (P＞0.05),but these parameters significantly changed before and after HVHF (P＜0.05).(3)The average level of serum bilirubin was sharply decreased after HVHF for 24 h following HD-PBD(P＜0.05).(4)After HD-PBD plus HVHF,there was a marked reduction of the plasma levels of TNF-?,IL-6 and IL-8.Conclusions HD-PBD plus HVHF,a newly proposed modality for patients with liver failure,can effectively decrease serum bilirubin,TBA,BUN,Scr,ammonia and cytokines,and adjust water-electrolyte as well as acid- alkali balance.It is a low-cost,safe,simple and convenient therapy.

OBJECTIVETo evaluate the impact of specific siRNA on survivin gene in transfected leukemia cells.

METHODThe small interfering RNA (siRNA) targeted survivin mRNA was synthesized in vitro and was transfected into K562 cell by Hiperfect into human leukemia cell line K562, which has high survivin expression level. The level of survivin mRNA expression was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR) with SYBR GREEN I. The apoptosis index of cytotrophoblasts were determined and analyzed by FCM (Annexin V-FITC/PI staining methods). The cell proliferation was examined by MTT at 48 h and 72 h after transfection.

RESULTThe level of mRNA expression was significantly inhibited by the siRNA 48 h and 72 h after transfection, the suppression rate of survivin mRNA separately reached 85.21%, 94.35% mensurated by quantitative RT-PCR with SYBR GREEN I, cell proliferation was inhibited significantly by 45.02% and 50.88%, respectively, the apoptotic rate detected by Annexin V-FITC assay reached 12.28%and 21.55%, respectively.

CONCLUSIONThe chemosynthesized siRNA targeting survivin could significantly down-regulate survivin mRNA. Survivin siRNA was able to inhibit the proliferation of leukemia cell line K562. Survivin may become a new target for leukemia gene therapy.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Gene Silencing ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; K562 Cells ; RNA, Small Interfering ; pharmacology ; Transfection

The present study was aimed to investigate whether Bcl-2, Fas and Bax are involved in monocyte chemotacitic protein-1 (MCP-1)-induced apoptosis of human umbilical vein endothelial cells (hUVECs). hUVECs were cultured, and the purity was identified by immunofluorescence and immunohistochemistry with specific anti-von Willebrand factor (vWF) and anti-VEGF receptor-2 (KDR) antibodies. With 90% confluence hUVECs were serum-starved for 12 h, and then treated with different concentrations of MCP-1 (0.1, 1.0, 10, 100 ng/mL) for 24 and 48 h respectively. The expressions of apoptosis related proteins Fas, Bcl-2, Bax were detected by flow cytometry (FACS) and Western blot. As shown in our preliminary study, MCP-1 induced apoptosis of hUVECs in a dose-dependent manner at both 24 h and 48 h. FACS and Western blot analysis results in the present study indicated that MCP-1 promoted the expression of proapoptotic proteins Bax and Fas and inhibited the expression of antiapoptotic protein Bcl-2. These results suggest that MCP-1 may induce the apoptosis of hUVECs through evoking the imbalance between proapoptotic Fas/Bax and antiapoptotic Bcl-2 protein.
Apoptosis ; Atherosclerosis ; physiopathology ; Cells, Cultured ; Chemokine CCL2 ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; metabolism

OBJECTIVESTo identify and clone human genes transactivated by HCV F protein by constructing a cDNA subtractive library using the suppression subtractive hybridization technique.

METHODSSuppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV F protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1 (-)-F or with pcDNA3.1(-) empty vector as a control, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sized cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 or adaptor 2. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5 alpha. The cDNA was sequenced and analyzed in GenBank with blast search after PCR.

RESULTSThe subtractive library of genes transactivated by HCV F protein was constructed successfully. The amplified library contains 71 positive clones. Colony PCR shows that 56 clones contain 200-1000 bp inserts. Sequence analysis was performed on 28 clones randomly, and the full length sequences were obtained with using the bioinformatics method. Altogether 19 coding sequences were obtained, consisting of 17 known and 2 unknown.

CONCLUSIONSThe obtained sequences may be target genes transactivated by HCV F protein, and some gene coding proteins are those involved in cell cycle regulation, metabolism, and cell apoptosis.

Cloning, Molecular ; Hepacivirus ; genetics ; Humans ; Nucleic Acid Hybridization ; methods ; RNA, Messenger ; biosynthesis ; genetics ; Transcriptional Activation ; Viral Core Proteins ; biosynthesis ; genetics

OBJECTIVETo study the clinical and pathological features, diagnosis, therapy and prognosis of primary small intestine malignant tumor.

METHODSA retrospective analysis was performed on the clinical data from the 120 cases of primary small intestine malignant tumor.

RESULTSAbdominal pain, gastrointestinal bleeding, anemia, abdominal mass and jaundice were the main clinical features. The pathology was confirmed by abdominal X-ray, gastrointestinal barium, CT, MRI, endoscopy and surgical exploration. Most tumors originated in the duodenum (54.1%), and adenocarcinoma (55.8%) was the main pathological type. The median survival time of the patients was 19.2 months and the 1-year survival rate was 55.4%. Chemotherapy did not seem to significantly improve the 1-year survival rate of the patients (P=0.842).

CONCLUSIONPrimary small intestine malignant tumors lack specific clinical manifestations and surgical resection should be performed as early as possible.

Adenocarcinoma ; diagnosis ; surgery ; Adult ; Aged ; Aged, 80 and over ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Female ; Humans ; Intestinal Neoplasms ; diagnosis ; drug therapy ; surgery ; Intestine, Small ; pathology ; surgery ; Male ; Middle Aged ; Retrospective Studies

Amyloid β protein (Aβ) is closely involved in the pathogenesis of Alzheimer's disease (AD), and one of the main strategies for AD treatment is antagonizing the neurotoxicity of Aβ or even clearing the Aβ deposited in the brain. The present study was aimed to observe the effects of intrahippocampal injection of Aβ₃₁₋₃₅ on the spatial learning and memory of rats by using Morris water maze technique, and explore the neuroprotective effects and possible mechanism of [Gly14]-humanin (HNG) against Aβ-induced deficits in learning behavior. The results showed that bilateral intrahippocampal injection of 2.0 nmol Aβ₃₁₋₃₅ significantly increased the mean traveled distance of rats in searching for the hidden underwater platform and decreased the distance percentage in the target quadrant in probe test after withdrawal of platform, whereas pretreatment with HNG (0.2 nmol and 2.0 nmol) suppressed Aβ₃₁₋₃₅-induced increase in the traveled distance and decrease in swimming distance percentage. Application of Genistein (40 nmol), a specific tyrosine kinase inhibitor, almost completely blocked the antagonistic effects of HNG against Aβ₃₁₋₃₅. These results indicate that HNG can dose-dependently prevent against Aβ₃₁₋₃₅-induced impairment in spatial learning and memory of rats, and the neuroprotective effects of HNG might involve the activation of endogenous tyrosine kinase pathway, suggesting that up-regulation of the tyrosine kinase signaling by using HNG might be of great significance for the improvement of cognitive function in AD.
Alzheimer Disease ; physiopathology ; Amyloid beta-Peptides ; adverse effects ; antagonists & inhibitors ; Animals ; Brain ; drug effects ; Genistein ; pharmacology ; Memory ; drug effects ; Neuroprotective Agents ; pharmacology ; Peptide Fragments ; adverse effects ; antagonists & inhibitors ; Peptides ; pharmacology ; Rats ; Spatial Learning ; drug effects