Objective:To investigate the expression of HIF-1?and its relationship with angiogenesis in osteosarcoma.Methods: Osteosarcoma MG-63 cells were cultured in vitro under hypoxia and mimic hypoxia conditions.Thirty paraffin-embedded osteosarcoma tissues and 20 fresh frozen osteosarcoma specimens were collected.The mRNA and protein expression of HIF-1?and VEGF were detected by RT-PCR,Western blotting,ELISA,and immunohistochemistry methods.The mean vessel density(MVD)were also calculated.Results:The mRNA level of HIF-1?had no change under hypoxia and minic hypoxia conditions,whereas the protein expression was increased dramaticaly.The mRNA and protein expression of VEGF was significantly increased under hypoxia and minic hypoxia conditions.The positive rate of HIF-1?mRNA(90%)and VEGF(100%)in 20 fresh frozen tissues were higher than those of the para-tumor tissues(P

Objective To study the pharmacokinetics of linezolid in the patients with continuous renal replacement therapy ( CRRT ).Methods Each of 10 patients with CRRT was given a dose of 600 mg of linezolid with iv during 0.5 h.Plasma samples were collected at different time points after administration of drugs.The concentrations of linezolid in plasma were measured by HPLC.The software of DAS 3.0 was used to determine the pharmacokinetic parameters.Results Two-compartment model was the best to describe the linezolid concentration -time relation when given intravenolisly.The pharmacokinetic parameters of linezolid in 10 patients with CRRT were follows:t1/2 was (4.13 ±0.92) h, Cmax was (16.09 ±2.21)mg· L-1, AUC0-t was (80.05 ±17.89)mg· L-1· h-1.Conclusion There was difference on the pharmacokinetic parameters of linezolid between the patients with CRRT and the normal volunteers.

OBJECTIVETo explore whether hepatitis C virus core protein (HCV C) regulates the expression of NFAT1 to participate in the progression and malignant biological behavior of intrahepatic cholangiocarcinoma cells.

METHODSThe recombinant plasmid pEGFP-N(3)-HCV C and the empty vector pEGFP-N(3) were cotransfected with enhanced green fluorescent protein (EGFP) into RBE cells using liposome. Real-time PCR and Western blotting were used to examine the expression of NFAT1 mRNA and protein in the transfected RBE cells. MTT assay was used to evaluate the changes in the cell proliferation, and the cell cycle changes were analyzed by flow cytometry.

RESULTSHCV C transfection significantly enhanced the expressions of NFAT1 mRNA and protein in RBE cells (P<0.05) and promoted the progression of cell cycle into G(2)/M phase to accelerate the cell proliferation.

CONCLUSIONTransfection with HCV C gene up-regulates NFAT1 expression and promotes the cell cycle progression and proliferation of intrahepatic cholangiocarcinoma cells, suggesting the involvement of HCV C in the progression of intrahepatic cholangiocarcinoma.

Bile Duct Neoplasms ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cholangiocarcinoma ; pathology ; Gene Expression ; Humans ; NFATC Transcription Factors ; genetics ; Plasmids ; Transfection ; Viral Core Proteins ; genetics

Objective　To compare the clinical efficiency of intracardiac procedures on traditional cardioplegic arrested-heart and on-pu mp beating-heart for congenital heart disease (CHD) with severe pulmonary hyper tension. Methods　Among all 153 cases, 95 cases underwent operat ions on cardioplegic arrested-heart, while 58 on-pump beating-heart. In arres ted-heart group, 79 cases with ventricular septal defect (VSD), 13 with atria l septal defect (ASD) and 3 with patent ductus arteriosus (PDA) were examined whi le in beating-heart group, 43 cases with VSD, 10 with ASD, and 5 with PDA were examined. Results　There were 12 cases of operative death (12.6%) and 8 of tracheotomy (8.4%) in heart arrested group. No operative death and tracheotomy in beating-heart group. 141 patients were followed up for 3 months to 10 year s with good recovery. There were 2 cases of right heart function failure six yea rs later in arrested-heart group. Conclusion　Results sugges t that on-pump beating-heart technique is superior to traditional cardiopl egic arrested-heart for CHD with severe pulmonary hypertesion. The cause might be t hat on-pump beating-heart intracardiac operation is more effective in cardio pulmon ary protection.

OBJECTIVETo determine the safety and efficacy of local administration of lentivirus-mediated small interfering RNA (siRNA) targeting tumor necrosis factor-alpha (TNF-alpha) in murine air pouch model.

METHODSFrom May 2007 to April 2008 a siRNA targeting TNF-alpha and a missense siRNA were designed, and recombine lentivirus which coexpressed the green fluorescent protein (GFP) as a marker gene was constructed. Air pouches were established and stimulated by Ti-6Al-4V particles. Pouches were divided into 3 groups randomly. Lentivirus-mediated siRNA targeting TNF-alpha (TNF-alpha group) or lentivirus-mediated missense siRNA (MS group), or virus-free saline (control group) were injected into pouches respectively. Pouch membrane, peripheral blood, heart, liver, spleen, kidney, lung and brain were harvested at 28 d after transfection, and assayed for markers of inflammation using histological, molecular, immunological techniques and Xenogen in vivo imaging system (IVIS) 50 vivo bioluminescent assay system.

RESULTSXenogen IVIS 50 vivo image revealed strong expression of GFP localized in pouch areas and no expression in other parts of mice both in TNF-alpha group and MS group at 4 weeks after transfection, while no expression of GFP was found in control group. By RT-PCR and ELISA, the mRNA and protein levels of TNF-alpha in TNF-alpha group decreased by 81.6% and 82.6% respectively compared to control group (P < 0.01), and decreased by 78.9% and 84.0% respectively compared to MS group (P < 0.01), whereas TNF-alpha level in peripheral blood, heart, liver, spleen, kidney, lung and brain remained invariant (P > 0.05). Less inflammatory responses (thinner pouch membrane and decreased cellular infiltration) were observed in TNF-alpha group.

CONCLUSIONEfficient local delivery of lentivirus-mediated siRNA targeting TNF-alpha into modified murine air pouch can inhibit debris-induced inflammation effectively, with no systemic adverse effects.

Animals ; Disease Models, Animal ; Genetic Therapy ; Genetic Vectors ; genetics ; Inflammation ; therapy ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; RNA, Small Interfering ; genetics ; Random Allocation ; Transfection ; Tumor Necrosis Factor-alpha ; genetics

To study the diagnostic method of slight viral myocarditis in the field of forensic pathology, slight viral myocarditis model was induced in Balb/c murine by coxsackie virus B3. Organs of hearts, livers, spleens, lungs and kidneys were examined through routine pathological methods. Pathological changes at different levels of these organs were observed. The results indicated that viral myocarditis was a kind of disease with multiple organ alterations and that the pathological observation and comprehensive analysis of multiple organs was one of the useful methods for diagnosing slight viral myocarditis.
Animals ; Coxsackievirus Infections/pathology* ; Female ; Forensic Medicine ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis/virology*

The study was purposed to prepare the recombinant lentiviral vector pTK161 and pTK162 carrying B-domain-deleted canine factor (BDDcFVIII) gene, and to investigate whether the canine FVIII (cVIII) can be expressed in vitro. The BDDcFVIII gene was ligated behind PUB and 2OH1 promotors to create lentiviral vectors pTK161 and pTK162. Meantime lentiviral vectors pTK161' and pTK161' were produced by cloning a green fluorescent protein (GFP) into pTK151 and pTK152, which was driven by PUB and 2OH1 promotors respectively. Vector supernatant were prepared by using transfer calcium phosphate mediated-cotransfection of 293T cells. The virus vector, DeltaNRF packaging-plasmid, and VSV-G envelope-plasmid was assayed by titers and cFVIII activity in cell culture supernatant after infection into 293T cells. pTK161, pTK162, pTK161' and pTK161' were identified by restriction enzyme analyzing. The results showed that the lentiviral vectors pTK161, pTK162, pTK161' and pTK161' were successfully constructed, and the titers of pTK161' and pTK161' reached to 1.54 x 10(6) U/ml and 2.83 x 10(6) U/ml; the activity of cFVIII could be detected at 24 hours after infection of 293T cells by pTK161 and pTK162, and achieved the highest level at 72 hours later. The higher level of cFVIII activity was achieved by transfected with pTK162 than that of pTK161 (p < 0.05), which closed to the cFVIII activity in normal dog plasma. 1/4 of the highest level could be detected 6 weeks later. It is concluded that the prepared HIV1-based lentiviral vectors can infect 293T cells to express cFVIII effectively. The results provide the basis for further studying HIV-1-based lentiviral vector gene therapy for hemophilia A.
Animals ; Dogs ; Factor VIII ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; HIV-1 ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

This study was aimed to construct a lentiviral vector carrying human coagulant factor VIII (FVIII) and to investigate its expression in 293T cells. B-domain-deleted factor VIII gene fragment (BDDhFVIIIcDNA) was obtained by enzyme digestion and cloned into lentiviral vector pXZ208 to establish the expression vector pXZ208-BDDhFVIII. Recombinant viral particles were prepared by cotransfection with packaging plasmid delta NRF and envelope plasmid VSV-G using calcium phosphate precipitation method. 293T cells were transfected by viral supernatant. Coagulant activity of FVIII, BDDhFVIIImRNA and genome integration were assayed by one-step method, RT-PCR and PCR after transfection. The results showed that 293T cells could be transfected by recombinant virus. The transfection rate of 293T was 59.57%. After transfection, the cells expressed FVIII efficiently. Detection confirmed that the activity of FVIII was 12%, 43% and 87% respectively at 24, 48 and 72 hours after infection. BDDhFVIII transcription was detected by RT-PCR from the infected cells. The gene integration in the targeted cells was also observed. It is concluded that the successfully constructed lentiviral vector is able to generate high level expression of human FVIII in 293T cells, which may provide a potential application of gene therapy to haemophilia A.
Cell Line ; Factor VIII ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

OBJECTIVETo study the value of 3D visualization technique in breast-preserving surgery for breast cancer with immediate breast reconstruction using laparoscopically harvested pedicled latissimus dorsi muscle flap.

METHODSFrom January, 2015 to May, 2016, 30 patients with breast cancer underwent breast-preserving surgery with immediate breast reconstruction using pedicled latissimus dorsi muscle flap. The CT data of the arterial phase and venous phase were collected preoperatively and imported into the self-developed medical image 3D visualization system for image segmentation and 3D reconstruction. The 3D models were imported into the simulation surgery platform for virtual surgery to prepare for subsequent surgeries. The cosmetic outcomes of the patients were evaluated 6 months after the surgery. Another 18 patients with breast cancer who underwent laparoscopic latissimus dorsi muscle breast reconstruction without using 3D visualization technique from January to December, 2014 served as the control group. The data of the operative time, intraoperative blood loss and postoperative appearance of the breasts were analyzed.

RESULTSThe reconstructed 3D model clearly displayed the anatomical structures of the breast, armpit, latissimus dorsi muscle and vessels and their anatomical relationship in all the 30 cases. Immediate breast reconstruction was performed successfully in all the cases with median operation time of 226 min (range, 210 to 420 min), a median blood loss of 95 mL (range, 73 to 132 mL). Evaluation of the appearance of the breast showed excellent results in 22 cases, good appearance in 6 cases and acceptable appearance in 2 cases. In the control group, the median operation time was 283 min (range, 256 to 313 min) and the median blood loss was 107 mL (range, 79 to 147 mL) with excellent appearance of the breasts in 10 cases, good appearance in 4 cases and acceptable appearance in 4 cases.

CONCLUSION3D reconstruction technique can clearly display the morphology of the latissimus dorsi and the thoracic dorsal artery, allows calculation of the volume of the breast and the latissimus dorsi, and helps in defining the scope of resection of the latissimus dorsi to avoid injuries of the pedicled vessels. This technique also helps to shorten the operation time, reduce intraoperative bleeding, and improve the appearance of the reconstructed breast using pedicled latissimus dorsi muscle flap.

Objective To evaluate the effect of an integrated control strategy and to quantify the spatial-temporal variation of infected snails in the bottomland areas after the strategy was implemented.Methods Based on the geographic database of infected snail distribution at the village level during 2004-2010 in Anxiang county,Hunan province,spatial autocorrelation analysis and spatial scan statistics were applied to analyze the spatial-temporal characteristics on the distribution of infected snails.Results The number of embankments with infected snails in Anxiang county decreased from 23 in 2004 to 10 in 2010,while the rate of frame with infected snail in embankments decreased from 4.32‰ in 2004 to 0.12‰ in 2010.The spatial distribution of infected snails was nonrandom,only in 2004 and 2005 with Moran's I=0.21 (P＜0.10) and Moran's I=0.13 (P＜0.10) respectively.Data from the local spatial auto-correlation analysis showed that the number of villages with H-H types of auto-correlation model had been gradually decreasing.The results of SaTScan statistics appeared the same as from the local spatial auto-correlation analysis,showing that the number of areas with increased risk was decreasing.Conclusion The comprehensive measures with emphasis on infectious source control seemed effective for schistosomiasis control program.The current distribution characteristics provided us with evidence that the resource assignment could be more reasonably implemented so as to control schistosomiasis in a more effective way.