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

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

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.

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*

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.

The present study was aimed to investigate the mechanism of the granulocyte colony-stimulating factor (G-CSF) on the viability of the bone marrow mesenchymal stem cells (MSCs). MSCs were cultured by classical whole bone marrow adhering method, and the MSCs were analyzed for the cell surface differentiation markers CD34, CD133, CD90 and CD105 by flow cytometry (FCM). The ability of the MSCs to differentiate into osteocytes and adipocytes was tested in osteogenic and adipogenic mediums, separately. The effect of G-CSF (20 mug/mL) on the passage 3 MSCs viability was evaluated by MTT method, and the molecular mechanism of the G-CSF mediated effects was assayed through the pretreatment of the signal pathway inhibitors including 50 nmol/L wortmannin (phosphatidylinoesitol 3 kinase inhibitor), 50 mumol/L PD98059 [extracellular signal-regulated-kinase1/2 (ERK1/2) inhibitor], 30 mumol/L SB203580 (p38 mitogen-activated protein kinase inhibitor), 10 mumol/L H89 (protein kinase A inhibitor), 20 mumol/L Y27632 (Rho kinase inhibitor), 1 mumol/L rapamycin [mammalian target of rapamycin (mTOR) inhibitor], 10 mmol/L straurosporine [protein kinase C (PKC) inhibitor], 6 nmol/L G0697 (PKCalpha inhibitor) and 50 mumol/L Pseudo Z (PKCzeta inhibitor). Cultured passage 3 MSCs expressed CD90 and CD105 strongly, and showed the ability of multi-differentiation into osteocytes and adipocytes. G-CSF promoted the viability of MSCs, and the promotion was completely inhibited by PKC inhibitor straurosporine and partially inhibited by wortmannin, rapamycin, PD98059, SB203580 or G0697. However, its effect was not inhibited by H89, Y27632 and Pseudo Z. It is thus suggested that the promoting effect of G-CSF on MSCs viability was closely related to AKT-mTOR-PKC signal pathway, and PKC maybe the central role in the signal pathway.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Survival ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cells ; Humans ; Mesenchymal Stromal Cells ; cytology ; Signal Transduction

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 investigate the effects of recombinant human erythropoietin (rhEPO) on proliferation of human bone marrow-derived mesenchymal stem cells (MSCs) in vitro. The aspirates of the bone marrow from healty volunteers were seeded in culture medium. Then MSCs were isolated according to characteristics adhering to the plastics. After three passages in culture, bone marrow-derived adherent cells were identified by growing morphological features, cell surface antigens and differentiation into multi-lineages. Then P3-MSCs which had been identified were incubated with different concentrations of rhEPO (0.5, 1, 5, 10 and 50 U/ml). Subsequently, proliferation of MSCs was measured by MTT assay, as well as cell counts. At the same time, cell cycle was detected by flow cytometry (FCM). The results indicated that the expressions of CD90 and CD105 in P3 bone marrow-derived adherent cells were positive, while the expressions of CD34 and CD45 were negative, and these cells could differentiate into adipocytes, osteocytes and chondrocytes in induction media. MTT assay showed that the optical density (OD) of group treated with EPO was significantly higher than that in the control group (p<0.05), and the group treated with 50 U/ml EPO achieved the most predominant effects. The results of cell count were coincident with that of MTT assay. Furthermore, the cell cycle analysis by FCM revealed that rhEPO could relatively decrease the cell ratio in G0/G1 phase, and increase the cell ratio in S and G2/M phases. As compared with the control group, all those differences were statistically significant (p<0.01). It is concluded that erythropoietin can promote proliferation of human bone marrow mesenchymal stem cells in vitro, which may be correlated with the increased entry into S and M phases of cell cycle of MSCs adjusted by EPO.
Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Culture Media ; Erythropoietin ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Recombinant Proteins