Objective To investigate the value of automatic spectral imaging mode selection and adaptive statistical iterative reconstruction(ASIR) in upper abdominal enhanced CT with low contrast agent dose, and to optimize the combination of monochromatic energy level and ASIR percentage.Methods 100 patients underwent upper abdominal enhancement CT were randomly assigned into control group (n=50) and study group (n=50).In the control group, tube voltage of 120 kVp and contrast medium of 450 mg I/kg were used with the images reconstructed using 40% ASIR.In the study group, CT spectral imaging with automatic spectral imaging mode selection and contrast medium of 300 mg I/kg were used,and monochromatic images (40-65 keV,intervals of 5 keV) were reconstructed using 40%-60% ASIR (intervals of 10%), respectively.CT value, image noise and contrast-to-noise ratio of the liver, pancreas, aorta and portal vein and radiation dose were compared using two independent samples t test.Overall image quality was assessed by Mann-Whitney U test.Results All image quality indexes in 60 keV with 40% ASIR, 55-60 keV with 50% ASIR in the study group were equal to or better than the control group.There was no significant difference in radiation dose between control group and study group.Conclusion Combined automatic spectral imaging mode selection with ASIR, upper abdominal enhanced CT with low contrast agent dose could improve image quality compared to 120 kVp with 40% ASIR, without increasing radiation dose.

OBJECTIVETo study the effects of TGF-beta/Smad signaling on the growth and apoptosis of human rhabdomyosarcoma cell line RD.

METHODSBiosynthesized short hairpin RNA (shRNA) was transfected into RD cells by cation liposome vector to suppress Smad4 expression. The mRNA and protein expression of Smad4 in RD after shRNA-transfection were examined by RT-PCR and Western blot respectively. Immunofluorescent staining was used to detect the location of Smad2/3 in RD by laser scanning confocal microscopy. The viability of RD cells was examined by MTT method and (3)H-thymidine incorporation assay. The apoptosis of RD was examined by flow cytometry analysis and fluorescent staining.

RESULTSThe expression of mRNA and protein of Smad4 in RD were effectively suppressed by shRNA interference. Such suppression effectively interrupted the endogenous TGF-beta/Smad signaling and consequently blocked the translocation of Smad2/3. The interruption of endogenous TGF-beta/Smad signaling not only inhibited the growth of RD but also induced apoptosis of RD. Exogenous TGF-beta1 inhibited the growth of RD but did not influence the apoptosis of RD.

CONCLUSIONshRNA interference can effectively interrupt the TGF-beta/Smad signaling by suppressing the expression of Smad4. TGF-beta/Smad signaling subtly regulates the growth and apoptosis of RD.

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Cell Proliferation ; drug effects ; Cytoplasm ; metabolism ; Humans ; Protein Transport ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; pharmacology ; Rhabdomyosarcoma ; genetics ; metabolism ; pathology ; Signal Transduction ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad4 Protein ; biosynthesis ; genetics ; Transfection ; Transforming Growth Factor beta1 ; pharmacology

BACKGROUNDTransforming growth factor beta (TGF-beta) plays an essential role in the regulation of normal physiologic processes of cells. TGF-beta has been shown to regulate several mitogen-activated protein kinases (MAPK) pathways in several epithelial cells. However, the effects of TGF-beta on soft tissue sarcoma are seldom reported. Our previous studies suggested that there should be some other signal transduction pathways besides Smads, which are important to regulate the growth of human embryonal rhabdomyosarcoma (RMS) cells. In the present study, we examined the expression and functional relations of extracellular signal-regulated kinase 2 (ERK2) and Smad4 in human RMS tissue and a RMS cell line, RD.

METHODSRD cells and normal human primary skeletal myoblasts (Mb) were treated with TGF-beta1 to establish the expression profile of ERK2 at the mRNA and protein levels detected by RT-PCR and immunofluorescence. Immunohistochemistry was used to detect the expression of ERK2 and Smad4 in 50 tissue specimens of human RMS and 23 specimens of normal skeletal muscles. Follow-up of specimens was performed 6 months to 70 months later.

RESULTSRD cells and human RMS tissues showed the higher expression of ERK2 and Smad4 than the normal control, either the protein level or the mRNA level. And, exogenous TGF-beta1 stimulation can lead to higher expression of ERK2 and its nuclear translocation, so TGF-beta1 can also activated MAPK (ERK2) pathway, resulting in a sustained activation of ERK2 for at least 2 hours. Immunohistochemistry analysis, however, showed that there was no correlation between ERK2 and Smad4 protein. The overexpression of ERK2 and Smad4 had no indicative effects on histological subtypes, histological grading, gender, age, and prognosis.

CONCLUSIONSIn RMS, signaling of TGF-beta1 from cell surface to nucleus can also be directed through the MAPK (ERK2) pathway besides the TGF-beta1/Smads pathway. The activation of ERK2 by TGF-beta1 may be Smad4 independent. Moreover, there may be some other tanglesome relationships between the TGF-beta1/Smads pathway and the MAPK pathway which takes part in the development, invasion and metastasis of tumor cells.

Cells, Cultured ; Humans ; Mitogen-Activated Protein Kinase 1 ; physiology ; Muscle, Skeletal ; metabolism ; RNA, Messenger ; analysis ; Rhabdomyosarcoma ; metabolism ; Signal Transduction ; Smad4 Protein ; physiology ; Transforming Growth Factor beta1 ; pharmacology

Objective:To study the role of endoplasmic reticulum stress(ERS)on ecdysterone(EDS)influenced protective effect in H2O2induced myocardial injury.Methods: The H9c2 cells were randomly divided into control group(normal cells),H2O2 groups(the cells treated with H2O2at concentrations of 1×10-3,1×10-4,1×10-5mol/L,respectively);EDS group(the cells were exposed to H2O2and treated with EDS at concentrations of 25,50,100 μmol/L respectively).The cell activity and apoptosis were measured by MTT assay and flow cytometry,respectively.The protein levels of Bcl-2,Bax,cleaved-caspase-3,caspase-4,caspase-7, caspase-12,GRP78 and CHOP were deter mined by Western blot.The MDA content and SOD activity were detected by the MDA and SOD detection kits.Results:The cell activity was decreased,cell apoptosis was increased,the content of MDA was elevated,the activity of SOD was inhibited and the protein expression of GRP78 and CHOP were increased in H2O2group as compared with control group, which were all significantly reversed by EDS treatment(P<0.05).Conclusion: Ecdysterone exhibits a protective effect on the cardiomyocytes with H2O2induced injury by inhibiting endoplasmic reticulum stress.

OBJECTIVETo study the effect of silencing pyruvate kinase M2 (PKM2) on gambogic acid (GA)-induced apoptosis of human prostate cancer PC3 cells.

METHODSThree specific PKM2 siRNAs and one negative control siRNA (si-NC) were transfected into PC3 cells. The silencing effect of PKM2 siRNAs was determined by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, and the effects of PKM2 siRNA on the vitality and apoptosis of GA-stimulated PC3 cells detected by MTT and AO/EB double staining, respectively. The mRNA and protein levels of c-myc and cyclin D1 were analyzed by qRT-PCR and Western blot, respectively.

RESULTSAll the 3 PKM2 siRNAs effectively reduced the mRNA and protein expressions of PKM2, and PKM2 siRNA-1 exhibited the strongest silencing effect. At 24 h after transfection, the expression levels of PKM2 mRNA and protein were reduced by 70% and 85%, respectively (P < 0.05). Twenty-four hours of treatment with GA (0.5 micromol/L) following transfection with PKM2 siRNA-1 inhibited the vitality of the PC3 cells by 68%, increased their apoptosis, and significantly down-regulated the mRNA and protein levels of c-myc (50% and 35%) and cyclin D1 (60% and 20%) (P < 0.05).

CONCLUSIONInhibition of PKM2 sensitized PC3 cells to GA-induced apoptosis, suggesting that PKM2 may be a potential therapeutic target for sensitizing human prostate cancer to GA.

Apoptosis ; drug effects ; Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Small Interfering ; Thyroid Hormones ; genetics ; metabolism ; Xanthones ; pharmacology

OBJECTIVETo evaluate the feasibility and efficacy of Narcotrend (NT) monitor in monitoring the depth of anesthesia in severely burned patients with target-controlled infusion (TCI) of remifentanil hydrochloride and propofol during perioperative period.

METHODSEighty patients with severe burn hospitalized from February to November 2011, to whom eschar excision was performed within one week after injury, were enrolled. They were classified into II to III grade according to the American Society of Anesthetists classification, and their total burn area ranged from 31% to 50%TBSA, or full-thickness burn area from 11% to 20% TBSA. Patients were divided into trial group (monitoring depth of anesthesia with routine method and NT monitor) and control group (monitoring depth of anesthesia with routine method) according to the random number table, with 40 cases in each group. All patients received TCI of remifentanil hydrochloride and propofol to induce and maintain anesthesia. During the operation, the anesthesia level of NT monitor used in the trial group was maintained from grade D1 to E0, while the fluctuation of mean arterial pressure (MAP) and heart rate of patients in control group was maintained around the basic values within a range of 20%, and on the basis of which, concentrations of two narcotics were adjusted. Concentrations of remifentanil hydrochloride and propofol during maintenance of anesthesia were recorded. The duration from drug withdrawal to waking from anesthesia (including the duration from drug withdrawal to eye opening by calling and the duration from drug withdrawal to orientation recovery) of patients was recorded. Values of MAP and heart rate at admission into the operation room, loss of consciousness, 2 min after intubation, before operation, 2, 15, and 30 min after the beginning of operation, and the end of operation were recorded. The prediction probability (P(k)) of NT stage (NTS) and NT index (NTI) in trial group, and that of MAP and heart rate in control group for two durations from drug withdrawal to waking form anesthesia were recorded. The administration of vasoactive drugs and intraoperative awareness of patients in two groups were recorded. Data were processed with t test, analysis of variance, and chi-square test, and the relationship between NTS, NTI, MAP, heart rate and their corresponding P(k) for the duration from drug withdrawal to orientation recovery was processed with Spearman correlation analysis.

RESULTSMaintained target effect-site concentration of remifentanil hydrochloride and target plasma concentration of propofol of patients were obviously lower in trial group [(2.62 ± 0.35) ng/mL, (3.84 ± 0.22) µg/mL] than in control group [(2.95 ± 0.21) ng/mL, (4.16 ± 0.31) µg/mL, with t values respectively -5.113 and -5.324, P values all below 0.01]. The duration from drug withdrawal to eye opening by calling and the duration from drug withdrawal to orientation recovery were obviously shorter in trial group [(10.2 ± 0.7) min, (11.1 ± 1.0) min] than in control group [(11.3 ± 1.0) min, (13.1 ± 0.7) min, with t values respectively -5.740 and -10.806, P values all below 0.01]. The MAP (except for 2 min after intubation) and the heart rate of patients in both groups were lower at the time points from loss of consciousness to the end of operation than at the time of entering operation room (with F values respectively 12.074, 36.425, P values all below 0.01 in trial group and F values respectively 21.776, 35.759, P values all below 0.01 in control group). The statistically significant difference between two groups in MAP level was only observed at the time of loss of consciousness (t = 3.985, P < 0.01). MAP level was close in two groups at other time points. Heart rates of patients in two groups were close during perioperative period. P(k) values of NTS and NTI for the duration from drug withdrawal to eye opening by calling (0.937 ± 0.025, 0.899 ± 0.049) were obviously higher than those of MAP and heart rate for this duration (0.579 ± 0.057, 0.536 ± 0.039, F = 900.337, P < 0.01). P(k) values of NTS and NTI for the duration from drug withdrawal to the orientation recovery (0.901 ± 0.031, 0.868 ± 0.046) were significantly higher than those of MAP and heart rate for this duration (0.532 ± 0.060, 0.483 ± 0.044, F = 890.895, P < 0.01). NTS, NTI, MAP, and heart rate were respectively negative, positive, positive and positive in correlation with their P(k) values for the duration from drug withdrawal to the orientation recovery (with r values from -0.734 to 0.682, P values all below 0.01). There was no statistically significant difference between two groups in administration of vasoactive drugs. No intraoperative awareness occurred.

CONCLUSIONSApplication of Narcotrend monitor in monitoring the depth of anesthesia in severely burned patients during perioperative period with TCI of remifentanil hydrochloride and propofol is beneficial to reducing dosage of narcotics and shortening duration of recovery from anesthesia, and it can accurately predict the level of consciousness of patients at the time of withdrawal of anesthesia.

Adolescent ; Adult ; Aged ; Anesthesia, Intravenous ; Burns ; surgery ; Female ; Humans ; Male ; Middle Aged ; Monitoring, Intraoperative ; instrumentation ; methods ; Piperidines ; Propofol ; Skin Transplantation ; methods ; Young Adult

OBJECTIVETo modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference.

METHODSThe U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference.

RESULTSThe sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection.

CONCLUSIONThe siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research.

Gene Silencing ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Polymerase Chain Reaction ; RNA, Small Interfering ; biosynthesis ; genetics ; RNA, Small Nuclear ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

BACKGROUNDInfections caused by gram-negative bacteria (GNB) often lead to high mortality in common clinical settings. The effect of traditional antibiotic therapy is hindered by drug-resistant bacteria and unneutralizable endotoxin. Few effective methods can protect high risk patients from bacterial infection. This study explored the protection of adeno-associated virus 2 (AAV2)-bacteriacidal permeability increasing protein 700 (BPI(700))-fragment crystallizable gamma one 700 (Fc gamma1(700)) chimeric gene transferred mice against the minimal lethal dose (MLD) of E. coli and application of gene therapy for bacterial infection.

METHODSAfter AAV2-BPI(700)-Fc gamma1(700) virus transfection, dot blotting and Western blotting were used to detect the target gene products in Chinese hamster ovary-K1 cells (CHO-K1cells). Reverse transcription-polymerase chain reaction and immunohistochemical assay were carried out to show the target gene expression in mice. Modified BPI-enzyme linked immunosorbent assay was used to identify the target gene products in murine serum. The protection of BPI(700)-Fc gamma1(700) gene transferred mice was examined by survival rate after MLD E. coli challenge. Colony forming unit (CFU) count, limulus amebocyte lysate kit and cytokine kit were used to quantify the bacteria, the level of endotoxin, and proinflammatory cytokine.

RESULTSBPI(1-199)-Fc gamma1 protein was identified in the CHO-K1 cell culture supernatant, injected muscles and serum of the gene transferred mice. After MLD E. coli challenge, the survival rate of AAV2-BPI(700)-Fc gamma1(700) gene transferred mice (36.7%) was significantly higher than that of AAV2-enhanced green fluorescent protein (AAV2-EGFP) gene transferred mice (3.3%) and PBS control mice (5.6%). The survival rate of AAV2-BPI(700)-Fc gamma1(700) gene transferred mice treated with cefuroxime sodium was 65.0%. The bacterium number in main viscera, the levels of endotoxin and proinflammatory cytokine (tumor necrosis factor-alpha and interleukin-1beta) in serum of the AAV2-BPI(700)-Fc gamma1(700) gene transferred mice were markedly lower than that of PBS control mice (P < 0.01).

CONCLUSIONSAAV2-BPI(700)-Fc gamma1(700) gene transferred mice can resist MLD E. coli infection through expressing BPI(1-199)-Fc gamma1 protein. Our findings suggested that AAV2 mediated BPI(700)-Fc gamma1(700) gene delivery could be used for protection and treatment of clinical GNB infection in high-risk individuals.

Animals ; Anti-Bacterial Agents ; therapeutic use ; Antimicrobial Cationic Peptides ; Blood Proteins ; CHO Cells ; Cricetinae ; Dependovirus ; genetics ; Disease Models, Animal ; Escherichia coli Infections ; therapy ; Gene Transfer, Horizontal ; Genetic Therapy ; Mice ; Mice, Inbred BALB C ; Proteins ; genetics ; Receptors, IgG ; genetics ; Recombinant Fusion Proteins ; genetics

OBJECTIVETo identify DUOX2 gene mutation in patients with congenital goiter with hypothyroidism.

METHODFive patients who had transit congenital hypothyroidism with goiter were enrolled. The exons of DUOX2 gene were amplified and sequenced.

RESULTA heterozygous missense mutation C1329T in the exon 10 of the DUOX2 gene was found in one patient, predicted to result in a Tryptophan to Arginine substitution at codon 376. However no mutation was detected in the other patients.

CONCLUSIONp.Arg376Trp mutation in DUOX2 was found in newborns of congenital hypothyroidism. The alleles frequency of this mutation may contribute to the function loss of congenital hypothyroidism.

Child, Preschool ; Congenital Hypothyroidism ; complications ; genetics ; Dual Oxidases ; Exons ; Female ; Goiter ; complications ; congenital ; genetics ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; NADPH Oxidases ; genetics

OBJECTIVETo analyze occurrence, prevention and treatment of the complications of thoracoscopic assisted spine surgery.

METHODSRetrospective review of 182 patients who underwent standard thoracoscopic technique or video-assisted thoracic surgical procedure from October 1998 to August 2004. The treatment of thoracic diseases included debridement, decompression with (or) reconstruction. The total number of complications were recorded, and its mechanism, prevention and treatment were analyzed.

RESULTSComplications occurred in 16 patients, 12 cases of perioperative complications included 3 patients suffered from pneumonia, 3 pulmonary atelectasis, 2 patients' lung injured by trocar, 1 patient obtained transient monoplegia, 2 suffered from transient intercostal nerve pain and 1 had superficial incision infection. Long-term complications occurred in 4 cases: spinal tuberculosis relapsed 2 cases (one who had diabetes obtained relapse in 8 months of post-operation and another relapsed with complex spinal tuberculosis in 4 weeks postoperation), 2 patients suffered from kyphosis deformity and pain.

CONCLUSIONSThe type and incidence of complications with thoracoscopic spine surgery mainly depend on indication, operation procedures and anesthesia, only by limit surgical indication, ameliorate technique, obey surgical principle and consummate perioperative treatment can we obtain mini-invasive effect by thoracoscopic assisted spine surgery.

Adolescent ; Adult ; Decompression, Surgical ; adverse effects ; methods ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Spinal Diseases ; surgery ; Spinal Fusion ; adverse effects ; methods ; Thoracic Vertebrae ; surgery ; Thoracoscopy ; adverse effects