Objective To construct a recombinant adenoviral vector carrying and co-expressing human inhibitor of growth 4(ING4) and human interleukin-24(IL-24) mediated by poly( A)-Promoter[Ad-ING4-poly(A)-Promoter-IL-24, referred to as Ad-ING4-IL-24] and explore its effect on the growth of HepG-2 human hepatocellular carcinoma cellsin vitro. Methods The poly(A)-Promoter(hEFl-elF4g) (Sal Ⅰ and Not Ⅰ ), ING4 ( Bgl Ⅱ and Sal Ⅰ ), and IL-24 ( Xho Ⅰ and Xba Ⅰ ) fragments were amplified by PCRusing pORF-mbcl-2α, pcDNA3.0-IL-24, and pcDNA3.0-ING4 plasmids as templates and subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-ING4-poly (A)-Promoter-lL-24, respectively. The pAdTrack-CMV-ING4-poly (A)-Promoter-IL-24 transfer vector linearized with Pme Ⅰ digestion and pAdEasy-1 backbone vector was further cotransformed into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy-l-pAdTrack-CMV-ING4-poly ( A )-Promoter-IL-24 homologous recombinant plasmids were linearized with Pac Ⅰ digestion and transfected into the human embryonic kidney 293 (QBI-293A) cells by liposome, leading to formation of the recombinant adenoviruses Ad-ING4-IL-24co-expressing ING4 and IL-24. The Ad-ING4-IL-24 were amplified in QBI-293A cells and its titer was up to 3.5 × 109 PFU/ml. Adenovirus-mediated ING4 and IL-24 genes expression in HepG-2 cells was examined by RT-PCR and Western blot. The growth-suppressing and apoptosis-inducingg effect of Ad-ING4-IL-24 coexpressing ING4 and IL-24 on HepG-2 human hepatocellular carcinoma cells was assessed by MTT assay and FCM, respectively. Results DNA sequencing showed that the ING4, poly (A)-Promoter, and IL-24 fragments subcloned into pAdTrack-CMV plasmids were completely identical to those reported in GenBank.ING4 and IL-24 gene mediated by adenovirus could both successfully express in HepG-2 cells. Adenovirusmediated ING4 and IL-24 co-expression significantly suppressed HepG-2 hepatocellular carcinoma cell growth and induced cell apoptosis, and the effect of Ad-ING4-IL-24 group was more significant than AdING4 and Ad-IL-24 group. Conclusion The adenoviral vector co-expressing ING4 and IL-24 mediated by poly(A)-Promoter(Ad-ING4-IL-24) was successfully constructed. Ad-ING4-IL-24 had marked anti-tumor effect in suppressing HepG-2 human hepatocellular carcinoma cell growth and inducing cell apoptosis in vitro. Compared with Ad-ING4 and Ad- IL-24, Ad-ING4-IL-24 enhanced anti-tumor effect.

OBJECTIVETo study the clinical results of mitral valve repair in patients of primary infective endocarditis with mitral insufficiency.

METHODSFrom January 2004 to July 2007, 40 patients who had undergone valve repair procedure for infective endocarditis with mitral insufficiency were analyzed retrospectively. There were 26 male and 14 female patients, with an average age of (34.0 +/- 3.5) years old, including 6 patients of underlying heart disease, 34 patients of no previously underlying heart disease. There were 12 patients in NYHA functional class II, 19 patients in class III, 9 patients in class IV preoperatively. Preoperative echocardiography showed moderate to severe MR in all patients. The surgery was performed under extracorporeal circulation and moderate hypothermia. The distribution of anatomical lesion according to surgical findings were vegetation in 32 patients, leaflet prolapsed in 34 patients, leaflet perforation in 16 patients, chordal rupture in 32 patients, and annular abscess in 2 patients. The vegetations and infected tissues were debrided. The surgery consisted of complex methods to repair mitral valve, including direction leaflet closure in 5 patients, pericardial patch closure of leaflet perforation in 18 patients, chords reimplantation in 4 patients and chords transference in 6 patients, quadrangular resection in 12 patients, double orifice method in 17, closure of the commissure in 8, rings annuloplasty in 28 cases, and so on. There were 28 selective surgeries and 12 emergent ones. Patients were evaluated for early and long-term clinic and echocardiographic outcome before and after operation.

RESULTSThere were no early postoperative death. Mitral valve repair was feasible in 39 patients, one patient was transformed to mitral valve replacement during the operation. Postoperative echocardiography demonstrated no (n = 24) or mild (n = 15) mitral regurgitation at the discharge examination and observed significant reductions in left ventricular end diastolic [from (62 +/- 7) mm to (51 +/- 6) mm, P < 0.05] and end systolic dimensions [from (45 +/- 3) mm to (40 +/- 4) mm, P < 0.05] and left atrial dimensions [from (49 +/- 4) mm to (42 +/- 6) mm, P < 0.05]. Mean follow-up (25.6 +/- 3.2) months, freedom from recurrent moderate to severe MR, freedom from repeat operation or infective endocarditis, revealed patients were 36 cases in NYHY class I, 3 cases in class II.

CONCLUSIONMitral valve repair for mitral valve endocarditis is feasible with a satisfied clinical outcome, maintains valve competency with significant reductions in left atrial and left ventricular dimensions after surgery.

Adolescent ; Adult ; Child ; Endocarditis ; complications ; Female ; Follow-Up Studies ; Heart Valve Prosthesis Implantation ; Humans ; Male ; Middle Aged ; Mitral Valve ; surgery ; Mitral Valve Insufficiency ; etiology ; surgery ; Retrospective Studies ; Treatment Outcome ; Young Adult

OBJECTIVETo study the stereoselective glucuronidation of carvedilol (CARV) by three Chinese liver microsomes.

METHODSThe metabolites of CARV were identified by a hydrolysis reaction with beta-glucuronidase and HPLC-MS/MS. The enzyme kinetics for CARV enantiomers glucuronidation was determined by a reversed phase-high pressure liquid chromatography (RP-HPLC) assay using (S)-propafenone as internal standard after precolumn derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosylisothiocyanate.

RESULTSTwo CARV glucuronides were found in three Chinese liver microsomes incubated with CARV. The non-linear regression analysis showed that the values of K(m) and V(max) for (S)-CARV and (R)-CARV enantiomers were (118+/-44) micromol/L, (2 500+/-833) pmol/(min.mg protein) and (24+/-7) micromol/L, (953+/-399) pmol/(min.mg protein), respectively.

CONCLUSIONThese results suggested that there was a significant (P<0.05) stereoselective glucuronidation of CARV enantiomers in three Chinese liver microsomes, which might partly explain the enantioselective pharmacokinetics of CARV.

Carbazoles ; metabolism ; China ; Glucuronic Acid ; metabolism ; Glucuronides ; metabolism ; Microsomes, Liver ; metabolism ; Propanolamines ; metabolism ; Stereoisomerism

15 scores were randomly divided into therapeutic group (30 cases)and control group(30 cases).The two groups were treated with the same conventional therapy. From the 1st day of admission,Xuebijing injection 80 ml was given by intravenous drip once for every 12 hours for 7 days to the therapeutic group.The level of tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?),IL-6,IL-10,and APACHEⅡscore were tested on 1,3,5 and 7 days after admission.The case fatality and incidences rates of sepsis and MODS in both groups were recorded.Results Compared with the control group,APACHEⅡscore,TNF-?,IL-1?,IL-6 and IL-10 of the therapeutic group were lower significantly on the 3rd day after admission,especially on the 5th day(all P

BACKGROUND & OBJECTIVE: Dendritic cells (DCs) or DClike cells had been successfully induced in vitro from leukemia cells, which may provide a promising immunotherapeutic protocol for leukemia. This study was designed to investigate the efficiency of in vitro generation of dendritic cells from CD14+ acute myelomonocytic (M4) or monocytic (M5) leukemia cells and their ability of stimulating specific antileukemia T-cell response.METHODS: Bone marrow mononuclear cells (BMMNCs) were isolated from 5M4/M5 leukemia patients with high CD14 expression, and then divided into 3groups: adherent leukemia cells, nonadherent blasts, and total unfractioned blasts. CD14 expression of the 3 groups was evaluated by flow cytometry (FCM). When cultured with or without granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-α) for 7-10 days, monocytic leukemia cell-derived dendritic cells (Mo-LDCs) were identified through morphologic observation and immunophenotype analysis using FCM. The immune function of Mo-LDCs was detected through allogeneic mixed lymphocyte reaction (Allo-MLR) and cytotoxicity assay of antileukemia cytotoxic T lymphocytes (CTLs). The leukemic origin of Mo-LDCs was confirmed by chromosomal karyotype analysis combined with the aberrant expression of myeloid antigens. RESULTS: The amount of CD14+ cells, which could differentiate into CD83+ mature DCs under induction of the cytokine combination, was higher in adherent leukemia cells than in nonadherent blasts and total unfractioned blasts. Regarding each 3 cell groups of the same patient or the unfractioned blasts of various patients, initial CD14 expression was positively related to the yield of CD83 + DCs after induction (r=0.967, P=0.007). Mo-LDCs exhibited typical morphology and phenotype as mature DCs, induced potent proliferation of homogeneous T cells in Allo-MLR, stimulated the expansion of leukemia-specific CTLs, and continued to possess the cytogenetic abnormalities of the original leukemia, as well as the aberrant expression of myeloid antigens. CONCLUSIONS: In M4/M5 subtype of AML, CD14+ cells could differentiate into immune-competent Mo-LDCs under the induction of the cytokine combination. CD14 expression level may predict the DCs differentiation ability of monocytic leukemia. MoLDCs, which possess the classical phenotype and function of DCs, as well as the abnormal leukemic antigens, may be useful for the immunotherapy of M4/M5 AML.

OBJECTIVETo evaluate the clinical efficacy of posterior short-segment pedicle screw fixation combined reduction of central end-plate by end-plate expand clamp and vertebroplasty with calcium phosphate cement for the treatment of thoracolumbar burst fractures.

METHODSThirty patients with thoracolumbar burst fractures were treated with posterior pedicle screw fixation combined reduction of central end-plate by end-plate expand clamp and vertebroplasty with calcium phosphate cement. There were 22 males and 8 females with an average of 47 years (range from 25 to 71 years). Segment of fracture had 1 case in T11, 5 in T12, 14 in L1, 8 in L2, 2 in L3, 1 in L4, 1 in L5. According to Denis typing, 4 cases were type A, 25 type B, 1 type C, 1 type D, 1 type E. The relative anterior height of vertebral body and the vertebral angle were measured before and after operation and during the follow-up period (8 months after operation) through X-ray, the failure of internal fixation and recurrent kyphosis were evaluated during the follow-up period. Vertebral intracorporeal gap was measured by CT images after operation and the central end-plate fracture and reduction were observed by the reconstructed CT images (sagittal and coronary) before and after operation.

RESULTSAll patients were followed up from 8 to 15 months with an average of 12 months. Relative anterior height of vertebral body and vertebral angle were well restored after operation, and there was no significant change between after operation and 8 months after operation. Preoperative,postoperative and 8 months after operation, relative anterior height of vertebral body respectively was 40.1%, 98.2%, 97.8%, vertebral angle respectively was 18.30, 2.70, 3.20. No failure of internal fixation and recurrent kyphosis were found during the follow-up period. The vertebral intracorporeal gap was about 3.1% and reduction of central endplate fracture was satisfactory.

CONCLUSIONPosterior short-segment pedicle screw fixation combined with vertebroplasty with calcium phosphate cement can provide excellent reduction of post-traumatic segmental kyphosis and restore vertebral body height in the fracture level, prevent the failure of internal fixation which is an ideal method for the treatment of thoracolumbar burst fractures.

Adult ; Aged ; Bone Cements ; Bone Screws ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Lumbar Vertebrae ; injuries ; Male ; Middle Aged ; Spinal Fractures ; surgery ; Thoracic Vertebrae ; injuries ; Tomography, X-Ray Computed ; Vertebroplasty ; methods

OBJECTIVETo compare the outcomes of intracytoplasmic sperm injection (ICSI) with retrieved epididymal and testicular sperm for obstructive azoospermia and with ejaculated sperm for severe oligozoospermia and asthenospermia.

METHODSWe retrospectively analyzed 431 ICSI cycles, which were divided according to sperm sources into Groups A (n=287 in patients with severe oligozoospermia or asthenospermia using ejaculated sperm), B (n=109 in obstructive azoospermia patients with sperm retrieved by percutaneous epididymal sperm aspiration, PESA) and C (n=35 in obstructive azoospermia patients with sperm retrieved by testicular sperm extraction, TESE). Comparisons were made among the three groups in the rates of embryo implantation, fertilization, pregnancy, cleavage, and miscarriage.

RESULTSGroup A showed statistically significant differences from Groups B and C in the rates of embryo implantation and pregnancy (18.46% vs. 25.23% and 28.76%, 31.23% vs. 42.16% and 39.39%, P < 0.05). But no significant differences were seen in the rates of fertilization, cleavage and miscarriage among the three groups (P > 0.05).

CONCLUSIONThe rates of embryo implantation and clinical pregnancy are higher in patients with obstructive azoospermia than in those with severe oligozoospermia or asthenospermia after ICSI with ejaculated sperm.

Azoospermia ; therapy ; Epididymis ; cytology ; physiopathology ; Female ; Humans ; Male ; Oligospermia ; therapy ; Pregnancy ; Pregnancy Outcome ; Retrospective Studies ; Sperm Injections, Intracytoplasmic ; methods ; Spermatozoa ; Testis ; cytology ; physiopathology

OBJECTIVETo study the relationship between enhanced degree in the cancer lesion and angiogenesis in the tumor through early manifestations in enhanced helical CT in patients with bladder cancer.

METHODSFifty-three patients with bladder carcinoma were examined by pelvic plain CT and helical CT scan at peak enhancement of cancer lesion. Histologic grade, vascular endothelial growth factor (VEGF) and micro-vascular density (MVD) were analyzed for each resected cancer lesion. Pearson and Spearman correlation tests were used to assess the relationship between CT enhancement and histologic grade, VEGF or MVD.

RESULTSIn early enhancing phase of helical CT, different degrees of enhancement were observed in 73 bladder cancer lesions. The difference between average CT attenuation and MVD in different histologic grade cancer lesions was statistically significant (P < 0.001). A positive correlation was found between the CT enhancement and MVD (gamma = 0.936, P < 0.001), histologic grade (gamma = 0.75, P < 0.001), but VEGF of bladder cancer did not correlate with the CT enhancement or MVD.

CONCLUSIONThe early enhancement of helical CT enhancement of bladder cancer, showing a positive relation to MVD and histologic grade, can reflect the tumor angiogenesis and blood supply.

Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; Radiographic Image Enhancement ; Tomography, Spiral Computed ; Urinary Bladder Neoplasms ; blood supply ; diagnostic imaging ; pathology ; Vascular Endothelial Growth Factor A ; analysis

This study was purposed to explore the expression of glucosylceramide synthase (GCS) in human leukemia cells and its relationship with multidrug resistance. RT-PCR was used to analyze peripheral blood samples from 53 leukemia patients with multidrug resistance/non-resistance, and to detect the expression level of GCS gene in HL-60 cells and HL-60/ADR cells, the expression level was compared with the level of mdr-1. The expressions of GCS protein and P-gp protein in HL-60 cells and HL-60/ADR cells were assayed by Western blot analysis. The results showed that the relative optical density ratio of GCS gene amplified bands in samples of leukemia patients with drug-resistance was significantly higher than that in samples of leukemia patients with drug non-resistance group (P < 0.05), meanwhile the significant enhancement of optical density value of GCS gene amplified bands accompanied by high expression of mdr-1 gene. Their correlation showed positive (P < 0.01, r = 0.6). The GCS mRNA and protein were overexpressed in HL-60/ADR cells, and their expression levels were obviously higher than that in HL-60 cells, meanwhile the expression of mdr-1 mRNA and P-gp also significantly increased in HL-60/ADR cells. It is concluded that the high level of GCS in leukemia patients possibly is associated with multidrug resistance of leukemia cells.
ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Female ; Glucosyltransferases ; metabolism ; Humans ; Leukemia ; enzymology ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Cells, Cultured

To investigate the crucial role of particle size in the biological effects of nanoparticles, a series of mesoporous silica nanoparticles (MSNs) were prepared with particle size gradients (50, 100, 150, 200 nm) with the traditional Stober method and adjusting the type and ratio of the silica source. The correlation between toxicity and size-caused biological effects were then further examined both in vitro and in vivo. The results indicated that the prepared MSNs had a uniform size, good dispersal, and ordered mesoporous structure. Hemolytic toxicity was found to be independent of particle size. At the cellular level, MSNs with smaller particle sizes were more readily internalized by cells, which initiated to more intense oxidative stress, therefor inducing higher cytotoxicity, and apoptosis rate. In vivo studies demonstrated that MSNs primarily accumulated in the liver and kidneys of mice. Pharmacokinetic analysis revealed that larger MSNs were eliminated more efficiently by the urinary system than smaller MSNs. The mice's body weight monitoring, blood tests, and pathological sections of major organs indicated good biocompatibility for MSNs of different sizes. Animal welfare and the animal experimental protocols were strictly consistent with related ethics regulations of Zhejiang Chinese Medical University. Overall, this study prepared MSNs with a particle size gradient to investigate the correlation between toxicity and particle size using macrophages and endothelial cells. The study also examined the biosafety of MSNs with different particle sizes in vivo and in vitro, which could help to improve the safety design strategy of MSNs for drug delivery systems.