Objective To establish a flow cytometry-based assay for the detection of monocyte-me-diated antibody-dependent cell-mediated cytotoxicity ( ADCC ) .Methods P815 cells double stained with PKH26 and carboxyfluorescein succinimidyl ester ( CFSE ) were used as target cells and coated with P 815 specific antibodies to form antigen-antibody complexes .The peripheral blood mononuclear cells were isolated as effector cells and co-cultured with the antigen-antibody complexes .The CD3-CD14+PKH26+CFSE-cell population were gated by flow cytometry .Optimized effector/target cell ratio and incubation time for killing assay were identified .Monocyte-mediated ADCC in 23 patients with chronic HCV infection and 22 healthy subjects were analyzed .Results The monocyte-mediated ADCC could be evaluated through analyzing the CD3-CD14+PKH26+CFSE-cells with flow cytometry .The optimized effector/target cell ratio was 10 ∶1 and the optimized time for incubation was 4 h.Monocyte-mediated ADCC was inhibited in patients with chronic HCV infection as compared with healthy subjects (P=0.009).Conclusion A flow cytometry-based assay for the detection of monocyte-mediated ADCC was established , which could be used as a fast , sensitive and safety method for the evaluation of monocyte-mediated ADCC during viral infections and the research and de-velopment of drugs .

Objective To explore the effects of pretreatment of bone marrow mesenchymal stem cells (BMSCs) by ultrasound-exposed microbubbles (UM) on both homing to ischemic myocardium and cardiac function after acute myocardial infarction (AMI).Methods Rats of AMI model established by ligation of left anterior descending coronary artery were divided into four groups randomized:phospho-buffered saline (PBS) group,stem cells treatment (SCT) group,ultrasound and stem cells treatment (USCT) group,and UM stem cells treatment (UMSCT) group,and each group was injected with PBS,stem cells,US-pretreated stem cells and UM-pretreated stem cells through the caudal veins after AMI respectively.Homing of BMSCs to the ischemic myocardium was examined by confocal microscopy at 48 h after implantation,and cardiac function was examined by ultrasonic cardiogram (UCG) after 4 weeks.Masson staining was used to examine the changes of local ischemic cardiac tissues,and immunohistochemistry was used to detect the density of local neo-capillaries (CD31).Results 1) The numbers of CM-Dil-positive cells counted under confocal microscopy in the ischemic myocardial tissues of each groups 48 hours after implantation were not the same:there was no significant difference of the numbers of positive cells between USCT group (19.67 ±2.08) and SCT group (18.67 ± 2.08).However,the number of positive cells in the UMSCT group (39.33 ±3.06) was larger than that in USCT group and SCT group (P ＜0.05).2) UCG examinations showed that there was no significant difference of left ventricular systole function between the USCT group [LVEF (44.92 ± 2.77)％,LVFS (22.83 ± 1.79)％] and SCT group [LVEF (42.28 ± 2.82)％,LVFS (21.52 ±1.88) ％,P ＞0.05],but both were better than that in PBS group [LVEF (20.52 ± 1.88)％,LVFS (9.55 ±0.85) ％,P ＜0.05].The left ventricular systolic function in UMSCT group [LVEF (61.85 ± 3.15)％,LVFS (32.74± 2.45)％] was significantly higher than that in USCT group and SCT group (P ＜0.05),while which was still significantly lower than that in pseudo-surgery group [LVEF (75.88± 4.52)％,LVFS (42.76 ± 2.88)％,P ＜0.05].3) Pathological examinations showed the percentages of AMI areas in the USCT group (35.9 ± 1.1％) were not different compared with that in SCT group [(36.5 ± 1.3)％,P ＞0.05],while both were significantly smaller than that in PBS group [(45.2± 1.4)％,P ＜0.05].The percentages of AMI areas in the UMSCT group [(25.8 ± 1.0)％] were significantly smaller than that in USCT group and SCT group (P ＜0.05).The density of neo-capillaries (25.9 ± 1.3) in USCT groups had no difference compared with that in SCT group (25.2 ± 1.3),while both were significantly higher than that in PBS group (17.6 ± 1.1,P ＜0.05);the density of neo-capillaries (33.2 ± 1.6) was significantly higher in UMSCT group than that in both USCT group and SCT group (P ＜0.05),which were examined by immunohistochemistry.Conclusions Homing to ischemic myocardium of BMSCs transplanted intravenously could be promoted by UM pretreatment,which stimulates development of capillaries,reduces AMI areas,and improves the cardiac function after AMI.

10.3969/j.issn.1008-9691.2013.03.011

Objective To explore the effects of ultrasound-exposed microbubbles (UM) on the expression of CXC chemokine receptor 4 (CXCR4) in bone marrow mesenchymal stem cells (BMSCs) and the mechanisms involved.Methods Mesenchymal stem cells were isolated from bone marrow taken from male Sprague-Dawley rats.They were divided into a control group,an ultrasound (US) group,an ultrasound-exposed microbubbles (UM) group,a UM plus catalase (UMC) group,a UM plus AMD3100 (UMA) group,and a UM plus anti-CXCR4 antibody (UMCX) group.The control group was not given any treatment.The US group was treated with 1 MHz ultrasound at 1 Watt per square centimetre for 30 seconds.The UM group was treated with ultrasound plus microbubbles.The UMC group was treated with catalase,microbubbles and ultrasound.The UMA group was treated with AMD3100,microbubbles and ultrasound.The UMCX group was treated with anti-CXCR4 antibody,microbubbles and ultrasound.Quantitative polymerase chain reaction (qPCR) and Western blotting were performed to determine the levels of CXCR4 mRNA transcription and the expression of BMSCs in the control,US,UM and UMC groups.Immediately,5 minutes and 15 minutes after the intervention,fluorescence intensities were observed in the cells labeled with Fluo-4/AM of the control group,US group and UM group under a fluorescence microscope.Migration assays were conducted to determine the chemotactic ability of the BMSCs with respect to stromal-derived factor-1α (SDF-1α) in all six groups.Results No significant differences were found in the levels of CXCR4 mRNA transcription and protein expression between the US and control groups(P＞0.05),but the levels in those groups and the UMC group were lower than those observed in the UM group.Fluorescence intensity in the cells of the US group was not significantly different from that in the control group (P＞0.05),but those levels were both significantly lower than that in the UM group (P＜0.05).There was no significant difference in the number of cells migrating to the SDF-1α between the US (22.4±2.2) and control group (20.5±2.3).However,the number of cells migrating to SDF-1α in the UM group (53.1±3.8) was significantly larger than that in the US group,the control group,the UMC group (35.2+3.1),the UMA group (32.5±2.8) and the UMCX group (30.7+2.9) (P＜ 0.05).Conclusion UM can increase mRNA transcription and the expression of CXCR4 protein in BMSCs,and promote BMSCs migration to SDF-lα.This may in part be mediated by an increase in calcium influx.

Objective To evaluate the analytical performance of a novel HBV DNA assay based on automated DNA extraction and real-time fluorescence quantitative PCR .Methods Analytic verification studies.Accuracy and lower limit of detection were assessed by determining a panel of HBV standard plasma of WHO.HBV standard plasma (genotype A, B, C and D) at 6 different concentrations were measured 18 times to evaluate precision and reproducibility .Pseudo-viral particles at high HBV DNA concentration were serially diluted to assess linear range .One hundred and forty-four clinical specimens were quantified for HBV DNA so as to evaluate the correlation between the new test and COBAS ? system. Results Quantification of HBV standard plasma showed acceptable accuracy , with each deviation between observed and expected values within ±0.35 lg IU/ml (-0.17-0.32 lg IU/ml).Intra-assay coefficients of variation ( CV) for genotype A , B, C and D were 3.87% -6.32%, 0.45% -14.68%, 0.16% -8.36% and 0.64%-13.01%respectively, and the inter-assay CV were 5.67%-9.69%, 1.28%-15.68%, 0.36%-9.05%and 1.69%-13.65%, separately.Linearity assessment exhibited an excellent dynamic range of linear quantification from 20 to 1.0 ×1010 IU/ml ( r =0.998, P <0.001 ) .And the satisfactory results obtained at 3 levels of HBV DNA concentration (10, 20, 50 IU/ml, respectively) confirmed the claimed lower limit of detection with 5/5 detectable rate at 20 IU/ml.Furthermore, good correspondence was observed between the new HBV DNA assay and the COBAS ? system with 100% ( 144/144 ) qualitative coincidence and significant correlation based on 104 positive data ( r=0.984, P<0.000 1).Conclusions The novel fully-automated real-time PCR assay displayed good analytical and clinical performance for highly sensitive detection of HBV DNA.It was well suited for monitoring antiviral responses as well as drug resistance according to current clinical practice guidelines for the management of chronic HBV infection .

OBJECTIVETo analyze the risk factors of neurological complications of posterior vertebral column resection in the treatment of severe rigid congenital spinal deformities.

METHODSThe clinical data of 88 patients with severe rigid congenital spinal deformities who underwent PVCR in Department Of Orthopaedics, Xijing Hospital, Fourth Military Medical University from June 2007 to November 2012 were collected. There were 39 males and 49 females at the average age of 16.9 years (range 6-46 years). To measure the Cobb angle and balance at preoperative, postoperative and follow up, and to record the operation report, neurological complications and at follow up. The relevant factors of neurological complications were analyzed by one-way analysis, including: age, Cobb angle, operation time, body mass index, pulmonary function, blood volume loss, resection level, number of vertebrae fixed, number of vertebrae resected, usage of cage or titanium mesh, preoperative neurologic function, the type of deformity and combination of spinal canal deformity, and further analyzed by multiariable Logistic regression analysis.

RESULTSThe average follow up was 42 months (range 19 to 83 months). The number of resected vertebrae average 1.3 (range 1 to 3), operative time average 502.4 min (range 165.0 to 880.0 min), estimate blood loss average 2,238 ml (range 100 to 11,500 ml) for an average 69.3% blood volume loss (range 9% to 299%). The average preoperative major coronal curve of 93.6° corrected to 22.2°, at the final follow-up, the coronal curve was 22.2° with a correction of 76.8%. The average preoperative coronal imbalance (absolute value) was 2.5 cm decreasing to 1.3 cm at the final follow-up. The average preoperative major sagittal curve of 88.2° corrected to 28.7°, at the final follow-up, the sagittal curve was 29.2°, average decrease in kyphosis of 59.0°. The average preoperative sagittal imbalance (absolute value) was 3.1 cm decreasing to 1.2 cm at the final follow-up. There were 12 patients (13.6%) developed a neurological complications. High rate of neurological complications was occurred in patients with operative time greater than 480 min, pulmonary dysfunction, blood volume loss greater than 50%, T7-T99 osteotomy and preoperative neurologic compromise (P=0.046, 0.000, 0.000, 0.033, 0.043).

CONCLUSIONSPosterior vertebral column resection can achieve satisfactory efficacy in treatment of severe spinal deformities. Pulmonary dysfunction and blood volume loss greater than 50% were significant risk factors of neurological complications.

Adolescent ; Adult ; Child ; Female ; Humans ; Kyphosis ; Male ; Middle Aged ; Neurosurgical Procedures ; Orthopedic Procedures ; Osteotomy ; Retrospective Studies ; Risk Factors ; Scoliosis ; Spinal Canal ; Spinal Diseases ; surgery ; Spine ; abnormalities ; surgery ; Treatment Outcome ; Young Adult