Expression of decoy receptor 3 and its signaling pathway in ankylosing spondylitis and its clinical significance / 中华风湿病学杂志

Objective:To investigate the expression and clinical significance of decoy receptor 3 (DcR3) and its signal pathway-related molecules in PBMCs of patients with ankylosing spondylitis (AS).Methods:Peripheral blood samples, clinical data and laboratory test results were collected from 100 patients with ankylosing spondylitis [50 patients with AS activity (ASA), 50 patients with AS stability (ASS)], 30 patients with osteoarthritis and 30 patients with gouty arthritis (as disease control group), and 60 healthy controls (HC). The mRNA expression levels of DcR3 and its signal pathway related genes (DR3, TL1A, Fas, FasL, LIGHT, LIGHTR, LTβR) were measured by real-time fluorescence quantitative polymerase chain reaction. Measurement data among the three groups in normal distribution were analyzed by t test or one-way analysis of variance, pairwise comparisons using LSD- t test, non-normal distribution data were analyzed by Mann-Whitney test or Kruskal-Wallis H test, χ2 test was used for correlation analysis of categorical variables. Correlation analysis between variables were analyzed using Spearman correlation analysis. Results:① By comparing the AS group, disease control group and HC group, the expression levels of DcR3 mRNA and DR3 mRNA in the AS group were lower than those in disease control group and HC group, and DcR3 mRNA and DR3 mRNA in disease control group were lower than those in the HC group {DcR3mRNA: [6.21 (3.89, 10.70)]×10 -4vs [9.51 (5.89, 16.65)]×10 -4vs [17.81 (11.27, 24.20)]×10 -4, H=55.28, P<0.001; DR3 mRNA: [41.05 (24.09, 66.95)]×10 -4vs [58.28 (28.41, 94.38)]×10 -4vs [94.79 (54.07, 144.51)]×10 -4, H=37.10, P<0.001}. The expression level of TL1A mRNA in the AS group was higher than that in disease control group {[14.71(4.91, 42.22)]×10 -4vs [4.00(1.07, 16.60)]×10 -4vs [7.70 (3.52, 27.83)]×10 -4, H=17.71, P<0.001}; The expression level of Fas mRNA in AS group and disease control group was lower than that in HC group {[20.99(4.63, 62.89)]×10 -4vs [23.97(15.82, 38.99)]×10 -4vs [78.45 (27.32, 146.46)]×10 -4, H=31.17, P<0.001}. The expression level of FasL mRNA in AS group was higher than that in disease control group and HC group {[42.87(6.57, 91.21)]×10 -4vs [5.45(2.83, 10.32)]×10 -4vs [6.88 (4.57, 23.79)]×10 -4, H=46.42, P<0.001}. The expression level of LIGHTR mRNA in AS group was lower than that in disease control group {[52.66 (7.20, 143.21)]×10 -4vs [98.80 (53.11, 166.24)]×10 -4vs [63.47(40.85, 138.07)]×10 -4, H=11.96, P<0.001}. There were no significant differences in LIGHT mRNA and LTβR mRNA among all groups ( H=0.86, P>0.05; H=3.18, P>0.05). ②The expression levels of DcR3 mRNA, DR3 mRNA and Fas mRNA in ASA group and ASS group were lower than those in HC group. DcR3 mRNA in ASA group was higher than that in ASS group, and DR3 mRNA in ASA group was lower than that in ASS group {DcR3 mRNA: [7.28 (4.92, 16.56)]×10 -4vs [4.59 (2.49, 7.03)]×10 -4vs [17.81 (11.27, 24.20)]×10 -4, H=62.63, P<0.001; DR3 mRNA: [30.93(16.18, 66.66)]×10 -4vs [47.17(29.91, 67.40)]×10 -4vs [94.79(54.07, 144.51)]×10 -4, H=41.48, P<0.001; Fas mRNA: [20.04(3.29, 62.30)]×10 -4vs [22.49(5.63, 64.79)]×10 -4vs [78.45(27.32, 146.46)]×10 -4, H=23.54, P<0.001}. The expression levels of TL1A mRNA and LTβR mRNA in the ASA group were higher than those in the ASS group and the HC group {TL1A mRNA: [32.36(10.09, 97.84)]×10 -4vs [9.98(1.29, 21.63)]×10 -4vs [7.70(3.52,27.83)]×10 -4, H=21.14, P<0.001; LTβR mRNA: [6.13(2.16,20.06)×10 -4vs [2.13(0.53,8.04)]×10 -4vs [2.72 (1.24,5.73)]×10 -4, H=12.86, P<0.001}. The expression level of FasL mRNA in the ASA group and the ASS group was higher than that in the HC group {[60.70 (8.16, 106.16)]×10 -4vs [30.14 (5.37, 78.40)]×10 -4vs [6.88 (4.57, 23.79)]×10 -4, H=18.99, P<0.001}. The expression level of LIGHTR mRNA in ASS group was lower than that in HC group {[49.79(10.75, 168.48)]×10 -4vs [15.92(3.27, 105.91)]×10 -4vs [63.47(40.85, 138.07)]×10 -4, H=11.80, P<0.001]. There was no significant difference in LIGHT mRNA among all groups ( H=4.15, P>0.05). ③Spearman correlation analysis showed that DcR3 level was positively correlated with BASDAI score and hsCRP in AS patients ( r=0.52, P<0.001; r=0.35, P<0.01), and DR3 level was negatively correlated with BASDAI score, ESR and hsCRP level ( r=-0.28, P<0.001; r=-0.25, P<0.001; r=-0.31, P<0.001). TL1A was positively correlated with BASDAI score, ESR and hsCRP level ( r=0.23, P=0.046; r=0.26, P=0.015; r=0.25, P=0.017). Conclusion:DcR3 and its signal pathway-related molecules are differentially expressed in PBMCs of patients with AS, suggesting that they may participate in the occurrence and development of AS.

Studies on the differentiation of human urine derived stem cells into nucleus pulposus-like cells induced by human nucleus pulposus cell exosomes / 中华骨科杂志

Objective:To investigate the effects of exosomes of human nucleus pulposus cells (NPCs) on the differentiation of urine derived stem cells (USCs) into nucleus pulposus-like cells.Methods:USCs and NPCs were isolated and cultured in vitro. The exosomes of NPCs were extracted and detected by Western-blot. USCs cytoplasm was transfected with GFP lentivirus, while nucleus was transfected with DAPI dye. The NPCs exosomes were transfected with PKH26 dye. After co-incubation for 12 h, USCs and NPCs exosomes were observed by macroscopy. USCs differentiation was induced by NPCs exosomes and non-contact co-culture methods. The relative expression of marker gene mRNA of nucleus pulposus cells in each group and the absorbance at 450 nm wavelength were detected.Results:The isolated USCs had the ability to differentiate into osteocytes, adipocytes and chondrocytes with high expression of marker CD29 (99.57%), CD44 (97.46%) and CD73 (97.71%) and with low expression of negative proteins CD31 (0.59%) and CD45 (0.19%). The isolated NPCs highly expressed nuclear pulposus cell marker COL2A1, ACAN and SOX-9. The exosomes extracted from NPCs showed high expression of exosome marker CD63, CD81 and Tsg101. After 12 h co-incubation, NPCs exosomes fused with USCs membrane and appeared in the cytoplasm of USCs. At 3, 5 and 7 days of co-culture, the absorbance value of USCs cells in exosome group (0.44±0.004, 0.76±0.004, 0.82±0.006) was higher than that in co-culture group (0.39±0.022, 0.63±0.035, 0.69±0.012) ( P<0.05). The mRNA relative expression of USCs nucleus pulposus marker genes ACAN (1.80±0.31, 3.50±0.21, 5.35±0.31, 7.46±0.12), COL2A1 (1.43±0.15, 4.33±0.23, 6.89±0.22, 8.11±0.31), SOX-9 (2.21±0.13, 3.13±0.11, 3.96± 0.14, 4.52±0.26) and HIF-1α (1.45±0.16, 2.14±0.21, 4.31±0.41, 4.01±0.25) in exosomes group were significantly higher than those in the control group ( P<0.05) at the 3rd, 7th, 14th and 21st days. The mRNA relative expression of USCs nucleus pulposus marker genes ACAN (5.69±0.21, 6.69±0.13), COL2A1 (6.33±0.17, 7.89±0.15), SOX-9 (4.19±0.29, 4.38±0.12), HIF-1α (4.49±0.32, 4.96±0.26) in exosomes group were significantly higher than those ACAN (3.69±0.35, 5.13±0.23), COL2A1 (3.40±0.16, 6.79±0.19), SOX-9 (2.26±0.32, 3.69±0.26), HIF-1α (2.39±0.11, 3.96±0.13) in non-contact co-culture group ( P<0.05) at the 14th and 21st days. Conclusion:Human nucleus pulposus exosomes could induce differentiation of human USCs into nucleus pulposus-like cells in vitro. Compared with non-contact co-culture, exosomes have higher induction efficiency and can better maintain the proliferation activity of nucleus pulposus-like cells

Expression and clinical significance of interleukin-2/Janus kinase 3/signal transduction and transcriptional activator 5 in peripheral blood of patients with ankylosing spondylitis / 中华风湿病学杂志

Objective:To detect the expression of interleukin 2 (IL-2)/Janus kinase 3/signal transduction and transcriptional activator 5 (JAK3/STAT5) signaling pathway in peripheral blood of patients with ankylosing spondylitis (AS) and explore its mechanism in the development and progression of AS.Methods:Clinical data, peripheral blood and laboratory tests of 30 patients with active AS (ASA), 30 patients with stable AS (ASS) and 50 healthy subjects (HC) were collected. The mRNA expression levels of JAK3, signal transduction and transcription activator 5a (STAT5a) and signal transduction and transcription activator 5b (STAT5b) were detected by quantitative real-time-polymerase chain reaction (RT-qPCR). The expression levels of JAK3, STAT5a and STAT5b proteins and phosphorylated proteins were detected by Western-blot. Plasma IL-2 concentration was determined by enzyme-linked immunosorbent assay (ELISA). Two independent samples t-test or one-way analysis of variance were used for measurement data consistent with normal distribution, LSD- t test was used for pairwise comparison between the three groups, Mann-Whitney U test or Kruskal-Wallis H test was used for non-normal distribution, χ2 test was used for correlation analysis of categorical variables. Spearman correlation analysis was used for correlation analysis between variables, and receiver operating characteristic (ROC) curve was used to evaluate the value of JAK3, STAT5a and STAT5b mRNA expression levels in monitoring AS activity. Results:① The mRNA expression levels of JAK3, STAT5a and STAT5b were significantly different among the three groups ( F=65.98, P<0.001; F=21.15, P<0.001; F=13.67, P<0.001). JAK3 mRNA expression in ASA group (2.5±0.9) was significantly higher than that in ASS group (1.1±0.4) and healthy subjects (1.0±0.5), the difference was statistically significant (both P<0.001). The mRNA expression level of STAT5a in ASA group (1.4±0.3) was significantly higher than that in ASS group (0.9±0.3) and healthy subjects group (1.0±0.3), the difference was statistically significant (both P<0.001). STAT5b mRNA expression level in ASA group (1.5±0.6) was significantly higher than that in ASS group (1.0±0.4) and healthy subjects (1.0±0.4), the difference was statistically significant (both P<0.001). The expression level of JAK3 mRNA in HLA-B27 positive group (1.9±1.0) was higher than that in HLA-B27 negative group (1.4±0.6), and the difference was statistically significant ( t=-2.22, P=0.032). The phosphorylation levels of JAK3, STAT5a and STAT5b showed statistically significant differences among the three groups ( F=91.56, P<0.001; F=25.15, P< 0.001; F=178.59, P<0.001). The phosphorylation level of JAK3 protein in ASA group (1.04±0.08) was significantly higher than that in ASS group (0.568±0.019) and healthy subjects (0.536±0.064), the difference was statistically significant (both P<0.001). The phosphorylation level of STAT5a protein in ASA group (1.166±0.096) was significantly higher than that in ASS group (0.923±0.018) and healthy subjects (0.911±0.017), the difference was statistically significant (both P<0.001). The phosphorylation level of STAT5b protein in ASA group (0.81±0.05) was significantly higher than that in ASS group (0.21±0.03) and healthy subjects (0.24± 0.07), the difference was statistically significant (both P<0.001). The difference of plasma IL-2 concentration among the three groups was statistically significant ( F=3.32, P=0.040). The IL-2 concentration in the ASA group [(110±40) pg/ml] was significantly higher than that in the ASS group [(89±40) pg/ml] and the healthy group [(88±39) pg/ml], the difference was statistically significant ( P=0.044, P=0.016). ② Spearman correlation analysis showed that STAT5a mRNA expression level was positively correlated with platelets in AS patients ( r=0.353, P=0.006). JAK3 mRNA expression level in ASA group was positively correlated with IL-2 concentration ( r=0.766, P<0.001), and negatively correlated with estimated glomerular filtration rate ( r=-0.485, P=0.007). STAT5a mRNA expression level was positively correlated with erythrocyte sedimentation rate ( r= 0.680, P<0.001), and STAT5b mRNA expression level was positively correlated with hypersensitive C-reactive protein (CRP) ( r=0.823, P<0.001). ③ The ROC curve showed that JAK3 mRNA expression level predicted the area under ROC curve (AUC) of ASA with a 95% CI of 0.920 (0.853, 0.987), sensitivity and specificity of 86.7% and 90.0%, respectively. STAT5a mRNA expression level predicted the AUC 95% CI of ASA was 0.874 (0.787, 0.961), and the sensitivity and specificity were 96.7% and 66.7%, respectively. STAT5b mRNA expression level predicted the AUC 95% CI of ASA was 0.749 (0.617, 0.881), and the sensitivity and specificity were 73.3% and 80.0%, respectively. Conclusion:This study suggests that IL-2/JAK3/STAT5 may be involved in the pathogenesis of AS, and JAK3 mRNA can be used as a biological indicator to monitor the activity of AS disease.

Expression and clinical significance of Janus protein tyrosine kinase/signal transducer and activator of transcription signaling pathway in primary gouty arthritis / 中华风湿病学杂志

Objective:To explore the expression and clinical significance of Janus protein tyrosine kinase/signal transducer and activator of transcription (JAK3/STAT5) signaling pathway in peripheral blood mononuclear cells (PBMCs) of patients with primary gouty arthritis (GA).Methods:Peripheral blood, clinical data and laboratory tests were collected from 50 patients with acute gout (AG), 50 patients with intermittent gout (IG) and 50 healthy controls (HC). Quantitative real-time-polymerase chain reaction (RT-qPCR) was used to detect mRNA expression level of JAK3/STAT5 related genes (JAK3, STAT5a, STAT5b). Enzyme linked immune sorbent assay (ELISA) was used to detect interleukin-2 (IL-2) concentration in subject′s plasma. Measurement data among the three groups that was in accordance with normal distribution was analyzed by one-way analysis of variance, pairwise comparisons using LSD, non-normal distribution data was analyzed by Mann-Whitney test or Kruskal-Wallis H test, and correlation analysis between variables was analyzed using Spearman correlation analysis. Results:① The mRNA expression levels of JAK3, STAT5a and STAT5b were significantly different among the three groups ( F=50.13, P<0.01; F=7.573, P=0.000 7; F=12.14, P<0.01), of which the JAK3 mRNA expression level in the HC group [(606±65)×10 -4] was significantly higher than that in the AG group [(103±13)×10 -4] and IG group [(114±24)×10 -4], and the difference was statistically significant (both P<0.01), while the STAT5a mRNA expression level in the AG group [(89±9)×10 -4] was significantly higher than that in the IG group [(59±4)×10 -4] and HC group [(61±4)×10 -4], and the difference was statistically significant ( P=0.002, P=0.003 9), and the expression level of STAT5b mRNA in HC group [(60±5)×10 -4] was significantly lower than that in AG group [(95±7)×10 -4] and IG group [(98±7)×10 -4], and the difference was statistically significant ( P=0.000 2, P<0.000 1). ② The difference of IL-2 concentration in plasma among the three groups was statistically significant ( F=22.87, P<0.01), and the serum IL-2 concentration in the AG group [(87.9±8.4) pg/ml] was significantly higher than that in the IG group [(32±4) pg/ml] and HC group [(44±4) pg/ml], and the difference was statistically significant (both P<0.01). ③ Spearman correlation analysis showed that the mRNA expression of STAT5a and STAT5b was positively correlated with the absolute value of neutrophils in patients with gout ( r=0.282, P<0.05; r=0.257, P<0.05). Conclusion:The IL-2/JAK3/STAT5 signaling pathway is involved in the occurrence and development of gout, suggesting that this pathway may play a key role in the pathogenesis of gout.

Problem-based learning combined with case-based learning teaching mode in improving the teaching quality of antibacterial drugs for pharmacy interns / 中华医学教育探索杂志

Objective:To evaluate the effect of special teaching on anti-bacterial drugs that applied the problem-based learning combined with case-based learning.Method:s A total of 100 pharmaceutical students who participated in internships in Qujing First People's Hospital of Yunnan Province from 2018 to 2019 were included. According to the career plan after graduation, the students were divided into experimental group (to be engaged in clinical pharmacy) and control group (to be engaged in drug dispensing). There were 50 people in the experimental group, who received the problem-based learning combined with case-based learning teaching mode on the basis of traditional theoretical teaching (basic theoretical knowledge teaching). There were 50 people in the control group, who received only the traditional teaching mode. After the internship, the quality of teaching was assessed through examinations and questionnaires.Result:s There were no significant differences in the overall scores and individual scores of the objective questions ( P>0.05). The overall scores and individual scores of the case analysis of the experimental group were significantly higher than those of the control group ( P<0.05). The enthusiasm and initiative, and ability of clinical thinking, innovative practice, communication, team work of the experimental group were significantly better than those of the control group ( P<0.01). Conclusion:The problem-based learning combined with case-based learning teaching method is superior to the traditional theoretical teaching mode, which can significantly improve the teaching quality of antimicrobials for pharmacy interns. It is worth being promoted in teaching.

Establishment of Muti-channel Taqman-Probe Fluorescence Quantitative PCR Identification MRSA Method / 现代检验医学杂志

Objective To establish the method of identifying MRSA with Taqman-fluorescence quantitative PCR basing on mecA/nuc/fem B three gene combined detecting.Methods Taking the coagulase positive MRSA,which isolated from the clinical samples and confirmed by VITEK 2 compact microbial analyzer,as the research obj ect,designed mecA/nuc/fem B specific PCR primers and Taqman fluorescent probe by bio-software PrimerPremier 5 and Designer Beacon 7,FAM,HEX and ROX markers were used to label the fluorescent probe at 5’,and the end of 3’was labeled with BHQ1,detected by fluo-rescence quantitative PCR instrment.Results ①1 g/dl gel electrophoresis results showed that the primer’s specificity of mec A/nuc/fem B were good,and molecular weight of the amplification band consistent with the expected molecular weight and no non-specific amplification band.②Three genes were obtained specific amplification in a single tube single channel and single tube multiple channel detection in PCR,and the three gene amplification effect in a single tube single tube single chan-nel and multichannel PCR similar.Conclusion Successfully established a method of multi channel Taqman-probe fluores-cence quantitative PCR identification of MRSA,mec A/nuc/fem B combined detection can effectively differentiate coagulase negative and positive MRSA,improve the accuracy of identification.

Construction of targeted umbilical cord derived mesenchymal stem cells and their distribution in the mouse spleen / 中国比较医学杂志

Objective To construct lentiviral vectors containing peptide P1-GFP fusion genes.Umbilical cord derived mesenchymal stem cells were infected with lentivirus carrying peptide P1 and GFP fusion genes.To inject the targeted umbilical cord derived mesenchymal stem cells into mice and to detect GFP expression in the spleen.Methods Umbilical cord derived mesenchymal stem cells were cultured with adhered tissues of umbilical cord smaller than 1 mm3 . Lentiviral vector containing P1-GFP fusion genes with engineering technology was constructed and infected the umbilical cord derive mesenchymal stem cells.Targeted umbilical cord derived mesenchymal stem cells were intravenously injected in the mouse tail vein and after 18 hours GFP expression was detected with immunohistochamical staining of the spleen tissues.Results Harvested umbilical cord derived mesenchymal stem cells grew well in culture medium. Green fluorescence on umbilical cord derived mesenchymal stem cells were observed under fluorescence microscope at 18 hours after infected with lentivirus.Green fluorescence intensity of umbilical cord derived mesenchymal stem cells was increasing over time and reached a peak at 72 hours.Umbilical cord derived mesenchymal stem cells highly expressed CD105 (90.0%)/CD44 (98%) and CD73 (85.0%)/CD90 (98.5%) molecules.GFP expression was detected in the spleen after intravenous injection of targeted umbilical cord derived mesenchymal stem cells in the mice 18 hours later.GFP expressing cells intimately contacted with lymphocytes.Conclusions Targeted umbilical cord derived mesenchymal stem cells contain P1-GFP fusion genes are constructed.Targeted umbilical cord derived mesenchymal stem cells can be targeted to mouse spleen and intimately contact with lymphocytes after intravenous injection.Our results lay the groundwork for further studies.

Relationship between positive rate of EBV and HCMV testing with patients′age in 179 cases of leukemia / 国际检验医学杂志

Objective To understand the relationship between the positive rate of Epstain‐Barr virus (EBV) and human cyto‐megalovirus (HCMV) nucleic acid detection with the age in the patients with aplastic anemia ,acute lymphoblastic leukemia and my‐eloid leukemia .Methods The patients clinically diagnosed as aplastic anemia ,acute lymphoblastic leukemia ,and myeloid leukemia after bone marrow puncture in the Navy General Hospital from January 2012 to December 2013were collected as the research sub‐jects .the nucleic acid test kit from DAAN Gene Company was adopted for conducting the EBV and HCMV nucleic acid testing and analyzing the relationship between the positive rate of the EBV and HCMV infection with the patient′s age .Results There were different testing positive rate among 3 kinds of hematonosis .As a whole ,the positive rate of HCMV was lower than that of EBV (15 .8% vs .43 .7% ) .The EBV nucleic acid detection positive rate had statistically significant difference among different ages of pa‐tients with aplastic anemia and myeloid leukemia .(P< 0 .01) .Conclusion For children patients with aplastic anemia disease and myeloid leukemia ,more attention should be paid to monitoring of EBV and HCM V nucleic acid .

Complications and prophylaxis strategies of posterior hemivertebra resection for congenital scoliosis / 中华外科杂志

<p><b>OBJECTIVE</b>To evaluate the surgical outcomes and related complications of posterior hemivertebra resection with transpedicular instrumentation in the treatment of congenital scoliosis caused by fully-segmented non-incarcerated hemivertebra.</p><p><b>METHODS</b>From January 2003 to January 2012, one hundred and forty consecutive cases of congenital scoliosis treated by posterior hemivertebra resection with transpedicular instrumentation were investigated retrospectively. Radiographs were reviewed to determine the type and location of the hemivertebra, the coronal curve magnitude, sagittal alignment, compensatory cranial curve and compensatory caudal curve preoperatively, postoperatively and at the latest follow-up. Operative reports and patient charts were reviewed to record operation time, fusion level and complications.</p><p><b>RESULTS</b>One hundred and fifty-one posterior hemivertebra resections in 140 patients aged 2 to 45 years (average 10.8 years) with non-incarcerated hemivertebra were evaluated. All the patients were followed up from 3 to 119 months (average 25 months). The average fusion level was 5.0 segments (2-11 segments). There was a mean improvement of 71.3% in the segmental scoliosis from 42.5° before surgery to 10.6° at the time of the latest follow-up, and a mean improvement of 66.8% in segmental kyphosis from 29.5° to 7.2° at the same periods. There were 14 complications (13 patients), 3 pedicle fractures, 2 rod breakages, 2 pedicle elongation, 2 removed implants for prominent implants, 2 delayed wound healing, 2 additional surgeries for curve progression, 1 prolonged respiratory support. There was no neurological complication.</p><p><b>CONCLUSIONS</b>Posterior hemivertebra resection with transpedicular instrumentation is a safe and effective procedure for congenital scoliosis patients.Neurological complication is rare, but implant-related complication still remains a challenge.</p>

Preliminary results of osteotomy with short fusion and dual growing rod technique for congenital scoliosis / 中华骨科杂志

Objective To evaluate the clinical outcomes of the hybrid technique of posterior osteotomy with short segmental fusion and dual growing rod technique for severe rigid congenital scoliosis.Methods Seven patients (2males,5 females) undergoing this hybrid technique for severe rigid congenital scoliosis in our hospital from 2006 to 2011 were retrospectively studied.The average age was 5.9 years (range,2-10).The Risser sign of all the patients was 0.The follow-up time was 59.4 months (range,36-83 months).The patients' charts were reviewed.The analysis included age at initial surgery and the latest follow-up,number and frequency of lengthening,and complications.Radiographic evaluation included measured changes in scoliosis Cobb angle,thoracic kyphosis,lumbar lordosis,trunk shift,length of T1-S1 and instrumentation.Results All patients were treated with posterior osteotomy with short segmental fusion and dual growing rod technique.There were 48 total surgeries,41 of which were lengthening procedures,for 7 patients.The average lengthening was 5.9 per patient.The mean scoliosis improved from 81.4° to 40.1 ° after initial surgery and was 41.1 ° at the final follow-up.The average T1-S1 length was of 1.12 cm per year.The Campbell's space available for lung ratio increased from 0.87 to 0.97.Conclusion Osteotomy with short fusion could help to improve the correction of the growing rod and eliminate the large asymmetric growth potential around the apex,with little influence to the length of the spine.Dual growing rod technique could maintain correction achieved at initial surgery while allowing spinal growth to continue.However,this technique is relatively more aggressive and technique demanding.And the patients need numbers of surgeries.

Perioperative complications and risk factors of one-stage posterior vertebral column resection for severe spinal deformity / 中华骨科杂志

Objective To investigate the perioperative complications and risk factors of one-stage posterior vertebral column resection (VCR) for severe spinal deformity.Methods From September 2004 to July 2012,39 patients with severe and fixed spinal deformity underwent one-stage posterior VCR,including 15 males and 24 females,aged from 3 to 53 years (average,16.9 years).There were 24 cases of kyphoscoliosis (mean coronal Cobb angle:85.1°,mean sagittal Cobb angle:92.9°),7 cases of scoliosis (mean coronal Cobb angle:81.1°),and 8 cases of kyphosis (mean sagittal Cobb angle:94.4°).Eleven patients had neurological compromise.The perioperative complications and related risk factors of 39 patients were retrospectively analyzed.Results All patients were followed up for 3 to 72 months (average,29.4 months).There were 15cases (13 patients) of perioperative complications.Neurological complications occurred in 6 patients (15.4％),among whom one patient presented complete paraplegia after surgery and 5 patients presented transient paresthesia or muscle weakness.Adults had a significantly higher incidence of neurological complications than teenagers.The incidence rate of neurological complications was 36.4％ in patients with preoperative neurological compromise,while 7.1％ in patients without preoperative neurological compromise.All patients with postoperative neurological complications had kyphosis before operation,and the incidence of neurological complications increased significantly in patients with severe kyphosis (Cobb angle ≥90°).Prolonged respiratory support was conducted in 4 cases.Rupture of the parietal pleura occurred in 3 cases (7.7％),cerebrospinal fluid leak in 1 case (2.6％),and pulmonary infection in 1 case (2.6％).Conclusion One-stage posterior VCR is effective in the treatment of severe and rigid spinal deformity.However,the perioperative complications,especially the neurological complications are common.The risk factors of neurological complications include preoperative neurological compromise,degree of kyphosis and the age of patients.

Rational design, expression and biological activity assessment of a novel peptide based on ricin toxin antibody / 中华微生物学和免疫学杂志

Objective To design and express a novel peptide based on ricin toxin antibody in E. coli, and to evaluate its biological activity. Methods Based on the crystal structure of ricin toxin A chain (RTA) and the RTA-rRNA interact in the complex model, the steric conformation of RTA was theoretical modeled and its functional domain was preliminarily determined. The humanized single-domain RTA antibody was designed rationally by computer-guidod molecular design method. Its coding sequence was ob- tained by overlapping extension PCR, and cloned into the pET-32a vector. The fusion protein was then ex-pressed in E. coli BL21 (DE3), identified by Western blot, and purified with Ni-NTA agarose. The binding and neutralizing activity of this novel peptide for riein was evaluated by competitive ELlSA assay and MTT assay. Results A recombinant human single-domain antibody expressing a polypeptide against RTA in the CDR3 loop was designed. The fusion protein was successfully expressed in E. coll. The purified protein can bind to ricin, and neutralize its activity in SP2/0 viability assay. Conclusion The success of the novel pep-tide based on riein toxin antibody provides a novel method to develop new generation of ricin antagonists.

Pathogenicity of Vibrio fluvialis Isolated from Seacoast / 中华医院感染学杂志

OBJECTIVE To study the pathogenicity of the Vibrio fluvialis isolated from the coastal seawater.METHODS Virulence experiment group:22 Kunming mice were divided into four subgroups in random:V.fluvialis was injected into abdominal cavity in the test subgroup.And Staphylococcus aureus and Escherichia coli were injected into the positive control subgroups,separately and aseptic physiological saline was injected into the negative control group.Wound infection group:22 SPF mice were divided into four supgroups in random after their legs were injured:the experimental supgroup(soaked in artificial seawater with V.fluvialis);the positive control groups(with S.aureus and E.coli,separately);the negative control group(soaked in aseptic artificial seawater).The general condition,blood routine,blood culture,organ culture and wound secretion culture of the mice were observed.The pathological analysis of the mice was taken after sacrifice on the 3rd day.RESULTS In virulence experiment group,among all the 7 mice′s blood culture of V.fluvialis supgroup,5 mice were found V.fluvialis positive after 12 h injection,and 2 mice kept on positive until 24 h.In wound infection group,pathological examination showed there were a large number of neutrophils distributed over the striated muscle of the injured sites and cellulitis formed.CONCLUSIONS The V.fluvialis isolated from the sea water has pathogenicity,and can cause wound) infection and septicemia when the concentration reached 106 CFU/ml.

Value of anti-survivin in early diagnosis of malignant tumors / 第三军医大学学报

Objective To establish a soluble expression of recombinant survivin in E. coli, and evaluate the role of anti-survivin in tumor diagnosis. Methods The recombinant survivin was screened by ampicillin resistance, identified by PCR and double digestion of endonucleases. The sequenced DNA of survivin was analyzed by BLAST. The survivin/Trx fusion protein expressed in E.coli BL21 (DE3) was induced with IPTG, identified by Western blot and purified with Ni-NTA agarose respectively. The anti-survivin antibodies in serum were detected with an indirect ELISA. By the methods above, 144 serum samples from cancer patients and 300 serum samples from normal subjects were analyzed. Meanwhile, the joint detection of anti-survivin, AFP, CEA, CA199, CA125 in blood sera from cancer patients was detected. Results After PCR and double digestion with endonucleases, the survivin gene was inserted into the prokaryotic expression vector PET 32a(+). After DNA sequencing, the constructed expression plasmid was transformed into competent cells E.coli BL21(DE3). Western blot identified that the recombinant survivin/Trx fusion protein was specific against survivin antibody. The purity of the protein was over 96% after purified by Ni-NTA agarose. Anti-survivin was expressed in the sera of different cancer patients, but the positive rate varied. Conclusion Prokaryotic expression plasmid PET 32a(+)/survivin was successfully constructed and highly purified survivin/Trx fusion protein was obtained. The detection results of anti-survivin, AFP, CEA, CA199, CA125 in blood serum show in the diagnosis of tumors, the joint detection can overcome the shortfall of each gene and enhance the diagnostic rate.

New type recombinant antibody fragment scFv multimer and cancer targeting / 生物医学工程学杂志

New type recombinant antibody single chain variable fragment (scFv) is formed by the joined VH and VL domains of immunoglobulin with the used of a polypeptide linker that is at least 12 residues in length. scFv is the smallest functional unit of antibody and has shown a fine prospect for the radioimmunoscintigraphy of cancer because of its special characteristics including increased tumour penetration and fast clearance rates compared with parent Ig. A scFv molecule with a linker of 3-12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (Diabody). Reducing the linker length below three residues can force scFv association into trimers (Triabody) or tetramers (Tetrabody) depending on linker length, composition and V-domain orientation. This review describes linker length and V-domain orientation of scFv, expression and stability of scFv multimers, size of scFv multimers and its effect on in vivo pharmacokinetics, flexibility and avidity of scFv multimers, in vitro application of multimeric murine scFv, multispecific scFv multimers and cancer targeting.

Immune responses of dendritic cells after loaded with cytotoxicity T lymphocyte epitope based peptide of human alpha-fetoprotein (hAFP) / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the immune responses of lymphocytes after activated by dendritic cells (DCs) loaded with cytotoxicity T lymphocyte (CTL) epitope based peptide of human alpha-fetoprotein (hAFP, 218-226 LLNQHACAV).</p><p><b>METHODS</b>Get high purity DCs by cultured plastic-adherent monocytes isolated from healthy donor of HLA-A2(+) peripheral blood with granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days. Stimulate self-lymphocytes with DCs that loaded with CTL epitope based peptide of hAFP under the culture medium contains interleukin-2 (IL-2) for 7 days. Analyse IL-12 and TNF in culture medium and also the specific lysis activity of lymphocytes against four strains of primary hepatocellular carcinoma cells.</p><p><b>RESULTS</b>After stimulated by DC loaded with CTL epitope based peptide derived from hAFP, lymphocytes appeared a good characteristics and the culture medium of activated lymphocytes contained a high level Th1 type cytokines of IL-12 and TNF. Activated lymphocytes not only specifically lysed HLA-A2(+) HepG2 line but also had the cytotoxicity against other three primary hepatocellular carcinoma cell lines and T2 target cell loaded with peptide of hAFP.</p><p><b>CONCLUSIONS</b>The results of this research supply the basic materials for the DC based vaccine with HLA-A2 restricted peptide epitope derived from hAFP against AFP positive primary hepatocellular carcinoma.</p>

Generation of CD14+ dendritic cells in vitro with GM-CSF and IL-4 / 生物医学工程学杂志

This study was conducted to get high quality and sufficient numbers of mature dendritic cells from healthy donor peripheral blood. The experiment began on culturing of plastic-adherent monocytes isolated from healthy donor peripheral blood with granulocyte-monocyte clony-stimulating factor (GM-CSF 150 ng/ml) and interleukin 4 (IL-4 800 U/ml) without fresh medium feeding and cytokines for 7 days. After 7 days, CD14+ monocytes not only differentiated into high purity DC but also expressed HLA-I and HLA-II molecules, costimulating molecules, adherent molecules and its progenitor marker CD14 molecule highly. These cells displayed all phenotypic and morphologic characteristics of mature dendritic cells and were most potent stimulatory cells in allogeneic mixed leukocyte reactions. The endocytosis ability of these DCs peaked at the third day in culture and decreased remarkably afterwards. These results provide evidence for the first time that CD14+ monocytes differentiated in vitro from peripheral blood monocytes exhibit dendritic cells characteristics and still express its progenitor marker CD14 molecules highly. The results of this experiments may facilitate further studies of CD14+ DC and its clinical applications.

Obtained DC from PBMC recovered from one time used no leukocytes transfusion apparatus / 中国免疫学杂志

Objective:Got high purity dendritic cells from PBMC recovered from one time used no leukocytes transfusion apparatus.Methods:Rinsed PBMC with natural salt water from one time used no leukocytes transfusion apparatus and induced PBMC with GM-CSF and IL-4.Results:Got 5.1?10 7 PBMC from every one time used no leukocytes transfusion apparatus and 4.7?10 6 DCs with a purity of 96.9% were obtained.These DCs express CD86(96.1%)?CD40(88.9%)?CD83(91.6%)?HLA-DR(92.9%)?HLA-ABC(99%)?CD54(97.5%) highly and a few number of these DCs could stimulate proliferation of allogeneic mixed leukocyte reactions strongly.Conclusion:The results of this research supplied basic data for further research of DC and its clinical application.