Objective: To analyze the risk factors of varicella zoster virus(VZV) infection after allogeneic hematopoietic stem cell transplantation(allo-HSCT) in children, and to provide reference for the diagnosis, monitoring and prophylaxis of VZV infection after allo-HSCT. Methods: A total of 367 patients, who underwent allo-HSCT in Pediatric Transplantation Center of Nanfang Hospital Affiliated to Southern Medical University from January 2012 to June 2015 were collected.Clinical characteristics and risk factors of the patients complicated with VZV after allo-HSCT were retrospectively analyzed. Results: Thirty-four patients (9.26%) were complicated with VZV infection after allo-HSCT.The median onset time was 96.5 d(19-326 d). Two of 34 patients relapsed 3 times, 1 case of them relapsed twice, 3 cases of them relapsed once, and 4 cases of 34 patients were complicated with VZV encephalitis.All cases were treated with antivirus drugs, infusion of immunoglobulin, reduction of immunosuppressant dosages, with external use of Acyclovir ointment.The median therapy time was 13 days (7-28 days). All of their herpes subsided, and neurological symptoms such as headache, vomiting and convulsion disappeared.VZV-DNA both in blood and cerebrospinal fluid turned negative.No patient had herpetic dissemination and visceral involvement, and no one died directly of VZV infection.Results indicated that age(χ²=6.863, P=0.009), underlying disease(χ²=14.793, P=0.022), type of HSCT(χ²=14.459, P=0.001) and resource of stem cell (χ²=20.585, P=0.002) were significant risk factors for VZV infection after allo-HSCT, while sex (χ²=0.106, P=0.745) and antithymocyte globulin in conditioning regimen(χ²=0.010, P=0.921) had no relation to it. Conclusions VZV infection mainly occur within 12 months after allo-HSCT and is prone to be complicated with VZV encephalitis.The effect of Acyclovir is good.Monitoring and prophylaxis of VZV infection after allo-HSCT should be strengthened in children with high risk factors.

Objective To observe the expression changes ofmiR-34a and its related target genes sirtuin 1 (Sirtl) and nuclear factor-E2-related factor 2 (Nrf2) in subependymal zone (SVZ) of rats after cerebral ischemia,and to explore the effect of miR-34a on proliferation of endogenous neural stem cells (NSCs) after cerebral ischemia.Methods Forty SD rats were randomly divided into control group,cerebral ischemia one d group,cerebral ischemia three d group and cerebral ischemia 7 d group (n=10).Middle cerebral artery occlusion models were established by thread embolization in the latter three groups,and the SVZs were taken one,three and 7 d after model making,respectively.Immunofluorescence staining was used to detect the expression of nestin,real-time PCR was used to detect the mRNA expressions ofmiR-34a,Sirtl and Nrf2,and Western blotting was used to detect the protein expressions of Sirt1 and Nrf2.Results (1) In SVZs of cerebral ischemia one d group,cerebral ischemia three d group and cerebral ischemia 7 d group,the number of nestin positive cells increased gradually ([2.013±0.526],[4.821 ±1.154],and [6.394±1.027]) cells/filed,with statistical differences (P＜0.05).(2) In SVZs of cerebral ischemia one d group,cerebral ischemia three d group and cerebral ischemia 7 d group,the expression levels of miR-34a mRNA increased gradually (0.295±0.145,0.701 ±0.075,and 1.136±0.018),the Sirt1 mRNA expression levels showed a downward trend (0.835±0.024,0.620±0.133,and 0.278±0.110),the Nrf2 mRNA expression levels obviously decreased (1.032±0.256,0.833±0.187,and 0.630±0.123),the Sirt1 protein expression levels showed a downward trend (0.832±0.018,0.731±0.156,and 0.454±0.132),and the Nrf2 protein expression levels obviously decreased (1.003±0.133,0.813±0.111,and 0.671 ±0.071),with statistically significant differences (P＜0.05).(3)Statistical correlation analysis showed that the number of nestin positive cells was positively correlated with miR-34a mRNA expression level and negatively correlated with Sirt1 and Nrf2 mRNA and protein expression levels after cerebral ischemia (r=0.887,P=0.003;r=-0.746,P=0.005;r=-0.521,P=0.013;r=-0.344,P=0.025;r=-0.863,P=0.000).Conclusions MiR-34a may regulate the proliferation of NSCs through Sirt1/Nrf2 pathway after cerebral ischemia.

Objective CRISPR/Cas9 genome-editing technique provides an novel method for whole genome editing in eukaryotic cells.Recently,we found that gene subtype library with smaller size and focused pur-pose is more economical and practical. In this study,we aimed to target kinases,a group of pivotal cell signal transducers,to construct a kinase knock-out library using CRISPR/Cas9 technique.The construction strategy wll al-so be discussed. Methods 10 sgRNA was designed for each kinase target.After oligo pool synthesis by semicon-ductor chip,the oligos were eluted from the chip. The oligo templates were amplified and cloned into Cas9 vector and transformed into Stble3 competent cells.Monoclonal colonies were selected for DNA sequencing. Results(1) GO analysis of 507 cell kinases showed that the cell kinases took part in a wide range of cell signaling.(2)The sgRNA pool with about 140 bp in length was successfully amplified by using oligo pool as the template and univer-sal PCR primers.(3)In 40 identified library clones,34 clones were sequenced successfully. Among them,the DNA sequencing results of 25 samples were completely consistent with the designed target sequences.But there are some mutations in the primers of 9 samples.Failure in bacteria shaking,DNA sequencing and other factors were ex-isted in the other clones. Conclusion The CRISPR/Cas9 kinase knock-out library can be widely used for screen-ing the important kinases which may mediate cell proliferation,metastasis,drug resistance and autophagy.This li-brary will play an important role in clarifying the development of disease associated with kinases.

Objective To explore the relationship between prevalence of Kashin-Beck disease (KBD) and ecological environment, and to broaden the perspective of KBD etiology. Methods In 37 counties of KBD areas in Gansu Province, information about the ecological environment and implementation situation of control measures (altitude, temperature, rainfall, evapo ration, frost free period, annual sunshine hours, population density, per capita income, the proportion of staple food, returning farmland to forest, to forestry and replant crops) and X-ray detection rate of KBD of 7-12-year-old children in 2012 - 2014 was collected. Using four quantile regression method, the regression model was introduced to analyze the 11 ecological factors which related to the pathogenesis of KBD. The effect of three points on X-ray detection rate of KBD was estimated. Results The X-ray detection rate of KBD was independent of altitude, temperature, evaporation, population density, per capita income and cash crops, and was dependent of rainfall, frost free period, annual sunshine hours, the staple food purchase ratio, and returning farmland to forest and grassland. No matter where in any place numbered, the higher rainfall (measure value:0.003 3 to 0.006 4), the longer frost free period (measure value: 0.029 2 to 0.043 8), the longer annual sunshine hours (measure value:0.001 6 to 0.001 8), and the higher staple food purchase ratio (measure value:0.019 7 to 0.027 6), the higher risk of X-ray detection rate of KBD; the higher returning farmland to forest and to grassland, the lower risk of X-ray detection rate of KBD (measure value: - 0.037 2 to - 0.013 3). Conclusion The X-ray detection rate of KBD is closely related to local ecological environment.

Objective To grasp the epidemic features of Kaschin-Beck disease in different ecotypic areas in Gansu Province,in order to provide ecological basis for Kashin-Beck disease control.Methods Totally 37 counties with Kashin-Beck disease were divided into four ecological areas,villages with historical serious condition in township and townships with historical serious condition in county were investigated.Clinical examination and Xray of right hand of all 7-12-year-old children in the villages were carried out,while investigation of prevention and control measures was performed.Results Among 27 966 children from four ecological areas,the clinical detection rate was 0.05％ (14/27 966) and the X-ray positive rate was 1.26％ (353/27 966),metaphyseal rate was 1.25％ (350/27 966),bone-side positive rate was 0.01％ (3/27 966) and no case of epiphyseal and carpal were discovered.The clinical detection rate in children in the four ecological areas was not statistically significant (x2() =7.757,P ＞0.05),the Loess Plateau-gully region of Longdong [0.09％ (10/11 604)] ＞ the Anyon area of Qinling of Longnan [0.04％ (3/7 969)] ＞ Alpine grassland meadow area of Gannan [0.02％ (1/4 021)] ＞ the Loess Plateau-hills region of Longzhong [0 (0/4 372)].The detected rate of X-ray in four ecological areas was statistically significant (x2 =18.133,P ＜ 0.05),the Anyon area of Qinling of Longnan [1.49％ (119/7 969)] ＞ the Loess Plateau-gully region of Longdong [1.41％ (164/11 604)] ＞ the Loess Plateau-hills region of Longzhong [0.89％ (39/4 372)] ＞ Alpine grassland meadow area of Gannan [0.77％ (31/4 021)].Comprehensive prevention and control measures on Kaschin-Beck disease were different in different ecotypic areas.Relocation,long-distance education and selenium supplement measures had not been implemented in the four ecological areas.Conclusion The epidemic situation of Kaschin-Beck disease is different in different ecotypic areas in Gansu Province,which maybe related to ecological environment,but is under a state of control.

Objective To evaluate the correlation between osteophytes size and lower limb alignment in the knees of patients with Kaschin-Beck disease (KBD).Methods A total of 300 clinically diagnosed patients with KBD were X-rayed on knee-joints which ranged from the distal half of femur to proximal half of tibia.Meanwhile some related parameters in the X film with the anteroposterior position (including osteophytes length,femorotibial angle,femorotibial joint space ratio of inner side to outer side) were measured by DICOM 2.0,a software of medical graphic measuring,then followed by calculating the osteophyte spur index.The association between femorotibial angle,femorotibial joint space ratio and osteophytes spur index was evaluated by Pearson correlation test.Results The average of femorotibial angle of all the tested knee-joints was (165.97 ± 4.02)°,which positively correlated with both the osteophyte spur index of the medial femoral condyle [(6.54 ± 3.12)％,correlation coefficient (r) =0.524,P＜0.01] and the osteophyte spur index of medial tibil plateau [(7.14 ± 3.40)％,r =0.578,P ＜0.01].The femorotibial joint space ratio was 0.61 ± 0.13,which positively correlated with both the osteophyte spur index of medial femoral condyle (r =0.531,P ＜0.01) and that of the medial tibil plateau (r =0.563,P ＜0.01).Conclusions The results of this study indicate that there is a positive correlation between lower limb alignment and osteophyte size of both the medial femoral condyle and the medial tibial plateau.This finding may be evidenced by the fact that the changes of lower limb biomechanics may contribute to formation and development of osteophytes in the kneejoint.

Objective To observe the condition of Keshan disease (KSD) in Gansu Province in order to provide a scientific basis for prevention and control of the disease. Methods In 2011, according to “The national Keshan Disease Monitoring Program (trial edition)”, based on searching of KSD cases, 12 villages of 12 towns in 6 counties of Gansu Province, were selected as surveillance sites. All the residents in surveillance sites were clinically examined and given 12-lead ECG tracings; suspected cases were taken anterior chest X-rays in the distance of 2 meters, while staple food and life conditions of the residents were surveyed. Diagnosis of KSD was based on“Keshan Disease Diagnostic Criteria”(WS/T 210-2011). Results Among investigated 5 484 residents, the incidence of abnormal electrocardiogram was 15.41%. Of the 196 X-ray films, 61 cases had enlarged heart(in which 22 of mild, 21 of moderate and 18 of severe). Two hundred and forty-three cases of KSD were detected, the general detection rate was 4.43%, in which 31 cases of chronic, the detection rate was 0.56%; 212 cases of potential, the detection rate was 3.87%. In all cases, 47 cases were under the age of 30, including 46 cases of potential and 1 case of chronic. The major abnormal electrocardiogram change of KSD cases was ST-T changes[22.68%(71/313)], followed by complete right bundle branch block[16.29%(51/313)], low voltage[12.46%(39/313)], and left anterior fascicular block[6.71%(21/313)]. Per-capita annual income in surveillance site was 1 763 Yuan;and the major staple food was flour, accounted for 87%, and the staple food rice accounted for 5%. Conclusion The detection rate of KSD in Gansu Province is mainly to potential, and there is a cosiderable portion of patients under the age of 30;monitoring and investigation should also be strengthened in the younger age of KSD cases detected village.

OBJECTIVETo prepare a monoclonal antibody (mAb) against the fusion protein preM/EIII of West Nile virus (WNV) for clinical detection of WNV.

METHODSSp2/0 cells were fused with the spleen cells of BALB/c mice immunized with the recombinant fusion protein preM/EIII expressed in E. coil to obtain the hybridoma cell line that secreted preM/EIII mAb. The hybridoma cells were injected into the peritoneal cavity of BALB/c mice and the ascites was collected and purified. The specificity and titer of the obtained mAb were determined using ELISA and Western blotting.

RESULTSOne hybridoma cell line secreting preM/EIII mAb, named ME1, was obtained. The titer of the purified mAb was 10(-6). Identified as a mAb of the Ig subclass IgG1, ME1 was capable of specific reactions with preM/EIII protein and WNV without cross-reactions with other viruses such as JEV, SLEV, YFV and DENV. The accuracy of clinical testing of MNV with ME1 was 97.78%.

CONCLUSIONThe mAb against preM/EIII obtained have a high specificity and accuracy in clinical testing of MNV and can be used in clinical diagnosis of MNV infection.

Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Cross Reactions ; Hybridomas ; immunology ; Mice ; Mice, Inbred BALB C ; Viral Fusion Proteins ; immunology ; West Nile virus ; immunology

Objective To explore the diagnostic value of GP-/3 protein in gene detection in the patient of primary hepatic carcinoma, to discuss the joint roles of serum GP73 and AFP, and provide a novel method for the diagnosis for PHC and screening for high-risk population. Methods ELISA was used to detect the serum level of GP73 and AFP in 73 cases of PHC, 13 cases of hepatic cirrhosis, 32 cases of hepatitis and 62 cases of health people. SYBR Green real time fluorescence quantitative PCR was used to detect the relative value of GP73 mRNA in the peripheral blood cells of each group. Comparative Ct method was used to evaluate the relative expression levels. Eight cases of normal liver tissues and 8 cases of PHC tissues were detected at the same time to compare the relative expression levels. Results Kruskal-Wallis test showed that the serum levels of GP73 and AFP had significant differences between four groups(H value were 89. 6 and 52.0, P ＜ 0. 01) and the whole blood GP73 mRNA had no significant differences(H =4. 33, P ＞ 0. 05). Mann-Whitney test showed that the serum levels of GP73 had significant differences among PHC groups[166. 7 (162. 7-231.8) μg/L] and liver cirrhosis[57. 3 (46. 6-113. 6) μg/L], hepatitis[29. 6(26. 2-54. 5) μg/L], health group[25.1 (20. 8-29. 4) μg/L] (U value were 246, 297, 349, P ＜ 0. 01).The A FP levels of the four groups were 380. 9 (258.5-503.2) μg/L, 3.8 (1.3-14. 5) μg/L, 5. 1 (2. 4-7. 8)μg/L and 2. 8(2. 2-5.7) μg/L. It also showed significant differences (U value were 246,419 and 790,P ＜0. 01). The GP73 mRNA expression of PHC liver tissues(12. 64) was significant higher than normal liver tissues (1.00). The critical values for GP73 and AFP was determined to be 123. 2 μg/L and 10. 6 μg/L through the 8OC curves. Under the critical value the sensitivity of GP73 and AFP were 65.8% and 53.4% ,and the specificity of CP73 and AFP were 95.3% and 92. 5% respectively. Joint detection could increase the sensitivity up to 79. 5%, and achieve the high specificity of 90. 7%. Conclusions As a new diagnositic marker of primary hepatic carcinoma, GP73 protein has the very good sensitivity and specificity. The GP73 mRNA in the whole blood sample could not be used for the diagnosis of PHC. But it woule be a good molecular marker for diagnosis of PHC in the liver tissue sample. The joint detection of GP73 and AFP could improve PHC diagnostic performance, and provide an effective approcach to the PHC high-risk screening.

Objective To investigate the distribution of high pathogenicity island(HPI)in multiple-drug-resistance gram-negative bacilli and analyze the protein sequence.Methods To amplify thefyuA-irp2 gene cluster of the 84 isolates by multiple polymerase chain reaction(PCR),the product was subsequently sequenced.Results The positive rate ofirpl,irp2,irp3,irp4 and fyuA was 40.48％,41,67％,5.95％,O％and 16.67％,respectively.Theamino sequence offyuA comefromEC06748,Kp7151 and PAE7 was usedto compare with AL590842,there are 100％identities.Amino sequence ofirp2 come from Kp49 and Kp51 have 99％identities with AAA27636.1,but amino sequence of irp2 come from EC04 and EC07 only have 90％identities with 1176840.The GenBank accession number is FJ211852 and FJ211851.Amino sequence ofirpl come fromKp 10,Kp49 and Kp51 have 99％identities with AL590842。and amino sequence ofirp3 come from EC03,Kp51,Kp10 and Kp49 have 97％identities with CAA73128.There are the same mutation among the same species,and different mutation among different species.Conclusion There was different extant mutant lost in thefy～t-i,v2 gene cluster in multiple-drug-resistanee gram-negative bacilli.