Aim To explore the role of cytoplasmic FKBP52 in AAV-mediated transduction.Methods Murine embryo fibroblasts(MEFs)cultures from FKBP52 wild-type(WT),heterozygous(HE),and knockout(KO)mice were established.The role of FKBP52 in intracellular trafficking of AAV was analyzed by fluorescence-activated cell sorting(FACS)analyses,electrophoretic mobility shift assays(EMSA),southern blot,immunoprecipitations and western blot analyses.Results Conventional AAV vectors failed to transduce WT MEFs efficiently,and the transduction efficiency was not significantly increased in HE or KO MEFs.AAV vectors failed to traffick efficiently to the nucleus in these cells.Treatment with hydroxyurea(HU)increased the transduction efficiency of conventional AAV vectors by～25-fold in WT MEFs,but only by～4-fold in KO MEFs.The use of self-complementary AAV(scAAV)vectors,which bypass the requirement of viral second-strand DNA synthesis,revealed that HU treatment increased the transduction efficiency～23-fold in WT MEFs,but only～4-fold in KO MEFs,indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis.Following HU treatment,～59% of AAV genomes were present in the nuclear fraction from WT MEFs,but only ～28% in KO MEFs,indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs.When KO MEFs were stably transfected with an FKBP52 expression plasmid,HU treatment-mediated increased in the transduction efficiency was restored in these cells,which correlated directly with improved intracellular trafficking.Intact AAV particles were also shown to interact with FKBP52 as well as with dynein,a known cellular protein involved in AAV trafficking.Conclusion These studies suggest that FKBP52,being a cellular chaperone protein,facilitates intracellular trafficking of AAV,which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

ObjectiveTo observe the clinical effect of the treatment of clearing liver fire,removing dampness,strengthening spleen and tonifying kidney in treating middle-aged and old woman with chronic urinary tract infection.Methods82 cases were randomly divided into two groups.42 cases in treatment group were treated with herbal prescription of clearing liver fire,removing dampness,strengthening spleen and tonifying kidney,and 40 cases in control group were treated with sensitive antibiotics.The clinical effect,safety and changes of immune index were compared between two groups.ResultsIn the treatment group,the recent cure rate was 76.8％,the total effective rate was 90.5％ and the complete cure rate was 61.9％; while in the control group,the recent cute rate was 50％,the total effective rate 65％ and the complete cure rate 27.5％.It showed significant difference between two groups (Z=2.740,P＜0.05).There was no significant difference between two groups in hepatic and renal function,serum and urine β2-MG level before and after treatment and it showed a better clinical safety in treatment group.The level of IgA,IgG and IgM increased after the treatment in treatment group.Comparing with the control group,the results showed significant differences(t=9.1,10.5 and 11.2 respectively,P＜0.01).In treatment group the level of CD4+was increased,CD8+was decreased,and the ratio of CD4+/CD8+ was increased obviously after the treatment.The differences before and after the treatment and between groups were significant(t=13.3,8.6,P＜0.01).In control group the level of CD4+、CD8+、CD4+/CD8 +did not change significantly.The differences before and after the treatment had no statistical significance (P＞0.05).ConclusionThe treatment with herbs of clearing liver fire,removing dampness,strengthening slpeen and tonifying kidney had better effect,less side effect and good research prospect.

Objective To investigate the role of ski in proliferation of astrocytes and the molecular mechanisms in rats. Methods Astro-cytes were obtained from cerebral cortex of a three-day old rat and cultured in vitro. siRNA targeted to ski and negative control sequences were prepared. The astrocytes were divided into ski-siRNA group, siRNA negative control group and untreated control group, while the spe-cific siRNA targeting ski negative control sequences were transfected into astrocytes with Lipofectamine? RNAiMAX Reagent. The protein levels of ski, glial fibrillary acidic protein (GFAP) and Cyclin D1 were determined with Western blotting. The proliferation of astrocytes were measured with CCK8 assay. The cell-cycle of astrocytes were analyzed with flow cytometer. Results The protein level of ski (F=38.611, P<0.01), GFAP (F=7.547, P<0.05) and Cyclin D1 (F=3.901, P<0.05) reduced in ski-siRNA group, the proliferation of astrocyte was significantly inhibited since twelve hours after culture (F>30.507, P<0.01), and less cells were in S phase and more in G1/G0 phase (F>48.425, P<0.01), compared with the control groups. Conclusion ski knocking down by siRNA significantly inhibits the proliferation of astro-cytes, which may associate with the down-regulation of Cyclin D1 expression.

Objective To research the possible influence of operation and human chorionic gonadotropin(HCG) therapy on the development of antisperm antibody (AsAb) and serum levels of estradiol (E_2) and testosterone (T) of cryptorchidism boys after orchiopexy. Methods Forty cryptorchidism boys were studied who were divided into HCG treatment group and without HCG treatment group. Twenty boys who underwent inguinal operation were studied as the operation control group and 20 normal boys as the control group. The serum were collected for detection AsAb, E_2,and T. Results There were no significant difference in serum levels of E_2 and T among the three groups(P 0.05 ). Conclusions The level of AsAb is associated with the destroy of blood barrier and the disorder of immune modulation and endocrine. Treatment with HCG has no effect on AsAb level.

Objective To explore the expression and the changes of ski with time in the injured spinal cord in rats. Methods Sixty adult female Sprague-Dawley rats were randomly divided into sham group (n=30) and injury group (n=30), each group were further divided into 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks subgroups, with 6 rats in each subgroup. Spinal cord injury at T10 was established with modi-fied Allen's technique (10 g × 25 mm) in the injury group. The hindlimbs behavior of rats was rated with Basso-Beattie-Bresnahan (BBB) scores 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks after spinal cord injury. Three rats in each subgroup were stained with HE staining to observe the pathological changes of the spinal cord and the formation of cavity. The other 3 rats were analyzed with im-munofluorescence staining of ski and semi quantitative analysis. Results The BBB scores of each time point were less in the injury group than in the sham group (P<0.05). Necrosis was the major pathological change in the injury groups 1 and 2 weeks after injury;cystic cavity completely formed 4 weeks after injury, with dense scar tissue around it;there was no significant change in the cavity and scar 8 and 12 weeks after injury, however, the adjacent spinal cord was obviously thinner. Ski expressed little in the normal spinal cord, and expressed more and more after injury, peaked at 8 weeks and decreased then. Ski was mainly observed in white matter in the sham group and 12 weeks injury subgroup, which was in gray matter 2, 4 and 8 weeks after injury. Ski was highly expressed around the cavity in injury center and formed high expression band. Conclusion Ski expresses after spinal cord injury in rats, that may be associated with the activation and prolif-eration of astrocytes and the formation of glial scar.

AIM:To explore the time-dependent change of Ski protein expression in normal and activated astrocytes in rats.METHODS:The astrocytes were obtained from rat cerebral cortex and cultured in vitro.The astrocytes were treated with LPS and scratch injury for activation.Western blot analysis was used to determine glial fibrillary acidic protein (GFAP) and Ski protein levels in activated astrocytes at a series of time points.The indirect immunofluorescence staining method was performed to detect the location of Ski protein in the astrocytes.RESULTS:The protein of GFAP was naturally expressed in the astrocytes, beginning to increase after treated with LPS and scratch injury.Little protein expression of Ski in the normal astrocytes was observed.The Ski protein expression began to increase after treated with 1 mg/L LPS, peaked at 4 d (P<0.05) and then deceased, but was stills higher than that in the normal cells.The protein expression level of Ski after scratch injury was highly consistent with above mentioned.Ski was mainly observed in the nucleus of the normal cells and the cells treated with LPS for 6 d, while it was observed in the cytoplasm 2 and 4 d after treated with LPS.CONCLUSION:The protein of Ski is expressed in the astrocytes, and the expression level is increased in activated astrocytes,mainly located in the nucelus.Ski may plays an essential roles in the processes of activation and proliferation of astrocytes.

OBJECTIVETo measure uric acid of seminal plasma in fertile and infertile males.

METHODSOne hundred and sixty-three infertile males were divided into an obstructive azoospermic group (15 cases), a non-obstructive azoospermic group (36 cases), an oligozoospermic group (43 cases), and an asthenozoospermic group (69 cases). Twenty fertile males were included in the control group. Uric acid concentrations of seminal plasma in the fertile and infertile men were assessed by spectrophotometer, and sperm parameters were analyzed by computer-assisted semen analysis (CASA) system.

RESULTSUric acid concentration of seminal plasma in the control group was significantly higher than all the infertile groups (P < 0.01), and that of the obstructive azoospermic group significantly lower than the other infertile groups (P < 0.1), but no significant difference was observed among the other infertile groups (P > 0.05).

CONCLUSIONUric acid may play an important role in male reproduction because of its antioxidative property.

Adult ; Azoospermia ; metabolism ; Case-Control Studies ; Humans ; Infertility, Male ; metabolism ; Male ; Oligospermia ; metabolism ; Semen ; chemistry ; Uric Acid ; analysis

Objective: To observe the efficacy and safety related measures by blocking mother-to-child transmission of hepatitis B virus with high viral load and HBeAg positivity during pregnancy in Guizhou province. Methods: Outpatient and inpatient cases of the Department of Infectious Diseases and Obstetrics of Guizhou Medical University Affiliated Hospitals from May 2016 to July 2017 were retrospectively divided into intervention group, non-intervention group and non- hepatitis B pregnant women group; with 75 cases in each group. HBsAg and HBeAg were positive in the intervention group. Pregnant women with HBV DNA ≥10⁶ IU/ml were treated with anti-HBV therapy for 24 to 28 weeks of gestation until delivery. According to oral drugs, they were divided into tenofovir (TDF) group or telbivudine (LDT) group, non-intervention group (HBsAg and HBeAg positive), HBV DNA positive pregnant women, pregnant women with no anti-HBV drugs, non-hepatitis B pregnant women (normal pregnant women without HBV infection). Infants and young children born to the three groups of women were immunized with the national viral hepatitis B action plan. The gestational weeks and Apgar scores at birth, delivery mode, feeding mode, sex and 7-months-old age were observed and counted. Serum hepatitis B markers (HBVM) and HBV DNA were quantitatively detected. HBVM was detected by time-resolved fluorescence immunoassay (TRFIA), and HBV DNA was detected by real-time PCR (FQ-PCR). The changes of liver parameters, HBsAg, HBeAg, HBV DNA, adverse drug reactions and treatment response of pregnant intervention group before medication (12-24 weeks of gestation), 4 weeks of medication (28-32 weeks of gestation), 36-40 weeks of gestation (36-40 weeks of gestation) were statistically calculated. A t-test was used to compare the data between the measurements. Data measurements within the groups were analyzed using rank -sum test. Results: In the intervention group, therapeutic medications showed no differences in demographic and clinical characteristics between TDF group and LDT group, including liver parameters, HBsAg, HBeAg and log10HBV DNA level. Compared with pre-treatment (TDF group: 4.84 ± 2.01; LDT group: 5.08 ± 1.99), TDF and LDT were significantly lower at the end of pregnancy (TDF group: 3.06 ± 0.66; LDT group: 3.51 ± 1.20). P < 0.05); and the treatment response rate was 100%. There were no serious adverse events in the intervention group. Infants and young children (7-months-old) in the intervention group had negative HBsAg, HBeAg and HBV DNA. The mother-to-child transmission rate of HBV was zero, with blocking rate of 100%. In addition, both infants and young children had different degrees of hepatitis B protective antibodies (anti-HBs, M: 144.33), and their antibody titers were higher than that of non-intervention group (anti-HBs, M: 65.91) and non-hepatitis B pregnant women (anti-HBs, M: 58.43). The difference was statistically significant (P < 0.05), and there was no significant correlation between the use of antiviral and the way of delivery and feeding. Outcomes of mother-to-child transmission of HBV infection in infants and young children (7-months-old) delivered by three groups of pregnant women in the non-intervention groups had 20.0% (15/75)/ 17.3% (13/75) HBsAg/HBeAg positivity rate, and 17.3% (13/75) HBV DNA positivity rate. Overall, mother-to-child transmission rate of HBV infection was 20% (15/75). Furthermore, the relationship between mother's HBV DNA load and infant HBV infection in the non-intervention group showed mother's HBV DNA ≥10⁶ IU/ml. Conclusion In the non-intervention group, mother-to-child transmission of HBV occurred, and infected mothers HBV DNA was ≥106 IU/ml before delivery. This suggests that HBeAg positive and high load HBV DNA replication were independent risk factors for mother-to-child transmission of hepatitis B. Therefore, prenatal drug intervention and postpartum standard immune blockade are necessary for high-risk pregnant women with hepatitis B to achieve zero mother-to-child transmission of hepatitis B in real- clinical practice.