Objective To construct a replication-deficient recombinant adenovirus expression vector of human heparanase (hpa). Methods The hpa gene was cloned at the downstream of CMV promoter of the adenoviral shuttle plasmid pDC315 in sense direction, and the resultant recombinant plasmid pDC315-hpa was transfected into HEK293 cell together with plasmid pBHGlox (deltaE1,3) containing adenoviral genome, then the replication-deficient recombinant adenovirus expression vector of hpa (Ad-hpa) was obtained, and it was identified by infection test, electronic microscope observation and PCR amplification. Results After purification and concintration,the titer of Ad-hpa reached 5?10 10pfu/ml. Virus particles could be found in virus concintration solution, and replication of virus was observed in HEK293 cells was observed under transmission electron microscope. Both adenovirus and hpa special fragment could be amplified from Ad-hpa by PCR, whereas hpa special fragment could not be amplified from the control. Conclusion The replication-deficient recombinant adenovirus expression vector of hpa was constructed successfully. This study established a foundation for further study on hpa vaccines and gene therapy for carcinoma.

Objective To investigate the changes in biologic behavior of dendritic cells (DCs) after being modified by human telomerase reverse transcriptase (hTERT) gene. Methods In order to obtain the hTERT gene modified DCs, DCs were transfected with a replication-deficient recombinant adenovirus expression vector of hTERT. Then the expression to hTERT and PCNA was assessed by by Western blot, mature markers on DCs surface were detected by flow cytometry, and the proliferative capacity was determined by MTT method. Results Immunohistochemical staining and Western blotting showed that the expression of hTERT was upregulated obviously in DCs after being modified by hTERT gene. Flow cytometry indicated that the expression of CD83 and CD86 remained unchanged. The DC growth curve showed that the number of DC-hTERT was increased slightly in the first 2 weeks, meanwhile the number of DC/rAd-LacZ and DC was decreased obviously. Although the number in 3 groups was all decreased in the third week, the number of DC/rAd-hTERT was still greater than the other control groups. It was also found that the expression of PCNA was increased in DC/rAd-hTERT compared with that of immature DC, mature DC or DC/rAd-LacZ by Western blot. Conclusion After rAd-hTERT modification, life span of DC in vitro is extended and proliferative capacity is enhanced.

Based on the circuit board, we draw its circuit diagram. By analyzing the circuit diagram, we maintain medical device power supply and achieve the troubleshooting goal quickly.
Durable Medical Equipment ; Electric Power Supplies ; Maintenance

Objective To investigate the relationship between NF-kB activity of pancreatic acinar cells(PAC) and blood inflammatory cytokines ( IL-2, IL-6, IL-10, TNF-? and ICAM-1) in rat's ANP. Methods Fourty rats were randomly divided into two groups: ANP model group and contrast group. ANP was induced by retrograde injection of 5% sodium taurocholate into the pancreatic duct. NF-KB activity in the cell nuclear and IkBa activity in the cell spasm of PAC were measured by EMSA and Western-blot. Inflammatory cytokines were measured by ELISA. Results ANP model's NF-KB activity increased [(31.4?5.7) ?mol/L vs. (8.3?2.4) ?mol/L.(39. 4 ? 6. 4) ?mol/L vs. (10.7 ?2.6) ?mol/L. (33. 8?6.0)?mol/Lvs. (11. 5 ?2. 7) ?mol/L.(25. 7 ?4. 9) ?mol/L vs. (9.4 ?2.6) ?mol/L](P

Objective To evaluate the role of nuclear factor erythroid 2-related factor 2 (Nrf2)/ antioxidant response element (ARE) signaling pathway in reduction of myocardial ischemia-reperfusion (I/R) injury by ischemic preconditioning in the rats.Methods Healthy male Sprague-Dawley rats,weighing 250-300 g,aged 4-6 months,were used in the study.Their hearts were excised,and retrogradely perfused with K-H solution at 37 ℃ in a Langendorff apparatus.Forty isolated hearts were randomly divided into 4 groups (n=10 each) using a random number table:control group (group C),I/R group,ischemic preconditioning group (group IPC),and ischemic preconditioning +Nrf2/ARE signaling pathway blocker luteolin group (group IPC+L).After 20 min of equilibration,the hearts were continuously perfused for 100 min in group C.After 20 min of equilibration,the hearts were subjected to 40 min ischemia at 32 ℃ followed by 60 min of reperfusion in group I/R.In group IPC,ischemic preconditioning was induced by 6 cycles of 10 s ischemia followed by 10 s reperfusion starting from the time point immediately after 20 min of equilibration,and then the hearts were subjected to 40 min ischemia at 32 ℃ followed by 58 min of reperfusion.In group IPC+L,after 20 min of equilibration,the hearts were perfused with K-H solution containing lueolin 50 μmol/L for 3 min before ischemia,and the other treatments were similar to those previously described in group IPC.Left ventricular developed pressure (LVDP),left ventricular end-diastolic pressure (LVDEP),heart rate (HR),and the maximum rate of increase of left ventricular pressure (+dp/dtmax) were recorded at the end of equilibration and reperfusion.At the end of reperfusion,left ventricular myocardial tissues were obtained for examination of the ultrastructure of myocardial cells and for determination of the expression of Nrf2,heme oxygenase-1 (HO-1),quinone oxidoreductase 1 (NQO1),and superoxide dismutase 1 (SOD1) mRNA and protein (by real-time polymerase chain reaction and Western blot,respectively).Results Compared with group C,the HR,+ dp/dtmax and LVDP were significantly decreased,and LVEDP was significantly increased at the end of reperfusion in I/R and IPC+L groups,and the expression of Nrf2,HO-1,NQO1 and SOD1 mRNA and protein was significantly up-regulated in I/R,IPC and IPC+L groups (P＜0.05).Compared with group I/R,the HR,+dp/dtmax and LVDP were significantly increased,and LVEDP was significantly decreased at the end of reperfusion,the expression of Nrf2,HO-1,NQO1 and SOD1 mRNA and protein was significantly up-regulated (P＜0.05),and the pathological changes were significantly attenuated in group IPC,and no significant change was found in the parameters mentioned above in group IPC+L (P＞0.05).Compared with group IPC,the HR,+dp/dt and LVDP were significantly decreased,and LVEDP was significantly increased at the end of reperfusion,and the expression of HO-1,NQO1,SOD1 mRNA and protein was significantly down-regulated (P＜ 0.05),no significant change was found in Nrf2 mRNA and protein expression (P＞0.05),and the pathological changes were significantly aggravated in group IPC + L.Conclusion Ischemic preconditioning reduces myocardial I/R injury through activating Nrf2/ARE signaling pathway in the rats.

10.3969/j.issn.1000-3606.2013.06.019

OBJECTIVE: To evaluate the effects of intact canal wall tympanoplasty with mastoidectomy (ICWT) with titanium ossicular prostheses. METHOD: A retrospective review was performed on 31 patients who underwent ICWT from 2008 to 2011. Patients' postoperative hearing results and complication rates were evaluated based on different types of ossicular prosthesis: partial ossicular replacement prosthesis (PORP), total ossicular replacement prosthesis (TORP). RESULT: The total effectiveness was 87.1%. No prosthesis was extruded. There was no significant difference in postoperative hearing results (average postoperative gain and ABG) between the two prostheses. In the low frequency (0.5 kHz), significant difference in ABG was found. CONCLUSION No significant difference in postoperative hearing results was found between PORP and TORP, which could be useful materials for tympanoplasty and obviously improve the hearing of otitis media patients after operation. As for the low incidence of postoperative complications in our short-term study, long-term follow-up visit is necessary.
Adult ; Cholesteatoma, Middle Ear ; surgery ; Female ; Humans ; Male ; Ossicular Prosthesis ; Retrospective Studies ; Titanium ; Tympanoplasty ; instrumentation ; methods

OBJECTIVE To evaluate the impact of mastoid obliteration withsingle-pedicle muscle flap covered by bone pate on vestibular stimulation. METHODS A retrospective study was performed on 59 patients who were treated for chronic otitis media with or without cholesteatoma by two techniques: canal wall down tympano-mastoidectomy(CWD) and subsequent mastoid obliteration(MO). The postoperative vestibular functions of all the patients in both groups were assessed by vestibular function tests and questionnaires. Finally, the data of examination a nd symptoms were a nalyzed. RESULTS After a minimum follow up period of 12 months, the rate of ear dry was 84％(22/26) for MO group and 55％(18/33) for CWD group(χ2=4.72, P <0.05). The dry ear time were 5.46±1.39 weeks for MO group and 8.67±2.3 weeks for CWD group(t =6.2529, P <0.05). When compared latent period of Caloric testing in the MO group (10.3±2.57)s and CWD group (12.7±3.33)s, significant differencewas found(t =3.1639, P <0.05). The postoperative caloric vestibular tests revealed an average nystagmus count of 52.96±20.82 beats per minute in the MO group and 69.94±18.98 beats in the CWD group(t =3.2688, P <0.05). By analyzing the questionnaire, 30％(10/33) of the patients who received CWD treatment reported vertigo by caloric stimuli such as wind, water compared with MO group(0)(χ2=7.45, P <0.05). The rate of suction cleaning induced vertigo was 48％(16/33) in CWD group and 23％(6/26) in MO group(χ2=3.17, P =0.075). CONCLUSION Our technique of mastoid obliteration with single-pedicle muscle flap covered by bone pate results in small cavities with complete epithelialization of all surfaces. Furthermore, obliteration of mastoid cavities provides protection to the labyrinthine organ and reduces postoperative vertigo to caloric stimulation.

Objective: To investigate the effect of pseudodeficiency alleles on the newborn screening of glycogen storage disease type Ⅱ(GSDⅡ) by using afluorometric enzymatic assay to determine acid α-glucosidase (GAA) activity in dried blood spot (DBS). Methods: A total of 30 507 newborns′ DBSs, obtained from Newborn Screening Center of Xinhua Hospital Shanghai Jiao Tong University School of Medicine from May to December 2017, were screened for GSD Ⅱ by fluorometric enzymatic assay of GAA activity. The suspected positive DBSs after the first and second screening were directly analyzed by Sanger sequencing of GAA to confirm the diagnosis. Retrospective analysis of 3 172 controls without GSDⅡand 36 GSD Ⅱ patients were conducted to investigate the carrier status of pseudodeficiency alleles. Statistical analysis of frequency of pseudodeficiency alleles were carried out by Chi-square test or Fisher exact probability test. Results: GAA activity of 30 507 newborns showed a positively skewed distribution.Twenty-nine cases of newborns, suspected to be GSDⅡwere confirmed to be normal with genetic analysis of the original DBSs. Among the 29 suspected positive cases, 24 cases were homozygous for pseudodeficiency alleles c.[1726A/A; 2065A/A], and the other 5 cases were c.[1726G/A; 2065G/A] heterozygote. The frequency of c.1726G>Ahomozygote in 3 172 non-GSD Ⅱcontrols was 2.08% (66/3 172), and c.1726G>A homozygote occurred in allelic conjunction with c.2065G>Ahomozygote. Frequency of c.[1726A; 2065A] haplotype in 3 172 controls was 3.2%(206/6 344). Frequency of c.[1726A/A; 2065A/A] homozygote in 36 GSDⅡpatients (16.67%, 6/36) was significantly higher than that in non-GSD Ⅱcontrols(2.08%, 66/3 172) (χ²=34.517, P<0.001). Conclusions Pseudodeficiency alleles show a high frequency in Chinese, which leads to a high false positive rate in the newborns screening of GSDⅡ.The afterword genetic analysis of the original DBS after the GAA activity screening could reduce the effect of pseudodeficiency alleles on the newborns screening of GSDⅡ.

OBJECTIVE: To assess the effectiveness and feasibility of carrier detection for Spinal muscular atrophy (SMA) by using digital PCR assay. METHODS: Peripheral blood samples were collected from 214 pregnant women who were routinely screened for SMA carriers, of which 204 were randomly selected samples and 10 were samples with known copy numbers of SMN1 exons 7 and 8. Samples with known copy numbers of SMN1 exons 7 and 8 were randomly mixed into the experiment to validate the performance of the digital PCR assay. RESULTS: The copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8 in peripheral blood samples were detected by digital PCR assay. The results of SMN1 exons 7 and 8 were compared with those of the quantitative PCR method to assess the reliability and clinical performance of the digital PCR assay. Among the 204 random samples, digital PCR has detected five samples with simultaneous heterozygous deletion of SMN1 exons 7 and 8, three samples with heterozygous deletion of SMN1 exon 8 only, and 196 samples with no deletion of SMN1 exons 7 and 8. Ten samples with known SMN1 exons 7 and 8 copy numbers were detected with the expected values. The digital PCR test results were fully consistent with that of the quantitative PCR. CONCLUSION The results of digital PCR for the detection of copy number variation of SMN1 exons 7 and 8 were consistent with qPCR. Digital PCR assay was able to clearly distinguish the copy number of the target genes, therefore can be used for SMA carrier screening. Moreover, it can also detect copy number of SMN2 exons 7 and 8, which can provide more information for genetic counseling.
Humans ; Female ; Pregnancy ; DNA Copy Number Variations ; Reproducibility of Results ; Muscular Atrophy, Spinal/genetics* ; Polymerase Chain Reaction/methods* ; Nucleic Acid Amplification Techniques ; Survival of Motor Neuron 1 Protein/genetics*