AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2 x 10(7) cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.
Animals ; Baculoviridae ; DNA, Single-Stranded ; Dependovirus ; Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Sf9 Cells ; Terminal Repeat Sequences ; Transfection

Objective To explore the clinical effect and lung CT change of long-term used of low-dose roxithromycin in treatment for bronchial expansion patients in stable phase. Methods 94 cases collected in the Department of Respiration, The Second Hospital Affiliated to Suzhou University from February 2011 to December 2012 were diagnosed as bronchiectasis, 34 cases in control group were given oral treatment for ambroxol 30 mg, three times one day, 60 cases in treatment group were added roxithromycin 75 mg on basis of control group, two times one day. Patients in two groups were both treated for 6 months. The therapeutic effect and the score of life quality and dyspnea scores in two groups were observed, and the changes of CT data were compared before and after treatment. Results After treatment, the life quality score and dyspnea score of two groups were all improved, but the treatment group was signiifcantly higher than the control group (P<0.05). The effective rate in treatment group was 86.67%, which was signiifcantly higher than 70.59%in the control group (P<0.05). After treatment, chest CT imaging score of patients in treatment group were improved, signiifcantly better than that in the control group (P<0.05). Conclusion Long-term low dose administration of roxithromycin can control and stable bronchiectasis symptoms, and improve signs and symptoms .

Objective To evaluate the therapeutic effect of ESWL combined with urinary stone decoction and ear acupoint pressure for the patients with renal and ureteral calculi.Methods A total of 110 patients with renal and ureteral calculi were randomly divided into the Chinese medicine (CM) and conventional groups, 55 in each group. Both groups were given ESWL treatment as basic treatment. Chinese medicine group were given the urinary stone decoction and auricular acupressure, and the conventional group used stone granules. Both groups were treated for 2 weeks. According to the photography of ultrasound, the changes of calculi, time of lithagogue, pain relief after operation and the effect rates of pain relief on abdomen and waist, of remission of haematuria, and of urinate pain. ResultsAfter treatment, the successful rates of operation in the CM group was 90.9% (50/55), and conventional group was 76.4% (42/55). There was significant difference between 2 groups (χ2=4.251,P=0.009). The time of stone removal in CM group was significantly shorter than that in the conventional group (7.3 ± 3.8 dvs. 11.2 ± 4.3 d,t=4.032,P<0.001). After 1st, 3rd, 5th, 7th days, the VAS pain scores in the CM group were significantly lower than those in the conventional group (4.1 ± 0.8vs. 5 ± 1.2,t=4.783;4.5 ± 0.9vs. 5.3 ± 1.3,t=4.492; 3.8 ± 0.8vs. 4.5 ± 0.8,t=4.503; 2.2 ± 0.8vs. 2.9 ± 0.9,t=3.691; allP<0.01). After 7th day, the rates of abdominal pain relief was 60% (33/55), pain relief was 79.1% (34/43), the remission of hematuria was 81% (17/21) in the CM group, and the control group were 40% (22/55), 59.6% (28/47), 52% (13/25). There were significant differences between 2 groups on these indices (χ2 values were 4.400, 3.982, 4.217,P values were 0.036, 0.046, 0.040).Conclusions The ESWL combined with urinary stone decoction and ear acupoint pressure could improve the symptoms of patients with kidney ureteral stones.

Objective To construct short hairpin RNA (shRNA) recombinant expression vector for herpes simplex virus typeⅡ(HSV-2) UL29 gene and observe its inhibitory effect on HSV-2. Methods Four interference target sites of HSV-2UL29 gene were selected to construct 4 groups of small hairpin RNA respectively,named shRNA recombinant expression vector. The expression vectors were transfected into HEK293 cells with liposome. HEK293 cells were infected with HSV-2 after expression vector being transfected. The viral titer was estimated by end-point titration assay. The level of transcription was estimated by Real-Time PCR method. The expressing effect of protein was detected by Western-blot. Results Recombinant expression vector pGPU6/GFP/Neo-shRNA was constructed successfully. The result of end-point titration assay showed that the viral titer was reduced comparing with blank control (P<0.05). The result of RT-PCR showed that inhibition rates were respectively 28.80%, 59.95%, 66.08%and 36.27% comparing with blank control, and there were significant differences (P < 0.05). The effect of UL29shRNA1461 group was the best one. The result of Western-blot showed that the expressing quantity of ICP8 was reduced. Conclusion Recombinant expression vector pGPU6/GFP/Neo-shRNA can interfere HSV-2 UL29 gene expression from different cell level in vitro, which can inhibit the replication of HSV-2 genome in HEK293 cells. Thus, RNA interference (RNAi) is conducive to the further exploration of viral therapy.

OBJECTIVE: To investigate the clinical features and therapeutic effects of sudden deafness after radiotherapy combined with chemotherapy in nasopharyngeal carcinoma patients. METHOD: Clinical data of 42 nasopharyngeal carcinoma patients suffered from sudden deafness after radiotherapy combined with chemotherapy were analyzed retrospectively. Among the 42 patients, 2 showed moderate deafness, 4 presented excessive deafness, 30 suffered from severe deafness, and 6 exhibited profound deafness. The audiogram pattern of 33 patients met with the type of high tone frequencies hearing loss, and that of the rest 9 cases showed hearing loss at all frequencies. All patients received medical therapy combined with hyperbaric oxygen therapy. RESULT: Of all the cases with hearing loss, 2 were cured, 2 showed excellent recovery, 9 came out partial recovery, and 29 showed no response to the treatment. The total effective rate was 30.95%. For the accompanied symptoms, none of the 30 cases of tinnitus were relieved, 3 out of 10 cases of aural fullness were cured, and the 5 cases of dizziness or vertigo were all improved. CONCLUSION The sudden deafness after radiotherapy combined with chemotherapy in patients with nasopharyngeal carcinoma is closely related to radiotherapy. The hearing loss is serious, and the therapeutic effects are not satisfactory.
Antineoplastic Agents ; adverse effects ; Carcinoma ; Dizziness ; etiology ; therapy ; Hearing Loss, High-Frequency ; etiology ; therapy ; Hearing Loss, Sudden ; etiology ; therapy ; Hearing Tests ; Humans ; Hyperbaric Oxygenation ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; drug therapy ; radiotherapy ; Radiotherapy ; adverse effects ; Retrospective Studies ; Tinnitus ; etiology ; therapy ; Vertigo ; etiology ; therapy

Objective:To evaluate the effect of preoperative panel reactive antibody(PRA)levels on long-term survival after kidney transplantation. Methods:Data on 1 162 patients underwent first kidney transplantation performed between January 2001 and June 2014 were included in our center. According to the preoperative PRA levels,the patients were divided into negative group( PRA≤10%) and positive group( PRA>10%) ,which were retrospectively analyzed. Results: The 1-,5-,10-year patient survival rates of the negative group calculated by Kaplan-Meier were 96. 8%,89. 4%,78. 6%,respectively,while the positive group were 93. 5%,81. 6%, 65. 4%. The 1-,5-,10 -year death-censored graft survival rates of the negative group were 95. 9%,84. 8%,63. 1%,respectively,while the positive group were 92. 3%,74. 1%,51. 9%. The log-rank test revealed that there was significant difference between the patient and graft survival curves (χ2 =9. 623/11. 019, P=0. 002/0. 001 ) . Cox multivariate analysis found that preoperative PRA levels were independent risk factors for reducing the patient or graft survival rates(P<0. 001). Logistic multivariate regression analysis confirmed the significant association between preoperative PRA levels and the risk of acute rejection ( OR=8. 25,95% CI=2. 86-5. 72, P<0. 001). The 5-,10-year creatinine values were significantly lower in the negative group compared to the positive group(all P<0. 05), while there was no difference in the 1-year. In addition, Logistic multivariate regression analysis confirmed the significant association between preoperative PRA levels and the production of donor specific antibody(DSA)(OR=6. 89,95% CI=4. 52-9. 17,P<0. 05). Conclusion: The detection of preoperative PRA is an important indictor predicting the sensitivity status of the recipients. The preoperative PRA positive recipients need careful monitoring and diagnosis of acute rejection and DSA after kidney transplantation.

This study compared two techniques of artery anastomoses,renal artery to the ex- ternal iliac artery (ESA) and to the internal iliacartery (EEA) in renal transplantation.The operation time and the incidence of anastomotic stenosis was cut down significantely in ESA group.The blood flow in grafts has no difference in two groups with normal renal function.The utilization rate of grafts with multiple arteries was higher (94.4%) in ESA group.Back bench surgery for artery repain,cold ischemia time and renal damage were reduced in ESA group.

BACKGROUND:Our previous studies have shown that salidroside can induce bone marrow mesenchymal stem cells directly into neuron-like cells, and Ca2+signal is one important way to achieve its biological signal transduction. OBJECTIVE:To investigate the role and mechanism of the calcium/calmodulin (Ca 2+/CaM) signaling pathway inducing bone marrow mesenchymal stem cells to differentiate into nerve cells. METHODS:Bone marrow mesenchymal stem cells were divided into two groups:control groups and salidroside groups. Salidroside groups were induced with different concentrations of salidroside (5, 10, 20, 50 and 100 mg/L) for 24 hours and 100 mg/L salidroside was added to culture cells for different time (12, 24, 48 and 72 hours). Western blot assay was used to detect the expression levels of neural cellmarker, microtubule-associated protein 2, and the important protein of Ca2+/CaM signaling pathway:CaM and calmodulin dependent kinase II (CaMK II). Then Ca2+/CaM signaling pathway specific blockers were applied to cells respectively for 30 minutes, including 500 μmol/L EGTA (Ca 2+chelator), 1 mmol/L Nifedipine(L-type Ca2+channel blocker) and 10 mmol/L LY294002 (PI3K inhibitor). Then, 100 mg/L salidroside was added and cultured for 24 hours. Western blot assay was used to detect the expression of neuron-specific enolase and CaM in the Ca2+/CaM signaling pathway. RESULTS AND CONCLUSION:(1) After inducing with salidroside, the expression of microtubule-associated protein 2 were upregulated (P<0.01), indicating that salidrosid can induce the neuronal differentiation of bone marrow mesenchymal stem cells. (2) After different concentrations of salidrosid induced bone marrow mesenchymal stem cells for 24 hours, the expressions of CaM and CaMK II were significantly upregulated in the 10 mg/L group ( P<0.01);For the 100 mg/L salidrosid that was added for cellinduction for different time, the expressions of CaM and CaMK II were significantly downregulated in 72-hour group (P<0.01). (3) After blocking extracellular Ca2+and PI3K signaling pathway, the expressions of neuron-specific enolase and CaM were higher than those in salidrosid groups (P<0.05). These results suggest that salidrosid can induce bone marrow mesenchymal stem cellto directly differentiate into nerve cells by inhibiting the Ca2+/CaM signaling pathway.

To study the in situ intestinal absorption kinetics of flrubiprofen in rats, the absorption of flurbiprofen in small intestine (duodenum, jejunum and ileum) and colon of rats was investigated using in situ single-pass perfusion method and the drug content was measured by HPLC. The effects of drug concentration on the intestinal absorption were investigated. The K(a) and P(app) values of flurbiprofen in the small intestine and colon had no significant difference (P > 0.05). Drug concentration (4.0, 10.0 and 16.0 mg x L(-1)) had no significant influence on the K(a) values (P > 0.05). However, when concentration was 4.0 mg x L(-1) and 10.0 mg x L(-1), significant effect on the P(app) values (P < 0.05) was found, but significant effect on the P(app) values was not shown between 10.0 mg x L(-1) and 16.0 mg x L(-1) (P > 0.05). The K(a) and P(app) values of flurbiprofen on the perfusion flow rate had significant difference (P < 0.05). Flurbiprofen could be absorbed at all segments of the intestine in rats and had no special absorption window. The absorption of flurbiprofen complies with the facilitated diffusion in the general intestinal segments, and accompany with the cytopsistransport mechanism probably. The perfusion flow rate had significant effect on the K(a) and P(app).

Objective To study the safety and efficacy of low dose Peginterferon-alpha-2a (PEG-INF-α-2a) combined with Ribavirin treating chronic hepatitis C in renal transplant recipients.Methods A total of 13 cases of HCV hepatitis were randomly divided into treatment and control groups.Seven cases in treatment group were given PEG-INF-α-2a (90 μg/week) and ribavirin (600mg/day) for 16 to 48 weeks,and the rest 6 cases in control group were subjected to general liver protection and anti-inflammatory treatment. All patients were followed up for more than 2 years.Results There were 5 cases getting early response in treatment group for 16 weeks,including four cases of complete response and no non-effects response patients. In 4 cases voluntarily receiving treatment for 48 weeks,1 case had facial muscle myalgia and increased Cr level at 35th week,humoral graft rejection was confirmed pathologically,and the treatment was terminated; 1 case had recurrence of HCV RNA replication and PEG-INF-α-2a was withdrawn at 38th week.As results,5 patients in the treatment group obtained complete response after two years,including 2 cases whose HCV-IgG had got negative,HCV RNA replication was significantly lower than in the control group,and the average Cr higher than in control group (P＞ 0.05). There were adverse reactions during this treatment protocol: fever,muscle myalgia,agranulocytosis, anemia and humoral graft rejection.Conclusion The efficacy of low lose PEG-INF α-2a combined with ribavirin is definite in the treatment of chronic HCV hepatitis in kidney transplant recipients.The 16-week treatment duration is reasonable.It is remarkable that PEG-INF-α-2a may cause humoral graft rejection and Cr crawling.