Adenovirus vectors are promising delivery systems for gene therapy. We established a new process for clinic trial of recombinant adenovirus vectors using a novel disposable bioreactor. The suspension HEK293 cells were inoculated into a 5 L disposable bioreactor with parameters control of pH, DO, agitation and temperature. After 6 days of a fed-batch culture, the final cell density reached 2.0 x 10(6) cells/mL. The culture was infected with Ad-IFNγ at an MOI of 30. The harvest was performed at approximately 48 h post-infection and crude viral lysate was obtained after 3 freeze/thaw cycles and centrifugation. The maximum titers of crude viral lysate was 1.49 x 10(13) Infectious units (IFU) and the bulk product specific was 3,800 IFU/cell. Purified Ad-IFNγ by anion-exchange chromatography and the final recovery of infectious unit reached 35.9%. The result demonstrates that an efficient and stable process of producing Ad-IFNγ using a disposable fed-batch bioreactor is established.
Adenoviridae ; growth & development ; Batch Cell Culture Techniques ; Bioreactors ; Cell Count ; Genetic Therapy ; Genetic Vectors ; HEK293 Cells ; Humans ; Virus Cultivation ; methods

[Objective] To investigate the effect of salvianolate injection on the levels of Serum interleukin-1β(IL-1β)and interleukin-18(IL-18) in acute exacerbated chronic obstructive pulmonary disease(AECOPD) patients. [Methods] Fifty-eight patients with acute exacerbated COPD from Respiratory Dep. of thrid Hospital Affiliated to Zhejiang Chinese Medical University, were randomly assigned to the treatment group(28 cases) and the control group(30 cases). Routine therapies for COPD were given to patients in the control group, while 20mg salvianolate injection was given to those in the treatment group by intravenous dripping, once daily for total 10 days. The levels of IL-18, IL-1β and blood gas analysis were evaluated before and 10 days after therapy. The levels of the cytokins were compared with 30 normal controls. [Result] There were no significant differences in the value of IL-1β,IL-18,arterial partial pressure of carbon dioxide(PaCO2),arterial partial pressure of oxygen(PaO2) before treatment between two groups. Compared with the blank control group, the levels of serum IL-1β,IL-18 in patient with AECOPD were increased significantly( P<0.01). When compared with before treatment and with the control group, the levels of IL-1β,IL-18,PaCO2 decreased more significantly in the treatment group after treatment.The PaO2 in treatment group was higher than in control group. There were significant differences between two groups.[Conclusions] Salvianolate injection can reduce the secretion IL-1β, IL-18 in patient with AECOPD, which maybe through certain anti-inflammatory effects to improve clinical efficacy of COPD pa-tients.

0 05) Conclusion Treatment of simvastatin 10 mg daily did no affect the stent restenosis and the in stent neointimal hyperplasia

Objective To explore the possible causes that result in serum-resistance of patients with syphilis. Methods Cerebrospinal fluid(CSF) routine and biochemistry, RPR, TPPA, and immunological function in 25 patients with syphilis serum-resistance were detected after giving the standard therapy, and radiographic examination of multiple internal organs was performed as well. All patients were treated with 24 million units of Penicillin G per day by intravenous drip for 14 days followed by muscle injection of Bicillin 2.4 million units weekly for 3 weeks after leaving hospital. Blood serum RPR was reexamined 3 and 6 months after treatment, respectively. Results Among 25 patients CSF-TPPA was positive in 11 cases, all patients' CSF-RPR were negative, CSF-WBC and CSF-Pr increased in 7 patients, serum IgM level increased and C3 decreased in 10 cases (40%), and CD_3+CD_8+ cell count increased and NK cell count decreased in 21 cases (84%). The regurgitation of bicuspid valve, tricuspid valve and aortic value was found in 3 patients. The RPR titers in all patients did not decrease after treatment according to neurosyphlis. Conclusion Asymptomatic neurosyphilis and immune disorder may be causes of serum-resistance of syphilis.

Objective: To further understand the role of folic acid supplements rivaling MTHFR gene silencing in pathogenesis of NCLP, RNA interference (RNAi) was applied to knock down MTHFR in mouse embryonic palatal mesenchymal (EPM) cells. Methods: MTHFR ShRNA expression vector were transfected into the primary cultured EPM cells. MTT was used to observe cell proliferation after MTHFR gene silencing. FCM was used to observe cell cycle after MTHFR gene silencing. Results: The results showed the cells proliferation had an inequality amelioration after using folic acid supplements in MEPM cells with MTHFR gene silencing. Using folic acid supplements rivaled the effect of MTHFR gene silencing had a dose-dependent manner. Using 20 μg/ml folic acid supplements could improve the cell proliferation to achieve normal level of cell proliferation. Conclusion: MTHFR gene is an important candidate gene of NCL/P. Using folic acid supplements could prevent teratogenic MTHFR gene silencing for embryonic palate development.

Objective To investigate the relationship between negative acute phase proteins and chronic urticaria (CU).Methods Fifty patients with CU were enrolled into this study,and divided into three grades,i.e.,mild (grade 1),moderate (grade 2) and severe (grade 3) according to symptoms.Twenty-eight health checkup examinees served as the control group.Immunoturbidimetry was performed to determine serum levels of prealbumin (PA) and transferrin (TRF).Enzyme-linked immunosorbent assay was conducted to measure serum levels of insulinlike growth factor-1 (IGF-1) and tumor necrosis factor-α (TNF-α).Statistical analysis was carried out to assess differences in these indices between these two groups,the relationship among these indices and between these indices and disease severity.Results Compared with the control group,the patients with CU showed reduced serum levels of PA ((229.99 ± 54.16) vs.(272.06 ± 36.42) mg/L,t =3.667,P ＜ 0.05) and IGF-1 ((177.23 ± 46.48) vs.(239.88 ± 45.16) μg/L,t =5.748,P＜ 0.05),but higher serum levels of TNF-α ((25.39 ± 11.01) vs.(14.13 ± 6.12) ng/L,t =4.989,P＜ 0.05),and similar serum levels of TRF ((2.48 ± 0.49) vs.(2.48 ± 0.25) g/L,P＞ 0.05).The serum level of PA showed a significant negative correlation with that of TNF-α (r =-0.312,P ＜ 0.05),as well as with disease severity (r =-0.635,P ＜ 0.01),whereas the serum level of TNF-α showed a significant positive correlation with disease severity (r =0.409,P ＜ 0.01),and no statistical correlation was found between the remaining indices (all P ＞ 0.05) in the patients with CU.Conclusions Serum levels of some negative acute phase proteins decrease and negatively correlate with disease severity in patients with CU.Acute phase response may be involved in the occurrence of CU.

Objective To evaluate the relationship of acute-phase response and coagulation/fibrinolysis with chronic urticaria (CU).Methods Fifty-three patients with CU and twenty-five healthy human controls were enrolled in this study.Venous blood samples were collected from all of these subjects.Then,enzyme-linked immunosorbent assay was carried out to measure the serum levels of interleukin-6 (IL-6) and amyloid protein A (AA),and immunoturbidimetry to determine the levels of serum high sensitive C-reactive protein (Hs-CRP) and plasma D-dimer.The patients with CU were classified into three groups according to the severity of clinical symptoms.The levels of serum IL-6 and plasma D-dimer were given in mean ± standard deviation,and those of serum AA and Hs-CRP in mean (25th percentile,75th percentile).Rank sum test and t test were performed to compare these parameters between the patients and controls,and Spearman rank correlation analysis was conducted to study the relationship among these parameters as well as between these parameters and symptom severity in these patients.Results The levels of serum IL-6,serum AA and plasma D-dimer were (10.70 ± 4.94) ng/L,4.92 (8.22,12.51) μg/L,and (222.32 ± 163.69) μg/L respectively in the patients with CU,significantly higher than those in the healthy controls ((7.49 ± 3.41) ng/L,2.11 (1.21,2.83) μg/L,(104.72 ± 43.12) μg/L,respectively,all P＜ 0.05),while no significant differences were observed between the patients and controls in the level of serum HsCRP (0.30 (0.10,1.40) mg/L vs.0.30 (0.10,0.55) mg/L,P ＞ 0.05).In patients with CU,the levels of serum IL-6 were unrelated to those of serum Hs-CRP,serum AA,or plasma D-dimer (all P ＞ 0.05),whereas a positive correlation was observed between the levels of serum Hs-CRP and AA (r =0.67,P ＜ 0.01),serum Hs-CRP and plasma D-dimer (r =0.49,P ＜ 0.01),serum AA and plasma D-dimer (r =0.38,P ＜ 0.01).Further more,the levels of serum Hs-CRP,AA and plasma D-dimer were significantly correlated with symptom severity in patients with CU (r =0.63,0.62,0.47,respectively,all P ＜ 0.01).Conclusions Acute-phase response coexists with the activation of coagulation system in patients with CU,suggesting a potential association between acute-phase response and coagulation system activation.

Objective To detect the expression and the biological activity of a recombinant adenovirus expression vector carrying human neurotrophin-3 (NT-3) receptor TrkC gene (Adeno-TrkC) in neural stem cells. Methods The expression of TrkC mRNA in 293 cells infected with Adeno-TrkC was detected by RT-PCR, and the expression of TrkC protein in neural stem cells infected by Adeno-TrkC was detected with immunocytochemistry and Western blotting. The effects of human neurotrophin_3 (NT-3) on the neural stem cells infected by Adeno-TrkC differentiating into neuron-like cells and astrocyte-like cells in vitro were observed. Results The transcription of TrkC mRNA and the expression of TrkC protein was detected in 293 cells and neural stem cells infected by Adeno-TrkC. This kind of TrkC was able to make more neural stem cells differenting into neuron-like cells in vitro with its ligand NT-3 and the percentage of neuron-like cell’s differentiation was 55^2% which was higher than that of other control groups.Conclusion TrkC protein is expressed in neural stem cells transfected with Adeno-TrkC and is able to make neural stem cells differenting into neuron-like cells. The present study may provide the basis on gene therapy of central nervous injury using further NT-3 and TrkC.

Objective To explore the effects of neurotrophin-3 genetically modified Schwann cells(NT-3-SCs) and neural stem cells(NSCs) combinative transplantation to promote the neurons'survival and axonal regeneration of the spinal cord transected rat. Methods After the transected spinal cord injury(SCI) model was established in SD rats,NT-3-SCs or LacZ report gene modified Scwhann cells(LacZ-SCs) were combinative transplanted with NSCs into the transected site.60 days later,fluorogold(FG) was injected into the caudal spinal cord fo the transected site.7days after FG injected,the sacrifice,cryosection and morphology serious studies were performed to observe the FG labeled neurons in the rostral spinal cord(RSC) of transected site,red nuclei(RN) and the sensorimotor cortex(SMC);The survival neurons in the L_1 Clark's nuclei(CN),RN and SMC were couut,and regenerated axons within or near the transected spinal cord observed. Results From more to less,the order of groups of the survival neurons in the CN,RN and SMC was NT-3-SCS & NSCs group,LacZ-SCs & NSCs group,experimental control group.In the NT-3-SCs & NSCs group and LacZ-SCs & NSCs group,there were 5-HT,CGRP and SP positive axons within or near the transected spinal cord.And some FGlabeled cells were found in RSC,RN and SMC. Conclusion Combinative grafting NSCs and NT-3-SCs could promote the neurons'survival and axonal regeneration of the spinal cord injured rat.

AIM: To explore the effects of neurotrophin-3 (NT-3)-genetically modified Schwann cells (NT-3-SCs) on differentiation of neural stem cells (NSCs) into the neuron-like cells. METHODS: The NSCs were co-cultured with NT-3-SCs. Report gene LacZ genetically modified Schwann cells (LacZ-SCs) and normal SCs respectively in vitro. 7 d later, the differentiation of NSCs was studied by immunohistochemistry, and the percentage of neuron-like cells was calculated. RESULTS: NSCs differentiated to the GFAP-positive cells (glial-like cells) and NF-positive cells (neuron-like cells) in vitro. Compared to the normal SCs, NT-3-SCs more efficiently promoted NSCs to differentiate into the neuron-like cells. The effect of LacZ-SCs was as the same to the normal SCs. CONCLUSION: NT-3-SCs promote NSCs to differentiate into the neuron-like cells. [