BACKGROUND: DNA enzyme targeting early growth response factor 1 (Egr-1) mRNA (ED5) can inhibit expression of downstream target genes by specificaly inhibiting expression of early growth response factor 1. OBJECTIVE:To investigate the effects of ED5 on the expression of plasma tissue factor after vascular baloon injury in rats and the mechanism of inhibiting neointimal hyperplasia. METHODS: Intimal injury models of the left carotid artery were made in rats. Then, ED5, MgCl2 and FuGene6 were injected into the injured vascular segment. RESULTS AND CONCLUSION:At days 3, 7, 14, 21, the expression levels of Egr-1 and tissue factor in plasma were significantly down-regulated in the ED5 transfection group compared with the MgCl2 and FuGene6 groups (P < 0.01); and neointimal hyperplasia was significantly inhibited by ED5 at days 7, 14 and 21 after modeling (P < 0.01). ED5 may inhibit neointimal hyperplasia folowing baloon injury of rat common carotid artery through down-regulation of tissue factors.

We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector. In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII. By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity. The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells were up to (149 +/- 23) ng x mL(-1) and (1.12 +/- 0.14) u x mL(-1) respectively greater than that of intein-fused wild type heavy (HCIntN) and light chain (IntCLC) co-transfected cells [(99 +/- 14) ng x mL(-1) and (0.77 +/- 0.13) u x mL(-1)] indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity. A cellular mechanism-independent BDD-FVIII splicing was also observed. It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.

Objective To observe the characteristics of cognitive impairment in patients with primary hypothyroidism.Methods A total of 90 primary hypothyroidism patients untreated with thyroid hormone were selected.All the 90 patients were divided into subclinical hypothyroidism group,mild or moderate hypothyroidism group and serious hypothyroidism group,and 30 patients in each group.The other 30 healthy volunteers were selected as controls.Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) were used for the testing their orientation,immediate memory,attention and calculation,delayed recalling,linguistic competence,visual space and execution,naming ability and abstracting power.One-way analysis of variance was performed to determine significant differences among four groups.Results The MMSE scores in subclinical hypothyroidism group,mild or moderate hypothyroidism group and serious hypothyroidism group((27.53 ± 2.16),(26.90±1.88) and (24.80 ± 2.10) respectively) were lower than those of the control (28.23 ± 1.33).The MoCA scores of the above hypothyroidism groups ((23.57 ± 3.33),(2 1.60 ± 2.81) and (20.53 ± 3.03) respectively) were also lower than that of the control (26.63 ± 2.31) (P ＜ 0.05).Except for orientation and immediate memory,statistical significances of the other cognitive function were existed between hypothyroidism groups and the healthy controls (P＜ 0.05).With the increase in severity of hypothyroidism,the abnormality of attention,calculation,linguistic competence,visual space and executive ability,naming ability and abstracting power were appearing gradually in hypothyroidism groups (P ＜ 0.05),and the scores were low(P＜ 0.05).Conclusion Defects of attention and calculation,delayed recalling,linguistic competence,visual space and execution,naming ability and abstracting power are existed in primary hypothyroidism patients.

Arthritis, Rheumatoid ; diagnosis ; therapy ; Evidence-Based Medicine ; Humans ; Medicine, Chinese Traditional ; methods

Objective To figure out the optimal parameters of a volumetric modulated arc therapy ( VMAT) plan for cervical and upper esophageal cancer by quality evaluation of VMAT plans with different parameters, and to provide a reference for the design of clinical VMAT treatment plan. Methods Ten patients with cervical esophageal cancer and ten patients with upper esophageal cancer were enrolled as subjects. The Nucletron Oncentra 4. 3 treatment planning system was used to generate plans for Elekta Synergy VMAT accelerator. Six VMAT plans were made with variation in the gantry angle ( 2°, 3°, and 4°), the maximum delivery time (80 s, 110 s, and 150 s), and the collimator angle (0° and 45°). The doses to the planning target volume and organs at risk were analyzed by paired t test. Results For cervical and upper esophageal cancer, the quality of VMAT plans with a collimator angle of 45° was better than those with a collimator angle of 0°(P=0. 003?0. 007). For cervical esophageal cancer, there was no significant difference in quality between VMAT plans with a maximum delivery time of 110 s or 150 s and those with a maximum delivery time of 80 s ( P>0. 05 );for upper esophageal cancer, there was also no significant difference in quality between VMAT plans with three different maximum delivery times ( P>0. 05 ) . For cervical esophageal cancer, the VMAT plans with a gantry angle of 3° had a better quality than those with a gantry angle of 2° or 4°(P=0. 010?0. 048). For upper esophageal cancer, the VMAT plans with a gantry angle of 3° had a better quality than those with a gantry angle of 4° ( P=0. 010?0. 048) . Compared with those with a gantry angle of 2° , the VMAT plans with a gantry angle of 3° had a slightly better dose distribution in the target volume ( P=0. 046 ) , but a slightly higher dose to lung tissue ( V25 and V30 , P=0. 007 and 0. 026) . Conclusions The optimal initial parameters of a VMAT plan for cervical and upper esophageal cancer are a collimator angle of 45°, a maximum delivery time of 80 s, and a gantry angle of 3°.

Objective To discuss the effect and possibility of NIPPV in ILD combined with respiratory failure. Methods A total of 37 cases of ILD combined with respiratory failure admitted by our hospital during Feb 2004 to Oct 2007 were divided into treatment group (20 cases) and control group (17 cases). Routine pharmaceutical intervention was adopted in both groups while NIPPV was given in the patients of treatment group. The symptoms、signs and the arterial blood gases were observed and analyzed. Results Among 20 patients,3 gave up treatment , 17 patients were successfully treated with the NIPPV therapy. There were significant improvement of PaCO2、PaO2、pH and respiratory rate ,heart rate after 2h NIPPV. Conclusion NIPPV is an effective method for ILD combined with respiratory failure,which can save the patients life and decrease the complications.

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.

BACKGROUND:Prefabricated customized bone flaps have the advantages of few trauma, good vascularization, ossification with predetermined shape, and can be used to restore bone defects with compromised blood bed.
OBJECTIVE:To establish animal models of mandibular reconstruction with prefabricated, customized bone flaps.
METHODS:After computed tomography scanning of nine rhesus’ head, customized meshes were made. After loading with recombinant human bone morphogenetic protein-2-incorporated demineralized freeze-dried bone al ograft (DFDBA) or coral ine hydroxyapatite (CHA), the constructs were implanted in latissimus dorsi muscle. Meanwhile, segmental mandibular defects were created, and the customized meshes loaded with DFDBA, CHA, or recombinant human bone morphogenetic protein-2-incooperated DFDBA and CHA were implanted in situ. At 13 weeks, prefabricated bone flaps with recombinant human bone morphogenetic protein-2-incorporated DFDBA or CHA were transferred to repair segmental mandibular defects. Clinical and histological analyses were used to evaluate the ossification and vascularization of the prefabricated implants in ectopic and orthotopic sites.
RESULTS AND CONCLUSION:Segmental mandibular defects were successful y restored with prefabricated bone flaps and recombinant human bone morphogenetic protein-2-incorporated CHA in situ, but other segmental mandibular defects remained with recombinant human bone morphogenetic protein-2-incorporated DFDBA, DFDBA and CHA in situ. Moreover, mandibles reconstructed with prefabricated bone flaps revealed more regenerated and homogeneous bone formation than other reconstructions. These findings suggest that the animal model of mandibular reconstruction with prefabricated, customized bone in rhesus monkey is applicable.

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.