Objective To express a mutated insulin gene in HepG-2 cell line to further research of insulin gene therapy. Methods Native human insulin cDNA was obtained from fetus pancreas with RT-PCR. Furin consensus cleavage sequence was introduced into proinsulin cDNA with site-directed mutagenesis (overlap extension PCR), and the new sequence was named as INS/furin. Subsequently, INS/furin was subcloned into the multiple clone sites of plasmid p(G1RE)3BP-1Luc. The new plasmid p(G1RE)3BP-11?furin was identified with the method of enzyme digestion by Hind Ⅲ and EcoR V. HepG-2 cells were transfected with the plasmid p(G1RE)3BP-11?furin by liposome-mediated method. The transfected HepG-2 cells were incubated for 48h in a glucose-containing medium (25mmol/L), and then the conditioned media were collected and HepG-2 cells were harvested respectively. The expression of INS/furin mRNA in transfected HepG-2 cells was examined by RT-PCR, the regained DNA was sequenced and insulin in conditioned media was investigated by radioimmunoassay. Results Two enzymes, Hind Ⅲ and EcoR V, digested p(G1RE)3BP-11?furin, and 2 fragments with length of 260 bp and 4 700bp, were obtained. The 260bp fragment was identified as insulin/furin, indicating that the target gene had been successfully inserted in specific sites. RT-PCR showed that insulin/furin mRNA was expressed in transfected HepG-2 cell, and the regained DNA was confirmed as insulin/furin by sequencing; while insulin was detected by radioimmunoassay in conditioned media. Conclusion The recombinant mammalian expression plasmid p(G1RE)3BP-11?furin has been successfully constructed, and transfected into HepG-2 cells, which therefore may efficiently secrete bioactive insulin.

Objective To evaluate the feasibility of liver senescence model with senescence accelerated mouse prone 8 (SAMP8), and to explore the possible mechanism of oxidative stress in the process of liver aging in SAMP8. Methods Male SAMP8 mice at the age of 9 months were chosen as research objects, and senescence accelerated mouse resistance 1 (SAMR1) mice were used for normal control. Histopathological changes in the liver of SAMR1 and SAMP8 mice were observed by hematoxylin-eosin (HE), Sudan Ⅳ and Masson staining. Senescence associated β-galactosidase activity was measured by histoehemical staining method, and the activities of superoxide dismutase (SOD), eatalase (CAT), glutathione peroxidase (GSH-Px) and the level of malondialdehyde (MDA) in liver homogenate were examined by chemical colorimetry. Results Compared with SAMR1 group, the liver degenerative changes of SAMP8 mice were observed by microscopy, such as extensive fatty degeneration, focal necrosis of hepatocytes and inflammatory cells infiltration. Meanwhile, senescence associated β-galactosidase-positive cells were significantly increased in SAMP8 group [(78.1±11.0) vs.( 23.9±8.8),t=10.887, P<0.01]. In addition, the activities of SOD, CAT and GSH-Px in liver homogenate were decreased [SOD: (214.8 ± 34.8) vs. ( 295.3 ± 29.7), t = 4.975,P<0.01;CAT: (23.0±4.0) vs. ( 36.3±8.3),t=4.084,P<0.01;GSH-Px: (524.0±74.2) vs. (648. 4±102.8) ,t=2. 776, P<0. 05]and the level of MDA was markedly increased ((2.3±0.2) vs. (1.8±0. 1),t = 6. 329, P<0. 01]. Conclusions SAMP8 mice is a feasible animal model for the study of liver senescence, and oxidative stress may play an important role in the process of liver aging in SAMP8.

Objective To evaluate the effect of health education on blood glucose level in patients undergoing renal transplantation.Methods Two hundred and four patients who underwent renal transplantation were randomly assigned to the control group(n =102)and the intervention group(n =102).Participants of the control group received traditional health education,and those in the intervention group received general health education.Food intake,physical exercises.disease-related knowledge,treatment satisfaction,fasting plasma glucose and 2 h blood glucose were assessed at 6 months.Results Daily diet,physical exercises,disease-related knowledge,treatment satisfaction,and blood glucose control were significantly improved in the intervention group(all P＜ 0.05).Conclusion General health education may contribute to the control of hyperglycemia and improve long-term survival of renal transplantation receivera.

Objective　To investigate the significance of TGF-β1, TGFRl and TGFR2 in the pathogenesis and prognosis in patients with myelofibrosis. Methods　The expression of TGF-β1 and its receptors (TGFR1 and TGFR2 ) in bone marrow tissues and the level of TGF-β1 in the blood of 23 patients with myelofibrosis were detected by SABC immunocytochemistry and ELISA repectively. Results　Expression of TGF-β1 and TGFR 1 was significantly higher in primary and secondary myelofibrosis patients than that of the control. No significant difference of TGFR2 expression was found between the groups of myelofibrosis and the control (P>0.05). The level of TGF-β1 in the blood of the patients with myelofibrosis was significantly higher than that of the control (P<0.01) and more obvious in secondary cases while TGF-β1 decreased nearly to the normal level when patients were in clinical remission. Conclusion　TGF-β1 and it's receptors may be involved in the pathogenesis of myelofibrosis and might be of importance for the prognosis of the patients with myelofibrosis.

Objective To investigate the nursing professional values of different nursing groups and probe into the influencing factors.Methods By cluster and stratified sampling,a total of 583 participants including nursing students and registered nurses were investigated by Nursing Professional Values Scale.Results Nursing students and registered nurses hold a higher level of nursing professional values.Nursing professional value scores of nursing students were significantly higher than registered nurses.There was statistical difference between different nursing groups.Working years and monthly income were important influencing factors.Conclusions Nursing educators and administers could choose effective methods according to the condition to improve nursing professional values.

Objective To determine the retest reliability and measurement error of walking energy consumption measurement in children with cerebral palsy in special school. Methods 13 children with cerebral palsy studying in Shanghai Pudong Special Education School from September to December in 2010 were enrolled in this study. They were asked to walk up and down continuously on a 50-meter-long walking pace in 6 minutes, while the distance of walking and the heart rate of both rest and walking were measured to calculate physical cost index (PCI). After 1 week, they took the 2nd measurement at the same time, the same location and the same condition. Then the results between the former and the later measurements were compared to identify the retest reliability and measurement error. Results The study showed a good retest reliability in heart rate of walking, distance of walking and PCI (ICC=0.77~0.83), but a low retest reliability in heart rate of rest (ICC=0.38). The study also showed a high measurement error in PCI. Conclusion PCI had good retest reliability and high measurement error, and was easily affected by the emotion of the children. So it was limited as a measurement of walking energy consumption in children with cerebral palsy.

Objective To investigate the impact of hypoxia on the expression of early growth response-1 (Egr-1) and monocyte chemoattractant protein-1 (MCP-1) in mouse aorta,and to probe the underlying mechanism involving receptor for advanced glycation end-products (RAGE).Methods 3-month-old C57BL/6 mice were subjected to hypoxia [(6.0±0.5) ％ oxygen] to establish the global hypoxia model(n=6 rats for each).Aortas were dissected,Egr-1 mRNA and MCP-1 mRNA were detected by real time RT-PCR,Egr-1 and RAGE proteins were tested by Western blot,and Egr-1 DNA binding activity was assayed by electrophoretic mobility shift assay (EMSA).For blockade of RAGE,mice were pretreated with soluble RAGE (sRAGE) for 1 h by intra-peritoneal injection before they were exposed to hypoxia.Mice with normoxia were used as controls.Results After 30 minutes of hypoxic exposure,Egr-1 mRNA in aorta was increased to (28.3±0.9)folds compared with normoxic controls (F=617.17,P＜0.01),and the induction persisted for at least 3 hours.After 45 minutes of hypoxic exposure,Egr-1 proteins in aorta was increased to (5.7 ± 0.3) folds compared with normoxic controls (F =57.18,P＜ 0.01); the enhanced DNA binding activity of Egr-1 by hypoxia was attenuated by pretreatment with anti-Egr-1 lgG.After 4 hours of hypoxic exposure,MCP-1 mRNA expression in aorta was increased to(4.0±0.3)folds compared with normoxic controls (F=30.68,P＜0.01).RAGE antigen was increased significantly within 30 minutes of hypoxic exposure,with the peak at 15 minutes; hypoxia-induced Egr-1 mRNA expression was significantly attenuated by pretreatment with sRAGE (3.3 ± 0.2) folds compared with normoxic controls (F =30.20,P＜0.01).Conclusions Hypoxia significantly induces Egr-1 and MCP-1 upregulation expressions in mouse aorta,and blockade of RAGE significantly attenuates hypoxia-induced Egr-1 expression.Thcsc findings suggest RAGE signaling is involved in hypoxia-induced vascular inflammatory stress,and highlight this receptor as a potential therapeutic target to protect tissues injured by hypoxia.

Objective To estimate the effect of itermittent subglottic secretions drainage(ISSD)on management of artificial airway to prevent tube-related pulmonary pneumonia.Methods One hundred ICU patients with intubation for artificial airway were divided equally into control and experiment group by random digits table.Both were managed with construction of artificial airway and besides the experiment group received ISSD. The two groups were compared in terms of incidence and occurrence time of catheter-related pneumonia, time for airway opening and ICU stay.Result The incidence and the occurrence time of pneumonia, time for airway opening and ICU stay time in the experiment group were significantly lower or shorter than those in the control group with statistical difference(all P<0.05).Conclusion ISSD is effective in decreasing the incidence of catheter related pneumonia, shortening the occurrence time of catheter related pneumonia and ICU stay time for the patients with artificial airway.

Objective: To investigate the clinical effect of acupuncture-moxibustion therapy on simple obesity due to spleen deficiency. Method: Sixty-eight cases of simple obesity due to spleen deficiency syndrome, including internal dampness due to spleen deficiency, qi deficiency of lung-spleen, yang deficiency of spleen-kidney, were randomly allocated into two groups, treatment group (36 cases) and control group (32 cases). The former group was treated with warm needling moxibustion method, and the latter was treated with electroacupuncture. The therapeutic effect and body weight (BW) were observed, respectively. Result: There was a significant difference between the two groups in the total effective rate (P<0.05); there was a significant difference between the two groups in the decrease of BW (P<0.01), furthermore, the long-term effect in warm needling moxibusiton group was better than that in the electroacupuncture group. Conclusion: The effect of needle wanning moxibustion therapy on simple obesity due to spleen deficiency is accurate, and it is worth to spread.