To study the possibility of enhancing IFN ? level in the lung of rats by gene transfection. The recombined IFN ? expression vector in eukaryotic cell was constructed, and it was transfected into lungs of immunocompromised rats. The expression of pLXSN IFN and pLXSN in the genomic DNA of the transfected cells was investigated, and the level and activity of IFN ? in the BALF and sera was assessed. The results showed that the sequence of the IFN ? gene in the constructed recombinant pLXSN IFN eukaryotic cell was the same as that of the IFN ? of rats found in the Gene Bank. The band of transfected plasmid was found when the eletrophoresis analysis of PCR product of genomic DNA of the transfected cells in BALF was done. It could still be the found 21 days after transfection. The level and activity of IFN ? in the BALF of the transfected pLXSN IFN group were markedly higher than those of transfected pLXSN group.On the other hand, the level and activity of IFN ? in sera showed no difference between the two groups. It was suggested that the constructed recombinant IFN ? exprossion vector in eukaryotic cell could be transfected into the cells of the lungs of rats successfully, inserted into the genomic DNA, resulting in active secretion of IFN ?, and prolongation of its expression.

Objective To establish a three-dimensional finite element model of thoracolumbar fractures treated by mono-segmental instrumentation in the fractured part for testing effect of such fixation mode on adjacent segments.Methods CT scanning data of T10-L2 were used to build a normal model at T10-L2 region,a fracture model at T12 segment as well as a mono-segment fixation or short-segment fixation model.Stress of discs and vertebral body adjacent to the fixed vertebrae were tested in axial compression,anteflexion,extention,lateroflexion,and axial rotation.Results The fracture model presented significant increase concerning stress of nucleus pulposus and annular fibrosus at T10-T11 segments and annular fibrosus at L1-L2 segments in anteflexion,extention,and lateroflexion when compared with the normal model.General raise range of the stress reached around 75％,but was dropped to 23％ after short-segment fixation and to 18％ after mono-segmental fixation.And again,stress of nucleus pulposus at L1-L2 segments was increased by 46％ or so,which was declined to 12％ after short-segment fixation and to 8％ after mono-segmental fixation.Stress at lower endplate of T10 and at upper endplate of L2 in the fracture model group were increased by 24％ and 43％ respectively when compared to the normal model,but both presented a notable drop after internal fixation.The latter was decreased to a level slightly higher than that of model group,namely 8％ more after short-segmental fixation and 4％ more after mono-segmental fixation; the former was decreased to a level even lower than that of control group,namely 2％ less after short-segmental fixation and 8％ less after mono-segmental fixation.Conclusion Mono-segmental fixation reduces adjacent disc stress in contrast to conventional short-segmental fixation and hence is an effective alternative treatment of monosegmental thoracolumbar fractures.

Objective To evaluate the association between serum 25-hydroxyvitamin D3 [25 (OH) D3],parathyroid hormone,and arterial stiffness in patients with type 2 diabetes.Methods Serum 25 (OH) D3 and parathyroid hormone(PTH) were determined in a cross-sectional sample of 258 patients aged 30 years and over.Arterial stiffness was assessed by pulse wave velocity(PWV) obtained with a VP-1000 pulse wave unit.Fasting plasma HbA1c,lipid profile,calcium,and high sensitive-C reactive protein were determined.Results (1)The prevalence of vitamin D3 deficiency was high(79.84％) in patients with type 2 diabetes.(2) Those with lowered serum vitamin D3 levels had raised PWV [(1610.76 ± 142.70 vs 1527.95 ± 58.02) cm/s,P＜0.05].(3) Multiple stepwise regression analysis showed that 25 (OH) D3 was an impact factor of PWV risk score,which was independent of age,duration of diabetes,and systolic blood pressure(β =-0.256,P＜0.01).(4) Serum PTH was positively correlated with PWV (r =0.210,P ＜ 0.05) and systolic blood pressure (r =0.229,P ＜ 0.05),but negatively correlated with 25 (OH) D3 (r =-0.153,P ＜ 0.05).Conclusions 25 (OH) D3 deficiency is common in patients with type 2diabetes,and a low serum 25 (OH) D3 level is significantly associated with increased arterial stiffness in these patients.The association of serum PTH with arterial stiffness may result via changes in vitamin D and blood pressure.

Objective To study the effects and mechanisms of resveratrol (Resv) on the expression of adiponectin receptors ( AdipoR1 and AdipoR2 ) in rat mesangial cells (MCs) cultured in intermittent high glucose medium.Methods The MCs cultured in normal glucose ( 5.6 mmol/L) medium were transfected by the plasmid vector of the FoxO1 short hairpin RNAs ( FoxO1 shRNA ),and then cultured in intermittent high glucose medium (5.6mmol/L or 30 mmol/L,alternately every 8 hours ) and resveratrol ( Resv 20 μmol/L).The MCs cultured in normal glucose (5.6 mmol/L) medium served as normal glucose control group (NG),and the MCs exposed to iutermittent high glucose medium were divided into five groups:intermittent high glucose group (IHG),IHG+Resv group,IHG+FoxO1 shRNA group,IHG+ Resv + FoxO1 shRNA group,IHG + Negative control group ( shNC ),each group was cultured for 72 h.The level of reactive oxygen species (ROS) was assessed by Fluorescence microplate reader.The mRNA levels of Sirt1,Foxo1,AdipoR1,and AdipoR2 were assessed by RT-RCR.The protein levels of FoxO1,phosphorylation FoxO1 ( p-FoxO1 ),AdipoR1,and AdipoR2 were assessed by Western blotting.Results ( 1 )Compared with NG,intermittent high glucose significantly decreased the mRNA expression of Sirt1,but markedly increased the level of p-FoxO1.Furthermore,the mRNA and protein expression levels of AdipoR1 were obviously decreased while the level of ROS was enhanced in IHG ( all P＜0.05 ).(2) Compared with IHG,the mRNA and protein expression levels of FoxO1 and AdipoR1 were inhibited,but the level of ROS was enhanced in group IHG+FoxO1 shRNA ( all P＜0.05 ).( 3 ) Compared with IHG,after treating with Resv,the mRNA expression level of Sirtl was increased,whereas the level of p-FoxO1 was decreased.Moreover,the mRNA and protein levels of AdipoR1 was increased while the level of ROS was lowered in group IHG+Resv (all P＜0.05 ).(4) Compared with group IHG+Resv,the mRNA and protein levels of FoxO1 and AdipoR1 were inhibited,but the level of ROS was increased in group IHG+Resv+FoxO1 shRNA (all P＜O.05).(5) In addition,the mRNA and protein expression level of AdipoR2 showed no significant difference among these groups ( P＞0.05 ).Conclusion ( 1 ) Resveratrol significantly increases the expression of AdipoR1 in MCs induced by intermittent high glucose.( 2 ) FoxO1 plays an important role in regulating the expression of AdipoR1 by resveratrol.

Objective To study the effects of inhibiting forkhead transcription factor O1 (FoxO1)expression by small interference RNA (siRNA) on the glucose utilization of insulin resistant HepG-2 cell line and the mechanism.Methods FoxO1 gene-targeted siRNA vector,which carried a red fluorescence,was constructed and then confirmed by DNA sequencing.HepG-2 cells were induced to a status of insulin resistance by being exposed to 10-6 mol/L insulin for 24 h.The study included 4 groups: HepG-2 cell group cultured with normal medium (group A) ; insulin resistant HepG-2 cell group (group B) ; insulin resistant HepG-2 cell group into which FoxO1 siRNA vector was transfected (group C) ; insulin resistant HepG-2 cell group into which Lipofectamine2000 was added (group D).The transfection efficiency could be estimated by observing the expression of red fluorescence.The expression of FoxO1 mRNA was analyzed by RT-PCR.The expressions of IRS-2 and tyrosine phosphorylation were detected by Western blot and immunoprecipitation.Results FoxO1 gene-targeted siRNA vector was built successfully.The expression of red fluorescence was the strongest at 48 h after transfection.Compared with group A,the glucose consumption and the expression of IRS-2 tyrosine phosphorylation of group B were decreased (P<0.01),and expression of FoxO1 mRNA was increased (P<0.05) in group B.There was no difference in the expressions of IRS-2 protein between A and B groups.Compared with group B,the expression of FoxOl mRNA was decreased (P < 0.01),and the glucose consumption and expression of IRS-2 tyrosine phosphorylation were significantly increased (P<0.05) in group C.There was no difference between group D and group B in glucose consumption, FoxO1 mRNA, IRS-2 protein and IRS-2 tyrosine phosphorylation expressions.Conclusions Inhibiting the expression of FoxO1 gene in insulin resistant HepG-2 cells seems to improve insulin sensitivity by feedback regulating the IRS-2 tyrosine phosphorylation expressions.

Streptozotocin-induced diabetic rats were treated with resveratrol for 12 weeks.Compared with group of diabetic rats without treatment,the levels of urine albumin,serum creatinine,and blood urea nitrogen were significantly decreased and pathological changes in kidney were improved in treatment group ( P＜0.05 ).The mRNA expressions of FoxO1 and catalase were higher and FoxO1 phosphorylation level in diabetic rats treated with resveratrol was lower than that in diabetic rats without treatment (P＜0.05),suggesting that resveratrol may protect the kidneys of diabetic rats via regulating expression of FoxO1.

AIM: To study the effect of IFN-? inhalation on the anti-infection ability of the lungs in the immunocompromised host. METHODS: The immunological factors in the immunocompromised rats and the immunocompromised rats administrated IFN-? via aerosol were investigated after 1, 3, 7 days when they were injected Candida albicans via tracheal The Canidda albicans count of the left lung was also determined after 7 days when injecting pathogen. RESULTS: The Canidda albicans count of the left lung in IFN-? group was significantly less than that of control group. The phagocyting and bactericidal percentages, Ia antigen expression percentages, the levels of TNF-?, IL-1? and IL-6 in the culture supernatant of the AM, the activity of IFN-? and TNF-? in BALF (except the TNF-? on 7 th day) in IFN-? group were markedly higher than those in control group. The expression of IFN-? and IL-1? pulmonary tissues in IFN-? group was higher than that in control group. The expression of TNF-? in IFN-? group was less than that in control group. The expression of IL-6 was no changes between two groups. The levels of IFN-?, IL-1? and IL-6 in the blood (except IL-1? on 3 rd day), and the killing ability of the lymphocytes in blood had no difference between two groups. CONCLUSION: Administration of IFN-? via aerosol obviously enhanced the anti-infection ability of the lungs in the immunocompromised host, but has no influence on the whole body cellular immunity.

Objective　To study the effect of IFN-γ inhalation via aerosol on cytokines of the immunocompromised rats. Methods　Immunocomprised rat model was established with cortisol acetate injection for 14 d and then Candida albicans fluid was injected by tracheal for establishing am immuno comprised with pulmonary infection model. IFN-γ was inhaled with aerosol 1 d before the bacterium injection and then for 1, 3 and 7 d respectively. The activity of TNF-α, and the levels of IL-1β and IL-6 in the supernatant of the cultured alveolar macrophage(AM), the activity of IFN-γ and TNF-α in bronchial alveolar lavage fluid (BALF), the expressions of IFN-γ,TNF-α, IL-1β, and IL-6 of the lung tissues, the level of IFN-γ,IL-1β, and IL-6 in the serum were investigated. Results　The activity of TNF-α, and the levels of IL-1β and IL-6 in the culture supernatant of the AM of the rats treated with IFN-γ were significantly higher than those of the control. The activity of IFN-γ and TNF-α in BALF was higher in the IFN-γ inhaled rats than in the control (except the activity of TNF-α on the 7th day). The expressions of IFN-γ and IL-1β in lung tissues was higher in the rats treated with IFN-γ than in the control. The expression of TNF-α in the rats treated with IFN-γ was less than that in the control rats. The expression of IL-6 had no difference between 2 groups. And no difference was found in the activity of IFN-γ, and the levels of IL-1β and IL-6 in the serum between 2 groups(except IL-1β on the 3rd day). Conclusion　Administration of IFN-γ via aerosol can obviously increase the activity or levels of some cytokines in the lung of the immunocompromised rats, but has no effect on them in serum of the immunocompromised rats.

To study the effects of forkhead transcription factor O1 (FoxO1) on the expression of type Ⅳ collagen and desmin in podocytes of diabetic rats.Streptozotocin-induced diabetic rats were divided into three groups:diabetic rats (DM group),rats transfected with blank lentiviral vectors (diabetes mellitus plus LV-pSC-GFP group,LV-NC group),and rats which were transfected with lentiviral vectors carrying constitutively active FoxO1 (diabetes mellitus plus LV-FoxO1-AAA group,LV-CA group).Rats which received an injection of diluent buffer served as normal control.At 2,4,and 8 weeks after transfection,the levels of urine albumin,blood glucose,blood urea nitrogen,and serum creatinine were measured.Realtime PCR and Western blotting were performed to measure the mRNA and protein levels of FoxO1,COL4A3,COL4A5,and desmin in the renal cortex.Moreover,light microscope and electron microscope were used to observe the structural changes in glomerulus and podocytes.Compared with LV-NC and DM group,in LV-CA group,there was a significant increase in the mRNA and protein levels of FoxO1,and a distinct decrease in the levels of urine albumin,blood urea nitrogen and serum creatinine of rats (except at the twoweek time point) (all P＜0.05),the mRNA and protein levels of COL4A3,COL4A5,and desmin were all decreased (all P＜0.05),and pathological changes in kidney were also improved.Upregulating the expression of FoxO1 by transfecting with constructed lentiviral vectors can definitely improve the abnormal expression of type Ⅳ collagen and desmin in podocytes of diabetic rats.

Objective To clarify the clinical features and genetic background of a kindred of primary pigmented nodular adrenocortical disease (PPNAD).Methods Detailed clinical characteristics and laboratory test results from a ten-year old girl diagnosed as PPNAD were collected.Seven members of her family were screened for Cushing syndrome and Carney complex,and their blood DNA was extracted and sequenced for PRKAR1A,PDE11A,PDE8B and CTNNB1 mutations with ABI3730.Results The girl presented with symptoms and signs of hypercortisolism,while no features of Carney complex were observed.Hypercortisolemia,suppressed corticotrophin and high urinary free cortisol level were revealed.Cortisol level could not be suppressed both in high and low dose dexamethasone suppression test.The diagnosis of adrenocorticotrophic hormone (ACTH)-independent Cushing syndrome was established.Image and pathology of adrenal glands were in accordance with PPNAD.Other family members showed no evidence of Cushing syndrome or Carney complex.DNA sequencing showed that the patient harbored a missense mutation,C18G.Her father and younger sister were proved to be carriers of this mutation.Conclusion A Chinese PPNAD family was identified clinically and genetically,and a novel missense mutation of PRKAR1A was found.