Objective To establish a system for rapidly detecting single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) using hybridization probes and melting temperature (Tm) curve analysis. This technique is suitable for population-based studies on the interaction between genetic factors and environmental exposures and the risk of Parkinson's disease (PD). Methods mtDNA was extracted from the blood. Rapid polymerase chain reaction (PCR) and melting curve analyses were performed with primers and fluorochrome-labeled probes on a LightCycler. Genotyping of 10 SNPs was based on the analysis of allele-specific Tm of detection probes. The results of melting curve analyses were verified by sequencing all 150 PCR products. Results Real-time monitoring showed optimal PCR amplification of each mtDNA fragment. The changes at nucleotide positions 1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708, 16391 from wild-type to mutant genotype resulted in 6.51, 8.29, 3.26, 7.82, 4.79, 2.84, 2.73, 9.04, 8.53, 9.52℃ decline in Tm of the detection probes respectively. Genotyping of all detected genes was verified by 100% correspondence with the results of sequencing. Conclusion A rapid and reliable detection system for identifying mitochondrial polymorphisms and haplotypes has ben developed based on hybridization probe technology. This method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and may prove useful for exploring the molecular etiopathogenesis of PD, identifying markers of genetic susceptibility, and protecting susceptible individuals from PD.

Objective To explore the effect of BPDE on the expression of N-Ras in the human bronchial epithelial cell line. Methods The levels of mRNA and protein expression in BPDE transformed 16HBE cells(16HBE-T) and untransformed control 16HBE cells(16HBE-N) were examined by using RT-PCR and Western blot. Locations and expression levels of protein in both kinds of cells were analyzed by immunocytochemical method. Results Compared with 16HBE-N, the levels of mRNA and protein of N-Ras significantly increased to 3.616 and 1.600 times in 16HBE-T. Immunocytochemical method showed N-Ras protein in 16HBE-T and 16HBE-N expressed in the cytomembrane and cytoplasm, but the expression level of protein in 16HBE-T was significantly higher than that in 16HBE-N. Conclusion The up-regulated expression of N-Ras oncogene may play an important role in the malignant transformation of 16HBE induced by BPDE

Background Many methods of ocular hypertension modeling have been used before,but these models remain short-duration ocular hypertension only.A new method of elevating intraocular pressure in mice by anterior chamber injection of polystyrene microspheres was reported abroad.However,this model is rarely used in China.Objective This study was to evaluate the application value of anterior chamber injection of polystyrene microspheres to establish glaucoma model in mice.Methods Forty-two SPF adult female C57BL/6L mice were divided into three groups according to random number table.Polystyrene microspheres (2 μl) were injected into the anterior chamber monocularly in the microspheres group,and the equal amount of PBS was used in the same way in the PBS group.No intervene was performed in the normal control group.The eyes of mice were examined by slit lamp microscope,and the intraocular pressure (IOP) was measured with TonoLab rebound tomometer in a 3-day interval after injection.Ocular histological sections were prepared 2 and 4 weeks after injection,and the anterior chamber angle was examined under the optical microscope.Neurons retrograde labeling was performed by 4％ fluorogold to calculate the survival number of retinal ganglion cells (RGCs) and the nerve fiber density was detected to assess the degree of RGCs and axon damage in retinal flat mounts,and the β-Ⅲ-tubulin-positive cells in the RGCs layer were examined by immnofluorescence method.The use and care of the animals complied with the instruction of Association for Research in Vision Ophthalmology (ARVO).Results IOP was significantly higher in the mice of the microspheres group than that in the normal control group or PBS group 2 and 4 weeks (all at P＜0.05).In the microspheres group,IOP reached peak in 2 weeks after injection and was significantly higher than that of 4 weeks after injection ([29.67±2.34] mmHg versus[15.71±1.23] mmHg) (all at P＜0.05).In 2 and 4 weeks after the anterior chamber injection of polystyrene,corneal edema was found under the slit lamp microscope,and under the optical microscope,microspheres accumulated at the anterior chamber angle.Additionally,in 2 and 4 weeks after injection,the number of survival RGCs was (4 542.82 ± 653.72)/mm2 and (3 623.12 ± 628.79)/mm2,respectively in the microspheres group,which showed significantly decrease in comparison with (6 979.33 ± 678.49)/mm2 and (6963.91 ±497.29)/mm2 in the normal control group (t =17.729,28.569,both at P＜0.05) and (6 843.21 ±573.42)/mm2 and (6 937.53±465.24)/mm2in the PBS group (t =16.975,29.145,both at P＜0.05).The number of RGCs was significantly less in the fourth week compared with second week after injection (t =6.951,P＜0.05).The β-Ⅲ-tubulin positive RGCs were (4 576.36± 479.64)/mm2 and (3 712.90 ± 660.31)/mm2 in 2 and 4 weeks in the microspheres group,respectively,which were significantly decreased in comparison with (6 725.94 ± 619.42)/mm2 and (6 741.90±663.60)/mm2 of the normal control group (t =18.811,22.182,both at P ＜ 0.05) or (6 757.85 ±463.59)/mm2 and (6 773.17± 471.35)/mm2 in the PBS group (t =18.953,22.605,both at P＜0.05),and in the microspheres group,β-Ⅲ-tubulin positive cells in the fourth week were decreased than those in the second week after injection (t=7.253,P＜0.05).The neural fiber density in the microspheres group in 2 and 4 weeks after injection was (193.08 ±32.75)/mm2 and (139.O0 ±38.24)/mm2,respectively,with a significant decline in comparison with (305.57±81.21)/mm2 and (297.46±52.60)/mm2(t=8.900,16.883,both at P＜0.05) of the normal control group or (312.63±70.62)/mm2 and (269.37±61.63)/mm2 of the PBS group (t=7.731,15.959,both at P＜0.05),and the neural fiber density was significantly lower in the fourth week than that in the second week after injection (t =7.442,P＜0.05).Conclusions Single injection of polystyrene microspheres into the anterior chamber can induce chronic ocular hypertension in mouse,which leads to the progressive damage of RGCs and neural fibers.This animal model shows a similar chronic pathogenic process to human glaucomatous eye.

OBJECTIVE:To explore the effects of bioactive compounds from entomogenous fungi(BCEF0083)on hip-pocampus neurons and mRNA expression of BDNF in chronic stress depression rat models.METHODS:The selected rats were randomly divided into control group,model group,moclobemide20mg/kg group,and BCEF0083100,50,25mg/kg groups.The change in hippocampus neuron numbers were observed by HE staining and mRNA expression of BDNF was detected with RT-PCR.RESULTS:BCEF0083could increase the neuron numbers and up-regulate the expression of BDNF mRNA in hippocampus.CONCLUSION:The antidepressant effect of BCEF0083in chronic stress depression rat models is probably re-lated to the increase in hippocampus neuron numbers and up-regulation of expression of BDNF mRNA in hippocampus.

OBJECTIVE: To study the antidepressant effects of total flavone of A( TFA) on post stroke depression ( PSD) in rats. METHODS: Middle cerebral artery occlusion ( MCAO) followed by isolation feed and irritable stimuli was used to induce PSD. Behaviours of rats were detected by open- field test . The effects of TFA on the indexes of hemorheology and brain homogenate lipid peroxidation were also tested. RESULTS: TFA could increase the crossing and rearing scores in open- field test, inhibit the elevation of whole blood viscosity as well as plasma viscosity at high- middle- low shear rates, increase the erythrocyte deformation, enhance the activities of Superoxide Dismutase ( SOD) and Glutathione Peroxidase ( GSH- Px) , and reduce Malondialdehyde ( MDA) content in brain homogenate in PSD in rats. CONCLUSIONS: TFA produces an antidepressant- like effect in PSD rats. This mechanism may be in association with attenuating hemorheological parameters and decreasing lipid peroxidative damage.

Objective To assess the usefulness of left ventricular contrast echocardiography in diagnosis of left ventricular myocardium noncompaction.Methods Contrast echocardiography was done in ten patients who were diagnosed or suspected with left ventricular noncompaction by common transthoracic echocardiography,for further study of the trabecular muscles extent,the continuity of the endocardium,the compact myocardium thickness,and the contrast agent in the trabecula recessus.Results By contrast echocardiography,noncompaction myocardium thickness can be perspicuously observed,the turgor of the contrast agent was vividly detected in the trabecular recessus.Especially for the measurement of compaction myocardium,the contrast echocardiography was more accurate than in the condition of the common echocardiography.Conclusions Left ventricular contrast echocardiography can be used in the diagnosis of left ventricular noncompaction,it was a good added method of conventional echocardiography.

Objective To compare the efficacy of insulin aspart and human soluble insulin on postprandial glucose control and blood glucose excursion in type 2 diabetic patients managed with insulin pump therapy. Methods All of 345 hospitalized type 2 diabetic patients were randomized divided into two groups. One group underwent insulin pump therapy with insulin aspart (aspart group, 173 cases),another group with human soluble insulin (humulin R group, 172 cases). Capillary glucose concentrations were measured at 9 time points,including preprandial,2 hours postprandial,bedtime (22:00),midnight(0:00) and 3:00 every day during the treatment. The change of blood glucose at each time point and the variation of postprandial blood glucose excursion was compared between the two groups. The frequency of hypoglycemia was also evaluated. Results After treatment, fasting blood glucose and post breakfast and post dinner blood glucose levels were decreased more significantly in the aspart group than those in the humulin R group. And a significantly smaller postprandial blood glucose excursion was shown in the aspart group compared with that in the humulin R group (P< 0.05). The time to achieve good glycemic control in the aspart group was (4.40 ± 2.16) d, significantly shorter than that in the humulin R group[(5.68 ± 2.29) d](P< 0.05). The incidence of hypoglycemia was significantly lower in the aspart group (P <0.05). Conclusion Insulin aspart results in better control of blood glucose and less glycemic variability compare with human soluble insulin in type 2 diabetic patients during delivery by continuous subcutaneous insulin infusion.

Objective To evaluate the association between vitamin D deficiency and diabetic nephropathy in type 2 diabetic patients.Methods A total of 594 patients with type 2 diabetes were enrolled from the inpatients of the Nanjing Medical University Affiliated Nanjing Hospital.Fasting serum lipid profile,25-hydroxycalciferol vitamin D and urinary albumin excretion rate were investigated.The relationship between nephropathy and vitamin D deficiency (＜ 20 μg/L) or insufficiency (20-＜ 30 μg/L) was analyzed.Results Nephropathy was found in 177 subjects (29.8％) with albuminuria in 141 and proteinuria in 36 subjects.Vitamin D deficiency was found in 180 subjects and insufficiency in 157 subjects.The proportion of vitamin D deficiency was higher in the individuals with nephropathy than those without nephropathy (36.2％ vs 27.8％,P ＜0.05).The urinary albumin excretion rate was significantly higher in the patients with vitamin D deficiency than those with normal vitamin D concentration [(123.0 ± 299.2)mg/24h vs (47.6 ±97.1) mg/24h,P ＜0.01].The prevalence of nephropathy was higher in the patientswith vitamin D deficiency than those with normal vitamin D concentration (35.6％ vs 26.1％,P ＜ 0.05),while the prevalence of proteinuria was higher in patients with vitamin D deficency (12.2％ vs 3.1％,P ＜0.01).Logistic regression analysis demonstrated that vitamin D deficiency was associated with nephropathy (OR 1.57,95％ CI 1.04-2.37),even after the adjustment for age,gender,hypertension,dyslipidemia,smoking status,use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (OR 1.78,95％ CI 1.12-2.81).The Vitamin D concentration was significantly negatively correlated with urinaryalbumin excretion rate (r =-1.783,P ＜ 0.001).Conclusions Type 2 diabetic patients have a high prevalence of vitamin D deficiency.Vitamin D deficiency is independently associated with diabetic nephropathy.

Objective To evaluate a new gene therapy for the treatment of experimental occlusive arterial disease Methods Magnetic nanospheres were produced, VEGF gene was cloned for subsequent construction of eukaryotic expression plasmid The magnetic gelatin microspheres used in targeted gene therapy were prepared by emulsion crosslinking method The microspheres were injected intrafemorally in rabbits through contralateral femoral artery, and the ischemic limb was placed in a magnetic field Angiography was performed on day 10 and day 30 respectively The capillary density and the capillary to muscle fiber ratio were determined histochemically Results Compared to the controls there was significant collateral artery development in VEGF transfected group The capillary density and the capillary to muscle fiber ratio were significantly higher for the VEGF transfected group than for the control group. The capillary density of control was (125?23)/mm 2, and in VEGF group was (298?27)/mm 2, P

AIM To study the antidepressant effects of bioactivecompounds from entomogenous fungi(BCEF) in mice models of depression. METHODS The antidepressant effects of Bioactive compounds from entomogenous fungi were examined in the learned helplessness model( forced swimming mice, tail suspension mice) and chronic unpredictable stress mice models. Spectrofluorometer and UV spectrophotometer were used to detect the activity of MAO, central monoamine neurotransmitter in mice brain mitochondria. RESULTS BCEF0083 25,50,100 mg?kg -1 could obviously shorten the immobility time in forced swimming mice, tail suspension mice and showed some extent of dose-effect relationship. BCEF0083 25,50,100 mg?kg -1 could obviously inhibite the activity of MAO-A,B on brain remitochondria in chronic unpredictable stress mice models and could rise the content of NE,5-HT,5-HIAA,DA in defferent degree. CONCLUSION The results suggested that BCEF0083 had antidepressant effects in mice depression models. The mechanisms of BCEF0083 antidepressant effects may be related with the inhibition of MAO-A,B activity and the increased content of central monoamine neurotransmitter.