Objective To examine the effect of Caffeine on the cultured cortical neuron apoptosis in neonatal rats.Methods The primary cerebral cortex neurons for cultures were obtained from neonatal mice 2-3 days after birth,Caffeine reconstituted at final concentrations 300μmol/L and 1 000μmol/L was added to the cell cultures and continuously co-incubated for 6-36 h,respectively after the cortical neurons were continuously cultivated 7 days after incubation under temperature of 37℃ incubator with 5% CO_2 and 100% relative humidity,the intracellular calcium concentration,mitochondrial membrane potential and apoptosis rate were determined by the flow cytometry.The activity of Caspase-9 was assayed by enzyme-labeled instrument,and Caspase-9 activity by the enzyme-1inked analyzer.Cell morphological changes were observed under electron microscope and fluorescent microscope after being stained with Hoechst 33258 fluorescent dye.Results Compared with the control group,the average increase in intracellular calcium fluorescence intensity was most significant(P<0.05),which elevated from the normal value 43.13±2.02 to 45.28±1.16 and 46.92±1.99,respectively at 6 h;mitochondrial membrane potentials were reduced most significandy(P<0.05).from the base value 443.58 ±11.77 down to 289.53±16.47 and 165.14±14.72,respectively at 8h.Caspase-9 activity was peaked(P<0.05),from the normal value 1.00±0.000 to 5.33±1.02 and 8.33±0.92,respectively at 10 h.The neuronal apoptosis ratio was increased significantly (P<0.05),from the normal value 4.94±1.74 to 15.98±2.03 and 18.70±2.09,at 36h.The apoptotic bodies were observed at 24 h after administration of 300 μmol/L and 1000 μmol/L Caffeine.Conclusion Caffeine may promote neuronal apoptosis in neonate mice.

Objective:To observe the influence of valsartan combined with Bailing capsules on urinary albumin excretion rate in early stage of type 2 diabetic nephropathy ( DN) , and explore its protection in early DN. Methods:Sixty patients with early DN were randomly divided into two groups. On the basis of diet control and blood glucose regulation, the control group (n=30) was given valsartan 160 mg, qd, while the prevention group (n=30) was treated by valsartan (160 mg·d-1) combined with Bailing capsules (2. 0g, po, tid), and the treatment course was 12 weeks. The urinary albumin excretion rate ( UAER) , mean arterial blood pressure ( MAP) , serum creat-inine ( Scr) and hemoglobin A1c ( HbA1c) were measured and compared before and after the treatment in the two groups. Results:UAER in the two groups was significantly reduced after the treatment compared with that before the treatment (P<0. 01), and that in the prevention group was obviously lower than that in the control group (P<0. 01). MAP in the two groups was significantly decreased after the treatment as well (P<0. 01), while there was no significant difference between the two groups. Scr and Hb Alc in the two groups showed no significant changes before and after the treatment (P>0. 05). Conclusion:Valsartan combined with Bailing capsules shows certain effects in the treatment of early stage of type 2 diabetic nephropathy by decreasing the urinary albumin excretion.

ObjectiveTo study the effect of heat stress on the cytoskeleton and cell cycle of human umbilical vein endothelial cell (HUVEC) in vitro.Methods HUVEC was cultured in vitro in 5%CO2 medium at 37℃ (control group) or 43℃ (heat stress group) for 1 hour. Coomassie brilliant blue R-250 staining was used to determine the effect of heat stress on the cytoskeleton. The cells in heat stress group were subsequently cultured at 37℃in 5%CO2 medium after heat stress for 1 hour, and cell cycle of HUVEC was determined at 0, 6, 12, 18 and 24 hours with flow cytometry.Results Under light microscopy normal cytoskeleton was observed in control group, but thicker and shorter cytoskeleton was found after a rise of temperature, and stress fibers were found in heat stress group. The DNA content of HUVEC at all time points in G0/G1 stage was 38.07%-55.19% after heat stress. The DNA content in control group was 48.57%, and it was 54.06%, 55.19%, 48.23%, 38.07%, and 41.03% at 0, 6, 12, 18, 24 hours in G0/G1 stage in heat stress group. DNA content in S phase was 35.33%-48.18%. The DNA content in control group was 44.62%, and it was 35.33%, 39.50%, 42.50%, 48.18%, and 47.99% at 0, 6, 12, 18, 24 hours in S stage in heat stress group. DNA content in G2/M phase was 5.31%-13.75%. The DNA content in control group was 6.81, and it was 10.61%, 5.31%, 9.27%,13.75%, and 10.98% at 0, 6, 12, 18, 24 hours in G2/M stage in heat stress group. It was demonstrated that compared with control group, the DNA content in G0/G1 stage was significantly increased when the HUVEC were separated from heat stress within 6 hours, and it recovered at a similar level as control group at 12 hours.Conclusion Heat stress can change the cytoskeleton of HUVEC, and cause stagnation at G0/G1 stage in cell cycle.

Objective:To find the correlation of wrist bone mineral density ( BMD ) to wrist synovitis and erosion , by comparing wrist BMD and ultrasonography .Methods: A number of 80 female RA pa-tients were examined by BMD measurement of the femoral neck , spine and non-dominant wrist using du-al-energy X-ray absorptiometry ( DXA ) .Synovitis of the wrist was examined by ultrasonography .The wrist joint ( radiocarpal joint , dorsal midline , and carpoulnar joint ) was assessed in the same side of DXA, with transverse and longitudinal scans for USGS synovial hypertrophy and proliferation , tenosynovi-tis,tendinitis and bone erosion .Colour and power doppler ultrasonography ( PDUS) were used to sum the synovitis score .Results:We found:( 1 ) In the study , 80 female RA patients were enrolled , the mean age was 54.6 ±13.3 (27.0-80.0) years, the disease duration was 48 (12-116) months, and the body Mass Index was 23.0 ±4.0 (14.8-31.2) kg/m2 .The Wrist BMD ( g/cm2 ) in RA significantly reduced, compared with normal controls (0.297 ±0.121 vs.0.420 ±0.180,P<0.01).(2) The Wrist BMD (g/cm2) exceeded in early RA compared with the established RA (0.326 ±0.103 vs.0.285 ± 0 .132 ,P<0 .01 );the positive rate of severe osteoporosis in wrist was lower in early RA compared with the established RA(47.8%vs.64.9%, P<0.05); the positive rate of bone erosion in wrist by ultra-sound was lower in early RA compared with the established RA (39.1%vs.56.1%, P<0.01).(3) The wrist BMD ( g/cm2 ) in RA with high disease activity reduced compared with moderate and low disease activity (0.267 ±0.140 vs.0.280 ±0.126) and (0.267 ±0.140 vs.0.320 ±0.103) respec-tively , P<0 .05 ) .The percentages of positive ACPA in the high and moderate disease activity groups were significantly higher than those in the remission group (85% vs.81.8% and 92.6% vs.81.8%, respectively).DAS28ESR was correlated with wrist BMD (r=-0.288, P<0.01).(4) A significant positive correlation was found between wrist and spine /femur BMD ( r=0.634, P<0.01, r=0.795, P<0.01), and a negative correlation between wrist and disease duration and DAS 28ESR (r=-0.286, r=-0.301,P<0.01).There was a highly significant positive correlation between wrist BMD and femur BMD (r=0.95,P<0.05).(5) RA patients in wrist osteoporosis group had higher RF positive rate and ACPA rate than wrist osteopenia group (75.5%vs.55.6%,P<0.05,100% vs.83.3%, P<0.05). The patients of BMD osteoporosis group had higher DAS 28ESR compared with osteopenia group (5.3 ± 1.8 vs.3.7 ±1.5, P<0.01).The percentages of synovitis (61.5%vs.51.7%, P<0.05), tendeni-tis (14.3%vs.10.0%, P<0.05) and bone erosion (54.2%vs.46.2%, P<0.05) in wrist by ultra-sonography in osteoporosis group were higher than those of osteopenia group .(6) The wrist BMD in ne-gative bone erosion group by ultrasonography was lower than that in positive bone erosion group [ (0.333 ± 0.107) g/cm2 vs.(0.264 ±0.125) g/cm2, P<0.01], also the PDUS score was higher than positive bone erosion group (4.53 ±1.40 vs.2.55 ±2.66,P<0.01).Compared with negative bone erosion group, the patients in positive bone erosion group had longer disease duration (96.0 ±104.7) months vs.(66.2 ±78.0) months, P<0.05), higher percentage of RF (81.0% vs.53.8%,P<0.01), ACPA (92.7%vs.79.5%, P<0.05).and higher DAS28ESR (5.4 ±1.8 vs.4.2 ±2.0,P<0.05). The percentage of wrist synovitis in positive bone erosion group was higher (75.6% vs.30.8%,P<0.01) than that of negative bone erosion group , and moreover , the percentage of severe osteoporosis in the wrist was significantly higher (75.0%vs.46.4%, P<0.01).(7) A stepwise multivariate linear regression model was constructed to explore the relationship between the different clinical factors studied and a low wrist BMD.Statistically significant variables were age (P=0.001), disease duration (P=0.017), DAS28ESR (P=0.021), and ACPA (P=0.05).Conclusion:This study shows a highly sig-nificant correlation between hand BMD with disease duration and disease activity , and female RA patients with high titer of ACPA have lower wrist BMD .ABSTRACT Objective:To find the correlation of wrist bone mineral density ( BMD ) to wrist synovitis and erosion , by comparing wrist BMD and ultrasonography .Methods: A number of 80 female RA pa-tients were examined by BMD measurement of the femoral neck , spine and non-dominant wrist using du-al-energy X-ray absorptiometry ( DXA ) .Synovitis of the wrist was examined by ultrasonography .The wrist joint ( radiocarpal joint , dorsal midline , and carpoulnar joint ) was assessed in the same side of DXA, with transverse and longitudinal scans for USGS synovial hypertrophy and proliferation , tenosynovi-tis,tendinitis and bone erosion .Colour and power doppler ultrasonography ( PDUS) were used to sum the synovitis score .Results:We found:( 1 ) In the study , 80 female RA patients were enrolled , the mean age was 54.6 ±13.3 (27.0-80.0) years, the disease duration was 48 (12-116) months, and the body Mass Index was 23.0 ±4.0 (14.8-31.2) kg/m2 .The Wrist BMD ( g/cm2 ) in RA significantly reduced, compared with normal controls (0.297 ±0.121 vs.0.420 ±0.180,P<0.01).(2) The Wrist BMD (g/cm2) exceeded in early RA compared with the established RA (0.326 ±0.103 vs.0.285 ± 0 .132 ,P<0 .01 );the positive rate of severe osteoporosis in wrist was lower in early RA compared with the established RA(47.8%vs.64.9%, P<0.05); the positive rate of bone erosion in wrist by ultra-sound was lower in early RA compared with the established RA (39.1%vs.56.1%, P<0.01).(3) The wrist BMD ( g/cm2 ) in RA with high disease activity reduced compared with moderate and low disease activity (0.267 ±0.140 vs.0.280 ±0.126) and (0.267 ±0.140 vs.0.320 ±0.103) respec-tively , P<0 .05 ) .The percentages of positive ACPA in the high and moderate disease activity groups were significantly higher than those in the remission group (85% vs.81.8% and 92.6% vs.81.8%, respectively).DAS28ESR was correlated with wrist BMD (r=-0.288, P<0.01).(4) A significant positive correlation was found between wrist and spine /femur BMD ( r=0.634, P<0.01, r=0.795, P<0.01), and a negative correlation between wrist and disease duration and DAS 28ESR (r=-0.286, r=-0.301,P<0.01).There was a highly significant positive correlation between wrist BMD and femur BMD (r=0.95,P<0.05).(5) RA patients in wrist osteoporosis group had higher RF positive rate and ACPA rate than wrist osteopenia group (75.5%vs.55.6%,P<0.05,100% vs.83.3%, P<0.05). The patients of BMD osteoporosis group had higher DAS 28ESR compared with osteopenia group (5.3 ± 1.8 vs.3.7 ±1.5, P<0.01).The percentages of synovitis (61.5%vs.51.7%, P<0.05), tendeni-tis (14.3%vs.10.0%, P<0.05) and bone erosion (54.2%vs.46.2%, P<0.05) in wrist by ultra-sonography in osteoporosis group were higher than those of osteopenia group .(6) The wrist BMD in ne-gative bone erosion group by ultrasonography was lower than that in positive bone erosion group [ (0.333 ± 0.107) g/cm2 vs.(0.264 ±0.125) g/cm2, P<0.01], also the PDUS score was higher than positive bone erosion group (4.53 ±1.40 vs.2.55 ±2.66,P<0.01).Compared with negative bone erosion group, the patients in positive bone erosion group had longer disease duration (96.0 ±104.7) months vs.(66.2 ±78.0) months, P<0.05), higher percentage of RF (81.0% vs.53.8%,P<0.01), ACPA (92.7%vs.79.5%, P<0.05).and higher DAS28ESR (5.4 ±1.8 vs.4.2 ±2.0,P<0.05). The percentage of wrist synovitis in positive bone erosion group was higher (75.6% vs.30.8%,P<0.01) than that of negative bone erosion group , and moreover , the percentage of severe osteoporosis in the wrist was significantly higher (75.0%vs.46.4%, P<0.01).(7) A stepwise multivariate linear regression model was constructed to explore the relationship between the different clinical factors studied and a low wrist BMD.Statistically significant variables were age (P=0.001), disease duration (P=0.017), DAS28ESR (P=0.021), and ACPA (P=0.05).Conclusion:This study shows a highly sig-nificant correlation between hand BMD with disease duration and disease activity , and female RA patients with high titer of ACPA have lower wrist BMD .

The influence of inner cell mass (ICM) and trophectoderm (TE) score on pregnancy outcomes in frozen-thawed blastocyst transfer cycles was analyzed. A retrospective analysis of 741 cycles of frozen-thawed blastosysts transfer was performed. All cycles were divided into four groups based on the number and morphological score of blastocysts: S-ICM B/TE B group (n=91), the single blastocyst transfer of ICM B and TE B; D-ICM B/TE B group (n=579), double blastocysts transfer of ICM B/TE B; D-ICM B/TE C group (n=35), double blastocysts transfer of ICM B/TE C; and D-ICM C/TE B group (n=36), double blastocysts transfer of TE B/ICM C. The pregnancy outcomes were compared among the four groups. As compared with D-ICM B/TE C group, the clinical pregnancy rate, implantation rate and multiple pregnancy rate were increased in D-ICM B/TE B group (74.96% vs. 57.14%, 57.43% vs. 37.14%, and 48.62% vs. 25%, respectively, P<0.05 for all). Clinical pregnancy rate and implantation rate in D-ICM B/TE B group were also higher than in D-ICM C/TE B group (74.96% vs. 50%, and 57.43% vs. 33.33%, both P<0.05). Multivariable Logistic regression analysis indicated that ICM score was a better predictive parameter for clinical pregnancy (OR=3.05, CI 1.70-5.46, P<0.001), while the trophectoderm score was a better one for early abortion (OR=0.074, CI 0.03-0.19, P<0.001). Clinical pregnancy rate and multiple pregnancy rate in S-ICM B/TE B group were significantly lower than those in D-ICM B/TE B group (46.15% vs. 74.96%, and 2.38% vs. 48.62%, both P<0.05), but there was no significant difference in the implantation rate between the two groups. It was suggested that the higher score of ICM and TE may be indicative of the better pregnancy outcomes. The ICM score is a better predictor of clinical pregnancy than TE, while TE score is a better one in predicting early abortion. Single ICM B/TE B blastocyst transfer in frozen-thawed cycles can also get satisfactory pregnancy outcomes.

Objective To study the effect of heat stress on the permeability,cytoskeleton and cell cycle of human skeletal muscle cell (HSKMC).Methods The HSKMC membrane permeability was detected by calcium ion inflow with flow cytometer,the cytoskeleton was stained by CBB 250,and the cell cycle was determined by flow cytometer.Results After 1 h of heat stress on human HSKMC cells under different temperature gradient,the median level of calcium ion was 91.63 in 43 ℃ heat stress group compared with 22.98 in 37 ℃ control group.As temperature increased,thicker and shorter cytoskeleton and stress fiber were shown under the high power lens of microscope.The DNA expression of skeleton cells at G0/G1 stage was 44.13-62.98 in groups under heat stress.Compared with normal control group,DNA expression was much higher in heat stress group,when HSKMC was cultured under 37 ℃ temperature for another 18 h,it kept decreasing DNA expression to a similar level as control group.Conclusions Heat stress can cause calcium iron inflow resulting in intracellular calcium overload,and affect the cytoskeleton leading to loss of normal web ordered arrangement and increased gap in HSKMC cells,which give rise to blocking cell cycle into G0/G1 stage.

Objective To explore the energy feature of the spontaneous neural activity in depressive patients with diurnal rhythm.Methods Twenty depressive patients with diurnal rhythm,20 depressive patients without diurnal rhythm and 20 gender-,age-,education-matched healthy controls were scanned with 3.0 T MRI Scanner.According to Results of ANOVA,group-wise two sample t-test was completed.The correlation analysis were conducted between ALFF of significant difference brain regions and the score of Hamilton rating scale for depression.Results The brain regions showing significant differences among three groups were located in the right parahippocampal gyrus(MNI:18,-36,-9;K=136),left parietal lobe(MNI:-54,-45,42;K=86),left cerebellum crus(MNI:-84,-60,36;K=86) and right cerebellum crus(MNI:9,-78,-36;K=91)(P<0.05,Alphasim correct).Compared with non-diurnal rhythm group,the diurnal rhythm group showed significantly increased ALFF in the right parahippocampal gyrus.Compared with healthy group,the diurnal rhythm group showed significantly increased ALFF in right parahippocampal gyrus,left parietal lobe,and significantly decreased ALFF in left cerebellum crus 1 and right cerebellum crus 2.Compared with healthy group,the non-diurnal rhythm group showed significantly increased ALFF in left parietal lobe,and significantly decreased ALFF in left cerebellum crus 1 and bilateral cerebellum crus 2.No correlation between ALFF of significant difference brain regions and clinical symptoms was found(P>0.05).Conclusion The diurnal rhythm observed in depression may be associated with aberrant activity of parahippocampal gyrus.

Objective:To verify the fracture risk assessment tool ( FRAX) to estimate the probability of osteoporotic fracture in patients with rheumatoid arthritis ( RA ) with or without bone mineral density (BMD), and identify associated risk factors of osteoporosis .Methods: In the study, 200 patients with rheumatoid arthritis aged more than 40 years in Peking University First Hospital from Dec .2009 to Dec. 2012 were recruited.Clinical information was obtained from a questionnaire of their case history and medical records.FRAX tool was administered.Their lumber spine and left femoral BMD were determined by dual energy X ray absorptiometry.The gender, age, disease duration, menopause status, body mass index ( BMI) and accumulative dose of glucocorticoid were obtained in retrospect .Correlation analysis was conducted between the BMD and clinical information .Results:The study population ( female, 77.5%) had a mean age of 59.4 years, in which 10 (13%) patients showed a normal BMD, 67 (87%) were osteopenia or osteoporosis , while 32 patients (16%) had fragile fracture.Compared with the patients with normal BMD, the subjects with low BMD had significantly older age , longer period for corticoids usage , higher day dose and accumulated dose of corticoids .The 10-year fracture risk of sustai-ning major osteoporotic fractures and hip fracture was higher .No significant difference was observed be-tween the 10-year fracture risks calculated with BMD and without BMD .The values of the different area under the receiver operating characteristic ( ROC) curve ( AUC) for major and hip fractures calculated in three ways:without BMD, with the femoral neck BMD, and with T-score.The best result was for FRAX tool for hip fracture with the T-score ( AUC 0 .899 ) .A stepwise multivariate linear regression model was constructed to explore the relationship between the different clinical factors studied and a low BMD . Three statistically significant variables for lumber BMD were pain on visual assessment scale ( VAS ) (P=0.02), fracture history (P=0.003) and a higher steroid accumulated dose (P=0.008).Three statistically significant variables for left hip BMD were age (P<0.001), fracture history (P=0.05) and lower BMI ( P=0.03) .Conclusion:Low BMD is a common complication in RA patients .Risk factors for major fracture and hip fracture are increased .There is a positive correlation between FRAX calculated with and without BMD or T score .FRAX with the femoral neck T score or BMD presents a discriminatory capacity better than FRAX without BMD , according to the AUC ROC .

Objective To observed the impact of selenium supplementation therapy on the thyroid perioxidase antibody(TPO-Ab) levels and serum oxidative stress[malondialdehyde(MDA, glutathione peroxidase(GPx), and superoxide dismutase(SOD)] in patients with Hashimoto′s thyroiditis. Methods 79 patients with Hashimoto′s thyroiditis were randomly divided into trial group(n=44) and placebo group(n=35) .The double-blind treatment was for 24 weeks. The thyroid hormone levels, serum TPO-Ab levels, and oxidative stress indexes(MDA, GPx, and SOD) of both groups were detected before and after treatment. Results (1)There was no change of thyroid hormone levels either before or after treatments of both groups(P>0.05). (2)TPO-Ab of the trial group decreased significantly after the treatment(P<0.05). While the placebo group has little change. Group with TPO-Ab≤200 IU/ml and the course≤1 year manifested the most obvious declines by 29.98% and 26.63% respectively. (3)The oxidative stress level of trial group significantly decreased after 24 weeks. There was significantly positive correlation between the oxidative stress indexes and TPO-Ab. However the placebo group was with little change. Conclusion Selenium supplementation may reduce the level of TPO-Ab titers and oxidative stress in patients with Hashimoto′s thyroiditis, especially for those with lower antibody titers and short course.

Objective To determine PD-L2 expression in human cervical squamous cell carcinomas and analyze its association with the clinical and pathologic characteristics of these cases ;observe the role of recombinant PD-L2 protein on apoptosis of active peripheral blood T lymphocytes of cervical carcinoma patients .Methods PD-L2 expression in cervical squamous cell carcinomas was determined by immunohistochemistry staining ,and the association between PD-L2 expression with the clinical and pathologic characteristics of these cases was analyzed .In vitro ,the peripheral blood T lymphocytes of cervical carcinoma patients were divided into blank group ,PD-L2 group and PD-L2+anti-PD-1 group respectively .After these active T cells were cultured 72 h ,their apop-totic rates were detected by flow cytometry .Results PD-L2 expressed in 53 .3% (32/60)cervical squamous cell carcinomas ,and it′s expression associated with lymph node metastasis of these cases (P< 0 .05) .In vitro ,PD-L2 promoted apoptosis of CD4+ T and CD8+ T lymphocytes ,the apoptotic rates were 17 .0% and 22 .4% respectively ,which were higher than 9 .0% and 16 .2% of blank group;however ,the apoptotic rates dropped to 11 .1% and 17 .5% in PD-L2+anti-PD-1 group .Conclusion PD-L2 aberrantly ex-presses in cervical squamous cell carcinomas and is associated with their lymph node metastasis .PD-L2 promotes apoptosis of T lymphocytes and depresses the anti-tumor immunity of cervical microenvironment ,and promotes lymph node metastasis of these cancers .PD-L2/PD-1 pathway may be a potential immunotherapy target of cervical squamous cell carcinomas .