Objective To observe the level of autophagy induced by TRAIL in TT cell line and identify the role of autophagy in TRAIL-inducing apoptosis of TT cell line.Methods The growth inhibition of TT cells was measured by MTT assay.MDC staining was used to identify the happening of autophagy.Annexin V/PI double staining was used to analyze the apoptosis rate of TT cells by flowcytometry.The protein expression of caspase-8 and Beclin1 was detected by Western blot.Results ① The growth inhibition ratio of TT cells induced by TRAIL at the concentration of 250,500,1000 and 2000 ng/ml was (3.02 ± 1.82)％,(4.87 ± 1.45)％,(7.51 ± 1.57) ％,(12.76 ± 3.23) ％ respectively,which suggested significant resistance of TT cells to TRAIL.② MDC-labeled green light vesicles was significantly increased after the treatment of TRAIL for 48 h.③ The apoptosis rate of TT cells induced by TRAIL at the concentration of 500 ng/ml and 1000 ng/ml after the pretreat ment of 3-MA for 4 h was(17.83 ± 1.54) ％ and(27.81 ± 1.79) ％ respectively,which was significantly higher than the apoptosis rate induced by TRAIL(3.70 ± 0.34) ％,(6.55 ± 0.59) ％ alone and that induced by 3-MA(7.71 ± 0.64) ％ (t =3.282,P ＜ 0.05 ; t =7.830,P ＜ 0.01).④ The combination treatment of TRAIL and 3-MA increased the cleavage of caspase-8 and down-regulated the expression of Beclin 1.Conclusion Autophagy induced by TRAIL may contributes to the resistance of TT cells to TRAIL,which can be reversed by the inhibition of autophagy.

OBJECTIVE:To study the improvement effects of baicalein against myocardial ischemia/reperfusion(I/R)injury in rats. METHODS:I/R injury model was induced by Langendorff method. Isolated heart of 40 rats were randomly divided into nor-mal group(continuous perfusion),model group(perfusion withdrawal 20 min)and baicalein high,medium and low concentration groups (K-H solution of baicalein 40,10 and 2.5 μmol/ml 10 min before perfusion withdrawal). The myocardial infarction rate, the activity of creatine kinase(CK)and lactate dehydrogenase(LDH)in coronary effluent liquid,SOD activity and MDA content, GSH/GSSG and apoptosis rate of cardiac muscle cell in myocardial tissue were detected. RESULTS:Compared with normal group, the myocardial infarction rate,apoptosis rate of cardiac muscle cell,the activities of CK and LDH and the content of MDA in myo-cardial tissue were increased in model group,while SOD activity and GSH/GSSG of myocardial tissue decreased(P<0.01). Com-pared with model group,the isolated myocardial infarction rate and the activities of CK and LDH in baicalein low and medium con-centration groups decreased,while SOD activity increased(P<0.05 or P<0.01);apoptosis rate of cardiac muscle cell and the con-tent MDA of myocardial tissue decreased significantly in medium concentration group,while GSH/GSSG increased (P<0.05 or P<0.01);those indicators had no significant change in high concentration group(P>0.05). CONCLUSIONS:Baicalein has cer-tain improvement effect on myocardial I/R injury in rats,and its mechanism may be associated with antioxidant and anti-apoptosis effect of baicalein.

Objective To evaluate the significance of the expression of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) as prognostic indicators for patients with stage Ⅱ colorectal cancer. Methods A total of 285 patients with stage Ⅱcolorectal cancer who underwent potentially curative surgery were enrolled in the study. A high CEA level was defined as a level exceeding 5 ng/ml and a high CA19-9 level was defined as a level exceeding 37 U/ml. Patients were followed up every 3 months to review CEA and CA19-9 levels. Two years later, the chest and abdominal CT examination were performed every 3 months, and then every 6 months until 5 years. Results Out of these 285 patients, 99 (42.60%) patients had high CEA levels, 25 (8.77 %) patients had high CA19-9 levels, and 12 (4.21 %) patients had both high CEA and CA19-9 levels. The overall survival rates of patients with both high CEA and CA19-9 levels were significantly worse than those of others (P< 0.05). During the study, 51 recurrences were diagnosed. There were 22 hepatic recurrences, 10 pulmonary recurrences, 9 local recurrences, 8 lymph node recurrences, and 2 peritoneal recurrences. CT, CEA and CA19-9 were the first abnormal examinations in 30, 5 and 8 recurrent patients, respectively. In 27 % of recurrent patients, the recurrence was detected earlier by CEA and CA19-9 than that by CT. Conclusions The detection of preoperative CEA and CA19-9 levels is useful for predicting the prognosis after potentially curative surgery in patents with stage Ⅱ colorectal cancer. For early detection of occult recurrence of colorectal cancer, tumor markers are relevant.

Objective To study the effect and related mechanism of celecoxib on tumor necrosis factorrelated apoptosis-inducing ligand(TRAIL) induced apoptosis of medullary thyroid cancer TT cell line.Methods MTT assay was used to measure the growth inhibition induced by TRAIL and celecoxib alone and their combination.TT cell cycle distribution was analyzed by flowcytometry.Hochest33258 staining and DNA ladder was used to detect the apoptosis of drug combination on TT cells.Western blot was used detect the protein change of cyclin A,Cdk2,caspase-8,c-FLIP,and RIP.Results ①MTT showed the growth inhibition ratio of TT cell intervened by the combination of TRAIL and celecoxib was 47.53％ ± 1.34％,which was much higher than that intervened by TRAIL(7.75 ％ ± 3.84％)and celecoxib alone.The differences had statistical significance (t test,F =5.234,P ＜0.01);②PI detection found the cells' number in G0/G1 phase in celecoxib group and combination group were increased compared to that in control group and TRAIL group(F =242.694,P ＜ 0.01);③Western blot indicated the expression of Cyclin A and Cdk2 were down regulated,there was no statistic significance;④ The apoptosis morph in nuclus was detected by Hochest33258 staining and showed the karyopycnosis and muclear fragmentation were increased in combination group with the apoptosis rate 24.23％ ± 2.91％,which was much higher than that in TRAIL(5.86％ ± 1.41％) and celecoxib(20％ ± 1.24％) (t test,F =1.824,P ＜0.01),the difference has statistic significance;⑤Western blot illustrated the active schizolysis of casplase-8 was higher and the expression of c-FLIP and RIP was down regulated in combination group.Conclusion celecoxib plays a positive effect on TRAIL-reduced apoptosis of medullary thyroid cancer TT cell line,which may due to the cell cycle arrest at G0/G1 phase,down-regulation of c-FLIP and RIP and subsequent activation of caspase-8.

Matrix_assisted laser desorption ionization_time of flight tandem mass spectrometry ( MALDI_TOF/TOF MS) and electrospray ionization_quadrupole_time of flight mass spectrometry ( ESI_Q_TOF MS) were used to confirm the structure of cyclic lipopeptide daptomycin fastly. First, the relative molecular weight 1916. 7107 of daptomycin was measured by ESI with error 0. 0007. The sample’s doubly charged peak m/z 809. 848 was selected as precursor ion for ESI_MS/MS analysis, and the exocyclic amino acid sequence C9 H19 CO_Trp_Asn_Asp was successfully matched. Second, the experimental conditions of cleaving daptomycin by lithium hydroxide ( LiOH) were optimized and the ring_opened process was monitored by MALDI_TOF/TOF MS. After obtaining ring_opened product with purity of above 95%, the MS/MS measurements by MALDI and ESI were carried out. The b+and y+of ring_opened product were completely matched, which confirmed the amino acid sequence of daptomycin. Finally, ESI_MS/MS conditions of ring_opened product were further optimized to obtain more low mass fragment ions for analyzing the structure of fatty acid chain and the cleavage pattern of fat chain in mass spectrometry was proposed. The method was fast, convenient, accurate and reliable for identifying cyclic lipopeptide compounds.

Objective To observe microvascular architecture and free radical metabolism in hippocampus after focal cerebral ischemia/reperfusion and to explore the effect of NBP (3-n-butylphthalide). Methods Fifty-four SD rats were ran?domly divided into NBP pretreatment group, ischemia/reperfusion group and sham operation group (n=18 in each group). The model of middle cerebral artery occlusion(MCAO)was established by suture method. The neurological scores were counted and the volume of infarction was measured;TA-Fe method was applied to observe the microvascular architecture of hippo?campus, Mivnt image analysis system was used to analyze the microvessel density(MVD)and the microvessel area density (MVA)of hippocampus quantitatively;The activity of SOD and content of MDA were measured by colorimetric method. Re?sults Compared to the ischemia reperfusion(IR)group, the neurological scores and the volume of infarction were decreased sharply in NBP group. What′s more, the activity of SOD, MVD and MVA were all enhanced but the content of MDA and the count of closed microvessels were both reduced(P < 0.01). Conclusion NBP can improve microvascular architecture of hippocampus and reduce the free radical injury. There is a protective effect on hippocampus of rats who suffered focal cere?bral ischemia reperfusion.

BACKGROUND:Simvastatin enhanced the expression of bone morphogenetic protein-2(BMP-2),which plays an anabolic role in bone metabolism and osteoblastic lineage differentiation.However,little is known about the molecular mechanism of simvastatin on regulation of bone marrow stromal cells differentiation.OBJECTIVE:To investigated the effect of simvastatin on osteoblastic differentiation of bone marrow stromal cells based on genetics level.METHODS:Bone marrow stromal cells derived from femur and tibia were cultured in different mediums with simvastatin or Vehicle for 7 days Following extraction and purification,mRNA was reverse-transcripted into cDNA.Fluorescence labelina was employed and the samples were then hybridized with oligonucleotide chip to screen the different genes,which were utillzed to analyze osteogenesis-related factors.Alkaline phosphatase and Von Kossa staining were performed at days 14 and 21,respectively.RESULTS AND CONCLUSIONS:At day 14,alkaline phosphatase-positive cells were more in the experimental group than control group.Von Kossa staining demonstrated that simvastatin could promote BMSCs osteoblastic differentiation and mineralization.Comparative analysis showed that 103 genes out of 22 575 rat genes had differential expression (≥2 fold or≤ 0.5 fold),and some genes were related to cell proliferation and ostoeblastic differentiation,including C/EBP δ,Cited,Ascl2,Ptpnl6,Wisp2,Tieg,etc.Simvastatin could induce osteoblastic differentiation of bone marrow stromal cells,involving in many osteogenetic-related genes.

To investigate the role of Toll like receptor (TLR) in the activation of dendritic cells (DC) during early Plasmodium yoelii infection of the lethal strain 17XL (P.y 17XL), susceptible BALB/c and resistant DBA/2 mice were infected by i.p.injection of the P.y l7XL-parasitized erythrocytes, and the parasitemia of individua1 mice was monitored by the microscopic examination of blood smear stained with Giemsa.Mice from norma1 and infected groups were sacrificed on 0,3 and 5 days post-infection to collect their spleen cells.And the expressions of TLR-9 and TLR-4 on the cell surface of DCs in spenonocytes of these two strains of mice were assayed by applying flow cytometry to quantitatively analyze the percentages of CD11c+TLR9+ DCs and CD11c+TLR4+ DCs. It was found that the population of CD11c+DCs expressing TLR9 was significantly increased on day 3 and peaked on 5 p.i. in BALB/c (P<0.01) and DBA/2 mice(P<0.01). However, there was no statistical significance between these two strains of mice. Meanwhile, there was no change on the population of CD11c+ DCs expressing TLR4 in BALB/c and DBA/2 mice. These results indicate that TLR9 may contribute to the DC activation during early stages of P.y17XL infection.

Objective To study correlation between systemic sclerosis(SSc)and plasma D-dimer and to reveal the probably rules of the fibrinolytic systems in SSc.Methods In 2013 January to 2014 January,cellected 32 patients with SSc and 35 with healthy controls to detected level of plasma D-dimer.Logistic and t student test were used for statistical analysis for correlation between SSc and pulmonary artery hypertension.Results When compared to healthy controls (0.28±0.04μg/ml),the level of plasma D-dimer were significantly increased in SSc patient (0.31±0.05μg/ml,t=1.997,P=0.008).After stratifying SSc patients according to disease subset,whereas patients with diffuse subset displayed substantially increased values (0.41±0.06μg/ml,t=2.051,P<0.001).The level of plasma D-dimer was associated with pulmonary artery hyper-tension (OR=4.38,95%CI=2.59~8.91,P=0.008).Conclusion Demonstrated that SSc patients with diffuse subset are characterized by increased plasma D-dimer values,reflecting a potential activation of fibrinolytic cascaded,which might finally predispose these patients to thrombotic complications and pulmonary artery hypertension.

Objective To investigate the change of genomic DNA of liver and spleen tissue for different age of the elderly,and provide the experimental data for aging-related research. Methods 35 livers and 33 spleens of autopsied samples preserved in refrigerator at-80 ℃ were divided into 3 groups according to age:age 65y to 79y,age 80y to 89y,age≥90y. The content of DNA in liver and spleen was determined by ultraviolet absorbent method. Results Compaired with age 80y to 89y (0. 310 ± 0. 286)mg/mL,the content of DNA in liver was significant higher at age 65y to 79y (1.464 ±0.488)mg/mL and age ≥90y(1.147 ±0.333)mg/mL(P<0.05);Compared with age 80y to 89y(0. 938 ± 0. 589)mg/mL,the content of DNA in spleen was significant higher at age 65y to 79y(1. 723 ± 0. 726)mg/mL and age≥90y(1. 688 ± 0. 963)mg/mL(P<0. 05). The content of DNA was significant lower in liver (0. 856 ± 0. 658)mg/mL than that in spleen (1. 414 ± 0. 852)mg/mL. Conclusion The content of DNA in human liver and spleen tissue may be decrease along with aging. The content of DNA in the group at age≥90y may be increase. There were some differences between different viscera tissue in content of DNA.