Objective To observe the changes of intracellular free calcium and the expression of CaM in the hippocampal neurons of posttraumatic stress disorder (PTSD) rats and to further investigate the neurobiological mechanisms. Methods The SPS-method was used to set up the rat PTSD models. A total of sixty male Wistar rats were randomly divided into 12 hours,1 day,4 days,7 days groups of SPS and normal control group. The intracellular free calcium was examined by fluorescence spectrophotometer. The expression of CaM was detected by using immunohistochemistry, Western blotting and RT-PCR. Results The intracellular free calcium level in the hippocampus of experimental rats was markedly increased 12 hours after SPS stimulation,and reached the peak after 1 day, then gradually decreased to normal level after 7 days. The expression of CaM in the hippocampus 1day after SPS was also the highest and then gradually decreased.Conclusion The lasting dysfunction of Ca~(2+)/CaM signaling cascades in hippocampal may play important roles in the pathogenesis of PTSD rats.

By molecular cloning technique, the expressing plasmids pDOR-SV40-Bcl-2 cDNA were successfully constructed. The reconstructed plasmids with lipofectamine were transduced into gastric cancer cell line SGC7901 and then the positive clones which contained the reconstructed plasmids were choosed by G418. Circa 150 positive clones were choosed from 2 x 10_5 gene transduced cells, which suggested that the transducing efficiency was more than 1%o; 2 positive clones were expanded and passage, then 1 drug-resistance cell strain (SGC7901 anBcl-2 cells) was obtained. The bloting results suggested, Bcl-2 cDNA were expressed in both gene transduced and not transduced cell strains, but the expressing level of mRNA and protein in gene transduced cell strain was very low than that in gene not transduced cell strain were positive by the means of Southern blot, Northern blot and Western blot. This results showed that normally in gastric cancer cells the expressing level of Bcl-2 gene was very high, and the cDNA fragment of antisense Bcl-2 were successfully transduced into gastric cancer cells, and in gene transduced cell strains the expressing of Bcl-2 gene was effi-cientlv blocked.

Objective:To investigate YKL-40-mediated inflammation in human bronchial epithelial cells and analyzed the soluble factors secreted by bronchial epithelial cells exposed to YKL-40 that were responsible for increasing proliferation and migration of primary normal human bronchial smooth muscle cells (BSMCs).Methods:YKL-40-induced inflammation was assayed in two human bronchial epithelial cells (BEAS-2B cell line and primary human bronchial epithelial cells ,namely HBECs).In addition,we treated BEAS-2B cells and HBECs with YKL-40,and added the conditioned culture media ( YKL-40-BEAS-2B-CM) and ( YKL-40-HBECs-CM) to BSMCs.The proliferation and migration of BSMCs were determined by premixed WST-1 cell proliferation reagent and QCM chemotaxis migration assay ,respectively.Results: Bronchial epithelial cells treated with YKL-40 resulted in a significant increase of IL-8 production,but have no effect about RANTES ,Eotaxin and TNF-α.YKL-40-BEAS-2B-CM and YKL-40-HBECs-CM induced IL-8 was found to further stimulate proliferation and migration of BSMCs ,and the effects were inhibited after neutralizing IL-8.Conclusion:Through investigating the interaction of airway epithelium and smooth muscle ,our findings implicate that YKL-40 may be involved in the inflammation of asthma by induction of IL-8 from epithelium,subsequently contributing to BSMCs proliferation and migration.Moreover, inhibition of IL-8 signaling is a potential therapeutic target for YKL-40-induced inflammation and remodeling of asthma.

Objective: To further understand the role of folic acid supplements rivaling MTHFR gene silencing in pathogenesis of NCLP, RNA interference (RNAi) was applied to knock down MTHFR in mouse embryonic palatal mesenchymal (EPM) cells. Methods: MTHFR ShRNA expression vector were transfected into the primary cultured EPM cells. MTT was used to observe cell proliferation after MTHFR gene silencing. FCM was used to observe cell cycle after MTHFR gene silencing. Results: The results showed the cells proliferation had an inequality amelioration after using folic acid supplements in MEPM cells with MTHFR gene silencing. Using folic acid supplements rivaled the effect of MTHFR gene silencing had a dose-dependent manner. Using 20 μg/ml folic acid supplements could improve the cell proliferation to achieve normal level of cell proliferation. Conclusion: MTHFR gene is an important candidate gene of NCL/P. Using folic acid supplements could prevent teratogenic MTHFR gene silencing for embryonic palate development.

AIM: To investigate the effects of bone morphogenetic protein 7 ( BMP-7 ) on the expression of transcription factor E2A and inhibitor of differentiation 2 (Id2) in the renal tubule epithelial cells(NRK-52E)exposed to high glucose, and to explore its possible mechanism of improving renal tubular fibrosis induced by high glucose.METH-ODS:The NRK-52E cells were divided into control group, high glucose (HG) group and high glucose with different doses of BMP-7 (10μg/L and 20μg/L) group.The cells in HG group and BMP-7 group were cultured for 12 h, 24 h and 48 h. The protein expression of Id2, E2A, E-cadherin,α-smooth muscle actin (α-SMA) and collagen-I was detected by Western blot.In addition, the mRNA expression of Id2 was detected by real-time PCR.RESULTS:Compared with control group, the mRNA and protein levels of Id2 and the protein level of E-cadherin were down-regulated, while the protein levels of E2A,α-SMA and collagen-I were up-regulated in HG group (P<0.05).Compared with HG group, the mRNA and pro-tein levels of Id2 and the protein level of E-cadherin were significantly up-regulated, while the protein expression of E2A,α-SMA and collagen-I was significantly down-regulated in 20 μg/L BMP-7 group ( P<0.05 ) .The correlation analysis showed that the Id2 protein level was negatively correlated with the E2A protein level (P<0.05).CONCLUSION:BMP-7 may intercept the process of renal tubule fibrosis induced by high glucose via promoting the expression of Id2 and inhibi-ting the expression of E2A at protein level.

Objective To investigate the expression and possible mechanism of miR-21 and Ski-related novel protein N( SnoN) in the renal fibrosis diabetic process.Methods The animal model was established by tail-vein injection of Streptozotocin,and the other group were normal control ( NC) group.After 10 weeks, the rats were sacrificed to measure biochemical parameters and renal index , and to observe the changes of pathomorphology by HE staining as well.Meanwhile, immunohistochemistry and Western blot were employed to examine protein ex-pression of E-cadherin,α-smooth muscle actin(α-SMA), fibronectin(FN), collagen-Ⅰ(Col-Ⅰ), collagen-Ⅲ(Col-Ⅲ), transforming growth factor-β1(TGF-β1), Smad3, p-Smad3(Ser423/425) and SnoN in the renal tissue. In addition, the expression of SonN mRNA and miR-21 were detected by qPCR.Results In DM group,the ex-pressions of Col-Ⅰ, Col-Ⅲ and FN in renal interstitium were increased ( P <0.05 ) , TGF-β1 increased (P<0.05),while E-cadherin decreased(P<0.05).Compared with NC group, the expression of α-SMA,p-Smad3 (Ser423/425) protein increased in DM group(P<0.05),while the protein level of SnoN decreased but the level of SnoN mRNA increased ( P <0.05 ) .Moreover, the level of miR-21 markedly increased in DM group ( P <0.05 ) .Conclusions TGF-β1 may up-regulate the expression of miR-21 but restrain the translational expression of SnoN, aggravating fibrosis.

ObjectiveTo identify the factors influencing the transfer of porcine endogenous retroviruses across the membrane of a new bioartificial liver (BAL),which is a necessary caution to consider for BALs carrying porcine hepatocytes.MethodsA novel porcine BAL composed of two circuits was constructed using a plasma filter with membrane pore size of 500 nm and a plasma component separator with membrane pore sizes 10 nm,20 nm,30 nm,and 35 nm.Co-cultured cells of porcine hepatocytes and mesenchymal stem cells or single porcine hepatocytes were incubated in the bioreactors.The BAL was continuously worked for 72 hours during which the supernatant was collected from the internal and external circuits every 12 hours.PERV RNA,reverse transcriptase (RT) activity,and in vitro infectivity from the supernatant were detected.ResultsIn the plasma filter group,the PERV RNAlevels were the same in both circuits,suggesting little to no effect of the plasma filter on the PERV RNA's crossing.With plasma component separators,PERV RNA was found in the external circuits at different times without positive RT activity and HEK293 cell infection,but its level was reduced significantly.The larger the membrane pore size was,the earlier and the more RNA was detected.ConclusionsThe membrane pore size,the treatment time and the viral level in the internal circuit are contributing factors influencing the transfer of PERV RNA across the membrane in a BAL.

ObjectiveTo evaluate the therapeutic efficacy and prognostic factors of intensity-modulated radiotherapy combined with chemotherapy for early-stage nasopharyngeal carcinoma patients in northwest China. MethodsFrom January 2006 to December 2009,58 patients with early-stage nasopharyngeal carcinoma were treated with IMRT in Xijing hospital,the clinical data were analyzed retrospectively.Survival rates was calculated by the Kaplan-Meier method and the differences was compared by the Logrank test.Univariate analysis method was use to identify all significant factors.ResultsThe follow-up rate was 100％.The follow-up time of 46 patients was more than 3 years.The 1-,2 and 3-year survival were 98％,94％ and 91％,respectively.The 3-year overall survival (OS),local recurrence-free survival (LRFS),distant metastasis-free surv ival (DMFS) for T1N0-1,T2N0 and T2N1 stage were 100％,100％,100％ and 74 ％,81％,87 ％,respectively ( x2 =5.74,P =0.01 ; x2 =4.95,P =0.03 ; x2 =4.24,P=0.04).The 3-year OS,LRFS,DMFS for IMRT combined with chemotherapy and IMRT alone were 100％,100％,100％ and 85％,85％,88％ respectively ( x2 =4.02,P =0.04; x2 =4.12,P =0.03 ; x2 =4.84,P =0.02).In T2N1 stage,IMRT combined with chemotherapy and IMRT alone were 100％,100％,100％ and 79％,79％,80％ respectively (x2 =5.28,P =0.03 ;x2 =4.84,P =0.04;x2 =4.72,P =0.04).In univariate analysis,N stage,clinical stage,IMRT combined with chemotherapy were significantly associated with the survival ( x2 =5.39,P =0.02 ; x2 =5.74,P =0.01 ; x2 =4.02,P =0.04).Conclusions In all early-stage nasopharyngeal carcinoma,T2N1 stage is a sub-group of high risk of distant metastasis.Combination of IMRT and chemotherapy may improve the LRFS,DMFS and OS in those patients.

Objective To study the relationship between the MDR1 gene expressions and CD56 antigen expression in patients with de novo acute myeloid leukemia(AML) and to explore the role of this two factors in clinical drug resistance and their correlation. Methods A real-time quantitative RT-PCR method was established for detecting MDR1 expression levels and three-color flow cytometry analysis using CD34/ SSC gating was used to examined CD56 antigen expression in 79 de novo AML patients. Results CD56 an-tigen was recorded in 19 out of 79 cases (24.1%) and particularly in those with M5 cytotypes. Moreover, CD56 expression was significantly associated with unfavorable cytogenetic abnormalities (P<0.05), Patients with t(8:21)had a significantly higher incidence (57.1%, 4/7) of CD56 expression than those with favora-ble karyotype(P<0.05). CD56~+ AML patients had a higher incidence of splenohepatomegalia and lactate dehydrogenase level than CD56~- patients(P<0.05). The median expression levels of MDR1 was statistical-ly higher in CD56~+ AML patients than that in CD56 patients(P<0.001). Patients with both high levels of MDR1 and CD56~+ had a significantly lower CR(complete remission) rate than those with both low MDR1 level and CD56 (58.8% vs 89.2%, P<0.01). Conclusion There is a linear correlation between MDR1 gene expression and CD56 expression in AML. Quantification of the MDR1 gene expression together with CD56 antigen expression is more effective to the judgement of prognosis in AML.

The study aims to develop an LC-MS/MS method for the simultaneous determination of amygdalin and paeoniflorin in urine samples, and to investigate their urinary excretion characteristics in healthy volunteers after intravenous infusion administration of Huoxue-Tongluo lyophilized powder for injection (HTLPI). The urine samples were extracted by methanol, and then separated on a Hedera ODS-2 column with a mobile phase of acetonitrile and 5 mmol · L(-1) ammonium acetate buffer solution containing 0.05% formic acid (20:80). Electrospray ionization source was applied and operated in the positive ion mode using MRM. The method exhibited good linearity over the concentration range of 0.03 -40 µg · mL(-1). The values on both the occasions (intra- and inter-day) were all within 15% at three concentration levels. No matrix effect and carry-over effect were observed. Amygdalin and paeoniflorin were stable in human urine under different storage conditions. Approximately 79.6% of the administered amount of amygdalin was excreted unchanged in urine within 24 h and which was 48.4% for paeoniflorin. The developed LC-MS/MS method can be applied to evaluate the urinary excretion of amygdalin and paeoniflorin.
Amygdalin ; urine ; Chromatography, Liquid ; Drugs, Chinese Herbal ; Glucosides ; urine ; Humans ; Monoterpenes ; urine ; Tandem Mass Spectrometry