Objective To construct the mouse CD40 RNA interfering(RNAi) lentiviral vector and prepare dendritic cells (DCs) with low expression of CD40. And to explore the mechanism of inducing T lymphocyte incompetence by blocking CD40/CD40L costimulatory pathway. Methods Mouse myeloid DCs were cultured in selective medium containing necessary cytokines for DC growth in vitro. CD40 RNAi gene was transfected into DCs with lentiviral vector. The expression levels of CD40 mRNA and protein were assayed by real-time quantitative PCR and flow cytometry respectively. The influences on DCs stimulating the proliferation ability of T lymphocyte were observed through mixed lymphocyte culture(MLC). Results Myeloid DCs have been harvested from mouse through cell culture in vitro. A mouse CD40 RNAi ientiviral vector was built successfully. The lentiviral titer was 8×10~9 TU/ml. The CD40 mRNA inhibition rate after infection was significantly higher(P<0.05). The CD40 protein expression of DCs was significandy lower(P<0.05). The result of MLC demonstrated that the index of stimulation to T lymphocyte of CD40 RNAi transfected DC significantly decreased compared with non-transfected DC and empty plasmid transfected DC(P< 0.01). Conclusion Large quantity of myeloid DCs with typical histological configuration are obtained through in vitro culture in selective medium which may activate naive T lymphocyte to generate immune response. While DCs were infected by CD40 RNAi lentiviral vector, CD40 protein expression was inhibited significantly. The DCs could hamper the activation of allogeneic T lymphocyte by blocking CD40/CD40L cestimulatory pathway and induce T cell allergy. This will make the good foundation for studying the immune tolerance of CD40 RNAi.

Objective To study the role of endogenous telomerase catalytic inhibitor PinX1 in the progression of colorectal cancer tissue. Methods The expression of PinX1、telomerase subunits(hTERT) and telomere binding protein(hTRF1) was detected by semi-quantitative RT-PCR. Surgically resected colorectal cancer specimens in 66 cases were studied. Results PinX1 expression is significantly correlated with histologic differentiation,clinical stage and lymph node metastasis of colorectal cancer( Fd =11.40, Fs = 4.22,t =-2.81,P ≤0.05). The level of PinX1 was negatively associated with the expression of hTERT( r =-0.553,P

Objective To investigate the effect of blocking CD40/CD40L costimulatory pathway by the lentiviral vector-mediated RNA interference on the survival of mouse cardiac allograft. Methods Mouse bone marrow-derived dendritic cells (DCs) were infected by CD40-RNAi lentiviral vector in vitro, and tolerogenic DCs (Tol-DCs) with decreased CD40 expression were prepared. Fluorescence real-time quantitative PCR and flow cytometry were used to analyze the expression of CD40 mRNA and DC surface antigens CD40, CD11c, MHC Ⅱ before and after infection. Mouse model of heterotropic abdominal heart transplantation was established. Seven days prior to heart transplantation, Tol-DCs with decreased CD40 expression were transfused into recipient mice intravenously (lentivirus infected DC group). Control group and non-infected DC group were assigned simultaneously. The survival of cardiac allograft was monitored and pathological grade of acute rejection 7 days after heterotropic abdominal heart transplantation was determined. Results The transcription of CD40 mRNA of DCs was down-regulated significantly at 48 h after CD40-RNAi lentiviral vector infection, and the inhibition rate was 80. 9%. The expression of CD40 protein was also significantly decreased as compared with control group (40. 07% ± 4. 03% ) ( P ＜ 0. 05 ).Compared to control group (8 ± 2 days) and non-infected DC group (9 ± 1 days), the survival time of cardiac allograft in CD40-RNAi lentivirus infected DC group (14 ± 4 days) was significantly prolonged (P＜ 0. 05 ), and the pathological grade of acute rejection decreased significantly ( P ＜ 0. 05 ).Conclusion Blocking CD40/CD40L costimulatory pathway could hamper the activation of allogeneic T lymphocyte, inhibit the acute rejection and prolong the survival of mouse cardiac allograft.

Objective To establish the quality control standard of Pinellia Heart-Sedating Granules. Methods TLC methods for identifition of Radix Pinellia and Radix Scutellariae were established. A simple HPLC was established for the determination of Berberine hydrochloride. Results The spots on TLC plates were clear without interference in the blank reference. The liner range of Berberine hydrochloride was 0.009375～0.3 ?g,r =0.9 999. The average recovery of Berberine hydrochloride was 101.46% and RSD was 2.88%. Conclusion The method is simple, accruate, reproducible and can be used as the quality control method for Pinellia Heart-Sedating Granules.

This study is to report the influence of conditions in spray drying process on physical and chemical properties and lung inhaling performance of Panax notoginseng Saponins - Tanshinone II A composite particles. According to the physical and chemical properties of the two types of components within the composite particles, three solvent systems were selected including ethanol, ethanol : acetone (9 : 1, v/v) and ethanol : acetone (4 : 1, v/v), and three inlet temperature: 110 degrees C, 120 degrees C, 130 degrees C to prepare seven different composite particle samples; each sample was characterized using laser diffraction, scanning electron microscopy (SEM), dynamic vapour sorption (DVS) and atomic force microscope (AFM), and their aerodynamic behavior was evaluated by a Next Generation Impactor (NGI). The results indicate that under the conditions of using the mixed solvent system of ethanol--acetone volume ratio of 9 : 1, and the inlet temperature of 110 degrees C, the resulting composite particles showed rough surface, with more tanshinone II A distributing in the outer layer, such composite particles have the best lung inhaling performance and the fine particle fraction (FPF) close to 60%. Finally it is concluded that by adjusting the conditions in co-spray drying process, the distribution amount and existence form of tanshinone II A in the outer layer of the particles can be changed so that to enhance lung inhaling performance of the drug composite particles.

AIM To determine the relationship between the elevation of endogenous inhibitor of nitric oxide synthase (NOS)N G,N G-asymmetric dimethylarginine (ADMA) and metabolic control in diabetic rats. METHODS Diabetes was induced in Sprague-Dawley rats by a single intraperitoneal injection of streptozotocin. At 72 h after injection, half of diabetic rats received insulin treatment for 8 weeks (20 U?kg -1?d -1,ih, bid). Serum levels of ADMA were measured by high-performance liquid chromatography. Thoracic aortic rings from non-diabetic age-matched control, untreated diabetic, and insulin-treated diabetic rats were tethered in isolated organ baths,contracted with 1 ?mol?L -1 phenylephrine, and challenged with either the endothelium-dependent vasodilator acetylcholine or the endothelium-independent vasodilator sodium nitroprusside. Serum concentrations of glucose, glycosylated serum protein, and malondialdehyde, derived from lipid peroxidation were also examined to estimate metabolic control.RESULTS Serum levels of ADMA significantly elevated in untreated diabetic rats compared with control rats. This elevation of ADMA was accompanied by impairment of relaxation response to acetylcholine but not sodium nitroprusside in aortic rings. Chronic insulin treatment not only prevented the elevation of serum ADMA, but also improved the impaired endothelium-dependent relaxation in diabetic rats. Serum levels of glucose, glycosylated serum protein, and malondialdehyde were significantly increased in parallel with the elevation of ADMA in untreated diabetic rats compared with control rats. These parameters were normalized after diabetic rats received insulin treatment. CONCLUSION These results provide the first evidence that the elevation of endogenous inhibitor of NOS in streptozotocin-induced diabetic rats is close related to metabolic control of the disease.

Phospho-histone H2AX(γH2AX) has been widely used in vitro genotoxicity evaluation of ionizing radiation, carcinogenic substances and cigarette smoke as an important biomarker of DNA double stranded breaks ( DSBs) . The study developed an enzyme-linked immunesorbent assay for detection of the content ofγH2AX in cells to evaluate the genotoxicity of cigarette smoke. The study exposed CHO cells with cigarette smoke total particulate matter ( TPM) and cigarette smoke condensate ( CSC) of different dose, dose-effect relationship between smoke exposure and DNA damage and the poisonous difference of cigarette smoke components has been investigated by detecting the content change ofγH2 AX in cells under different exposure time and dose. In addition, reactive oxygen species ( ROS) in cells were also detected to study the mechanism of cigarette smoke exposure induced DSBs. The experiment result showed that the required time for the level ofγH2AX in cells grew to the peak prolonged with increased exposure dose. Once the level of γH2AX in cells grew to the peak value, then it decreased slowly; the level of γH2AX in cells rised with increasing dose of cigarette smoke;The effect of TPM on the content change of γH2AX was more than CSC; Moreover, smoke exposure could induce concentration increase of ROS in cells, and a good correlation of content change of ROS and γH2AX in cells were found, free radicals in cigarette smoke may be one major cause of DSBs.

Objective To establish the mouse bone marrow-derived dendritic cells expressing FasL protein and explore the mechanism of inducing allogeneic mouse spleen T lymphocyte apoptosis. MethodsMouse myeloid DCs were cultured in selective medium zontaining essential cytokines for DC growth in vitro. The mouse DCs were transfected with liposome-mediated FasL gene. The levels of FasL mRNA before and after transfection were assayed by real-time quantitative PCR. The expression levels of FasL protein were assayed by flow cytometry(FCM)and Western blot. Non-transfected DC，empty plasmid transfected DC and FasL transfected DC were infused intravenouslY into allogeneic mouse. After 7 days, the apoptosis in spleen T lymphocytes was evaluated by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling)method and FCM. ResultsCultured in vitro, the mature myeloid DCs from mouse could be obtained.The expressions of FasL mRNA and protein in FasL transfected DCs were significantly higher. Through the detection of spleen T lymphocyte apoptosis with TUNEL，the apoptosis index(AI)was higher in FasL transfected DC(11.67±1.53)，compared with non-transfected DC(2.67±0.58)and empty plasmid transfected DC(3.33±0.58)，P＜0.01. ConclusionA large quantity of myeloid DCs can be obtained through in vitro culture in selective medium. The liposome-mediated FasL gene transfected DCs could successfully express high levels of FasL protein. Intravenous infusion of FasL gene transfected DCs could induce apoptosis of allogeneic mouse spleen T lymphocytes.

This is to report the study of degradation of earthworm extracts prepared by wet superfine grinding in simulated gastrointestinal environment. Enzymatic reactions were terminated by adjusting the solution pH or using membrane bioreactor principle. Earthworm protein concentration change was detected by Bradford method, the degraded state of protein was described with SDS-PAGE technology, and the degraded state of small molecule substances was detected by HPLC. The results showed that earthworm protein degraded completely in artificial gastric juice. High molecular weight protein degraded greatly in artificial intestinal fluid, while low molecular weight protein was not significantly degraded. Small molecular substances degradation did not degrade in artificial gastric juice, while they degraded obviously in artificial intestinal fluid, there is even new small molecule substance appeared. Finally it is concluded that the substance that having therapeutic effects in vivo may be some degraded peptide, amino acid and stable small molecules existed in artificial intestinal fluid.

ObjectiveTo explore the relationship between fat content of postmenopausal osteoporosis patients and bone mineral density (BMD).Methods144 female osteoporotic patients with the age of 50～75 years were choosen from guangdong province from October 2010 to January 2011.According to TCM syndrome differentiations,these patients were divided into kidney Yang deficiency group,liver-kidney Yin deficiency group,spleen and kidney Yang deficiency group and Qi stagnancy and blood stasis group.Four groups of patients were performed total body fat content detection.The data were analyzed by software SPSS 16.0.ResultsComparing of L1～4 average BMD in four groups showed:BMD of Kidney Yang deficiency group was maximum,followed by spleen and kidney Yang deficiency group,and the BMD of Qi stagnancy and blood stasis group was minimum.Comparison between the four groups showed statistical significance (P＜0.05).Comparing of body fat content Iin the four groups showed:the body fat content in the kidney Yang deficiency group was minimum,followed by spleen and kidney Yang deficiency group,and body fat content in Qi stagnancy and blood stasis group was maximum.Pair-comparison in kidney Yang deficiency group,liver-kidney Yin deficiency group,spleen and kidney Yang deficiency group showed no statistical significance (P＞0.05).While these three groups showed statistical difference when comparing with Qi stagnancy and blood stasis group (P＜0.05).The body fat content and L1～4 average BMD had negative correlation.ConclusionOsteoporosis in Qi stagnancy and blood stasis group was more serious than the other three other groups.Fat content was negatively related with bone density.