Abstract:Objective: To obtain the histone deacetylase 1(HDAC1) cDNA and construct the target gene of yeast two-hybrid system. Methods: About 1.45kb DNA fragment was amplified from the human Jurkat cell by RT-PCR. After being automaticly sequenced, the fragment was ligased with the vector pLexA to construct pLexA-HDAC1. The recombinant plasmid was transformed into yeast EGY48,and the expression of pLexA-HDAC1 in the yeast was observed. Results: The amino acid sequence encoded by RT-PCR product was the same as reported HDAC1,and about 1 mm white yeast clone grew in the selective medium after 3d. Conclusion:The HDAC1 cDNA has been obtained. pLexA-HDAC1 has nontoxic to yeast, which can serve as a target gene of yeast two-hybrid system.

Abstract:Objective：To study the chemical stability of leflunomide in a wide pH value range. Methods：A method to determine leflunomide by high performance liquid chromatography (HPLC) was established. The rate constants at different temperature and different pH values were determined. The pH values of maximum stability at different temperatures and the decomposed activation energy values for different pH values were obtained. Results：The pH values of maximum stability were pH2.85-4.56 at 20-40℃, the activation energy also showed maximum values within pH2.85-4.56. The most stable pH values of leflunomide aqueous solution moved to lower pH values with a rise in temperature. When temperatures were over 60℃, the pH values of maximum stability were below 1.80. Conclusion：The chemical stability of leflunomide in aqueous solution can be improved by modifying pH values.

Abstract:Objective：To prepare the transgenic mice harbouring 2 copies genome of hepatitis B virus(HBV) as a useful model for the studies of medical issues related to HBV. Methods：The HBV(adr subtype)transgenic mice were prepared by microinjection of complate 2.0 copy HBV genome into pronuclei fertilized eggs. The PCR assay, Southern-blotting, radioimmunity, immunohischemistry and ELISA methods were used to detect the HBV intergration，expression and replication in the transgenic mice. The dane particles were observed in the liver. The pathological change was tested with ALT,AST in the serum and the normal histological slides. Results：The 4 transgenic mice were obtained and the HBV DNA can be expressed and replicated in the transgenic mice. HBV genomic DNA in mouse line could be transmitted to next generations. No evidence suggested the existence of pathogenesis. Conclusion：The HBV transgenic mice were obtained and the virus genes can be expressed, the Dane particles can be formed in the liver.The mice are tolerant to HBV gene products. The transgenic mice are similar to human chronic HBV carrier. The structure and the function of the transgenic mice are stable.

Abstract:Objective: To study the effect of anti-CD3-anti-glioma bispecific antibody (BsAb) on human glioma growth in nude mice. Methods: There were 6 groups in these experiments, basic group was treated with BsAb, others were different control groups. Nude mice-human glioma model NHG-1 was used to observe the occurence, growth of the tumor and survival periods. Results: In the basic group, the average incubation period, average tumor cells cycle, and average survival periods were postponed compared with all control groups (P<0.05 or P<0.01). According to tumor growth trend, different control groups went into rapid growth at different time after several week′s slow growth. However, the basic group treated with BsAb lived in a slow growth rate in the whole survival. Conclusion: BsAb can decrease the growth rate of tumor in nude mice, and it may has potential usage in clinic.

Abstract:Objective:To observe the change of peripheral PMNS CD18 at the early stage of rat lung reperfusion injury.Methods:Twenty SD rats were divided randomly into 4 groups:control group,ischemia group,reperfusion 30 min group and reperfusion 60 min group.The left lung wet/dry mass(mW/mD) ratio,the pulmonary artery pressure and the level of circulating PMNS CD18 of animals in each group were measured. Results:The mW/mD ratio and the pulmonary artery pressure of both the reperfusion 30min and 60min group were higher than those of the control group(P<0.01),respectively.The level of PMNS CD18 of the reperfusion 60min group increased significantly compared with that of the reperfusion 30min group(P<0.01).The PMNS count of the circulating blood was lower in the reperfusion 60 min group than in the control group (P<0.05).Conclusion:The level of PMNS CD18 increases and the count of PMNS decreases at the early stage of lung reperfusion injury.

Abstract:Objective: To investigate the effects of oxymatrin (OM) and glycyrrhizin (GL) on hepatocyte apoptosis induced by cessation of phenobarbital(PB) treatment in mice. Methods: The Kunming mice were treated with OM (150mg/kg,ip) and GL (50mg/kg,ip) 3times daily during 36h after cessation of PB treatment. Hepatic DNA content and the ratio of liver/body mass of mice were observed to estimate the regressive rate of hyperplastic liver at 36h after withdrawing PB. Histomorphology and the end-labeling method termed TUNEL (TdT-mediated X-dUTP nick end labeling) were used to identify apoptotic cells and DNA in apoptotic cells. Results: Regression ratio of hepatic DNA content and liver/body mass in the positive control group was 32.4%and 16.3%, and 28.9%and 16.1%in the group treated with GL. Hepatic DNA content and liver/body mass in the group treated with OM produced little regression as compared with the negative control group. The results showed that OM markedly inhibited the regression of hepatic DNA content and liver/body mass, but GL showed no effect. Additionally, those mice in the positive control group and the group treated with GL produced a typical hepatocyte apoptotic change with a positive result of TUNEL assay. No hepatocyte apoptotic changes were found in the liver of mice treated with OM, and a negative result of TUNEL assay was presented. Conclusion: OM inhibits the regression of hepatic DNA content and liver/body mass by preventing hepatocyte apoptosis induced by cessation of PB treatment in mice.

Abstract:Objective: To observe the change of amino acid transmitter and gamma-aminobutyric acid (GABA) receptor in the hypothalamus of burned rats during early stage, and to investigate its effect on the cardiovascular parameter of burned rats. Methods: 30%TBSA Ⅲ degree burn model of rat was made. Concentration of amino acid transmitter in the hypothalamus of burned rats was determined by auto-analysis of free amino acid. Radioligand binding assay was adopted to determine GABA receptor. The drug was injected into lateral ventricle (LV). Cardiovascular parameter was measured with four leads physiological recording instrument. Results: Concentration of GABA was significantly elevated, concentration of glutamate was reduced and there was no change of other amino acid transmitter in the hypothalamus of burned rats during early stage. Although maximum binding of GABA receptor increased significantly, there was no change of dissociation costant. Picrotoxin injection into LV may be significantly elevate MAP,LVSP,±dp/dtmax of burned rats. There were also limited effects of kinic acid (KA) on these parameters. Conclusion: The change of central GABA and glutamate transmitter may be a effecting factor on cardiovascular function failure of burned rats during early stage

Abstract:Objective：To observe the relationship between pathologic oxygen supply dependency and energy metabolism in organ tissues, and to explore the significance of this dependency as to evaluate tissue oxygenation. Methods: The model of septic shock was established by cecum ligation and puncture(CLP). SD rats were randomized to 7 groups: pre-CLP, 2, 4, 5, 6, 7 and 8h after CLP. The parameters of oxygen kinetics were measured just before rats were killed. Liver, heart and kidney tissues were removed quickly and tissue phosphocreatine (PC) and adenosine triphosphate (ATP) were assayed using the luciferase enzyme system. Results： Biphasic relationship between systemic oxygen delivery (DO2) and oxygen consumption (VO2) was exhibited. DO2 decreased progressively and lowered to a value of critical DO2 (cDO2) at 6 h after CLP. Pathologic supply dependency of VO2 on DO2 existed below cDO2. At 5 h after CLP, PC levels were lowered (P＜0.05) in liver tissues. Reductions both in hepatic ATP and renal PC and ATP levels were present. Myocardial PC and ATP levels decreased significantly (P＜0.01) at 6 and 7 h after CLP, respectively. Conclusion： The existence of pathologic dependency of VO2 on DO2 was indicative of the defect of energey metabolism. That abnormalities in renal and hepatic tissues began to exist before cDO2 appeared. Further management to prevent cellular bioenergetic failure should be taken in early septic shock.

Abstract:Objective: To evaluate the effects of propofol on metabolism and ultrastructure of acute ischemia-reperfusion myocardium in an open-chest canine model. Methods: Eighteen hybrid dogs(11-14kg) were divided randomly into 3 groups: NS group (normal saline), 2ml/(kg.h)； HP group (high-dose propofol), 11.2 mg/(kg.h)；LP group (low-dose propofol), 5.6mg/(kg.h). These dogs were subjected to a 90min left anterior descending coronary artery (LAD) occlusion followed by 200 min of reperfusion. Before administration, 60min after administration, 90min after occlusion and 60, 120,180 min after reperfusion, lactate dehydrogenase(LDH) and superoxide dismutase(SOD) in great cardiac vein were detected. 200min after reperfusion myocardial ultrastructure was examined by electronic-microscope. Results: Compared with the value in HP and LP group, LDH in NS group was significantly higher, SOD was significantly lower(P<0.05) during reperfusion. Impairment of myocardial ultrastructure was most severe in NS group. Conclusion: Propofol can reduce myocardial ischemia-reperfusion injury in a canine model with its anti-oxidation and protection of chondriosome.

Abstract:Objective: To observe the protective effect of puerarin on cultured human aortic endothelial cells (ECs) injury caused by lipid peroxide. Methods:ECs were treated with diamide to stimulate and faciliate lipid peroxidation. The diamide concentration was 0.1×10-4 and 1.0×10－4mol/L. Then 0.3mg puerarin was added immediately or 0.5h later. Results: Puerarin decreased the LPO and TXA2, then the MTT increased and LDH release decreased. Conclusion: Puerarin reduces the injury of ECs caused by lipid peroxide.It might play an important role in antiatherongenesis.

Abstract:Objective: To observe the effects of radioimmunotherapy with 90Y-OSMcAb on human osteosarcoma in nude mice osteosarcoma model. Methods:The monoclonal antibody labeled with carrier-free 90Y by singcyclic DTPA-chelation. The carrier-free 90Y was eluted from 90Y generator. The radioimmuno-complex was proven to combine with osteosarcoma cell. Osteosarcoma cells from human osteosarcoma cell line were implanted into the nude mice which could grow and reproduce in nude mice bodies. The therapy effects with 90Y-OSMcAb and 90Y-Cl3 on human osteosarcoma in nude mice model were observed. Results: (1) The 90Y activity was higher while the contumination of 90Sr was less than 0.0031%; (2) The efficiency of 90Y-labeled antibodies was over 90%，the activity of labeled antibody maintained 82.3%; (3) Tumor size of all 90Y-antibody-treated model was significantly less than that of all 90Y-Cl3-treated model, the average survival time of the mice of the 90Y-antibody-treated group was significantly longer than that of the 90Y-Cl3-treated group; (4) In the tissue sections extensive necrosis was observed in all 90Y-antibody-treated tumor, the living tumor cells were immunohistochemically stainable with PcNA. Staining with PcNA in all 90Y-antibody-treated tumor was negtive, but positive in all 90Y-Cl3-treated tumor. Conclusion: OSMcAb post-labeled targets osteosarcoma structures. 90Y-OSMcAbs can successfully treat osteosarcoma in nude mice.

Abstract:Objective: To study the effect and mechanism of LHRH analogue nafarelin on the growth of BCaP-37 human breast cancer cell xenografts in nude mice. Methods: Nafarelin treatment began at next day or 2 weeks after tumor inoculation and each group had 3 different doses by s.c. per day for 4 weeks. Tamoxifen and another LHRH analogue D-Trp6 LHRH were used as positive control. The changes of xenografted tumor induced by nafarelin treatment were investigated with respect to its histology, cell DNA index (DI), FCM, serum level of E2 as well as the mass of tumor, uterus and bilateral ovaries. Results: (1) Chronic administration of nafarelin at doses of 500 or 1 000 μg/kg resulted in a significant inhibition of tumor growth and a decrease in DI and serum level of E2, apoptosis of tumor cells in nafarelin treated groups was also observed. (2) Tamoxifen had no inhibitory effect on the growth of xenografted tumor at 30 mg/kg. D-Trp6 LHRH had a potency comparable to nafarelin in suppressing the growth of BCaP-37 xenografted tumor. Conclusion: Chronic administration of nafarelin could inhibit the growth of xenografted BCaP-37 breast cancer significantly.

Abstract:Objective: To compare the bioavailability and pharmacokinetics of famotidine powder and tablets. Methods: Ten healthy volunteers were given a single oral dose of 40mg of famotidine power or tablet. The blood and urine drug levels were determined by reverse-phase HPLC method. Results: The data obtained from the HPLC were analysed automatically by a MCPKP program. The results showed that the curve of famotidine plasma concentration-time data was in line with the one-compartment open model. The relative-bioavailability of famotidine powder was 103%.The tmax,cmax,t1/2k and AUC of famotidine powder and tablets were(1.91±0.44 ), (2.54±0.40)h; (99.4±37.6),(87.0±29.5)ng/ml;(3.21±0.67),(2.49±0.56)h;(627±100),(607±177)h.ng/ml, respectively. Conclusion: The AUC and cmax of 2 formulations are bioequivalent. There is statistical difference in the tmax between 2 formulations.

Abstract:Objective: To study the characteristics of collagen and the preparation of poly-hydroxyethyl methacrylate (HEMA)-collagen membrane. Methods: TypeⅢ collagen extracted from calf fetus skin was mixed thoroughly with HEMA and ethylene glycol. The polymerization was induced by ammonium persulfate and sodium metabisulfite. Results: The poly(HEMA)-collagen membrane was an elastic, flexible, absorbent and adhesive film. The cumulative in vitro release profile of indometacin from the membrane showed that the porous sponges could be used as a good drug release system. Conclusion: The poly(HEMA)-collagen membrane is a good material to be used as a wound dressing for burns.

Abstract:Objective: To develop pulsed-release system of verapamil in the light of circadian rhythms of hypertensive patients. Methods: Factors affecting the lag time of the system were tested. Three swelling disintegrates (L-HPC,CMS-Na, CMC-Ca) of different contents were used. Various composition of out shell was tested through the uniform design and dealed with multiple regression. Two basic formulations with lag time 3 and 5h were decided. Results: The lag time 5h formulation: CMS-Na was the preferable disintegrant with a content of 15mg/tablet, out shell composition were chosen as following: PEG 6000 110mg, HCO 105mg, EVA 25mg each tablet, hardness 490kPa. Conclusion: A new press-coated tablet for the pulsed-release of drug after a programmable period of time is achieved in the in vitro dissolution test.

Abstract:Objective: To investigate relative factors for decline of residual renal function(RRF) in patients treated by hemodialysis and discuss protective measures of RRF. Methods: All patients were treated by bicarbonate hemodialysis. Twenty-four hour urine volume, rate of creatinine clearance, increase of body weight between 2 sessions of dialysis and complication of hemodialysis were observed during 6-12months. Results: The decrease of RRF associated with primary glomerular disease was quicker as compared with primary renal tubular intestine disease. The more volume of ultrafiltration is during hemodialysis, the faster decrease of RRF in chronic hemodialysis patients. RRF was more significantly decreased in groups adapted with 1.7m2 dialyzer than that in group with 1.1m2 dialyzer. Conclusion: High ultrafiltration by hemodialysis can induce decline of effective renal blood flow and result in ischemia and reperfusion injury, which may be the primary factor for decrease of RRF at the initial stage of hemodialysis. High effective dialysis had attenuant effect on osmotic diuresis by elimination of solute load and speed up decrease of RRF. Primary glomerular disease, especially on the active phase, also may contribute to decline of RRF.

Abstract:Objective: To select the better method for the examination of functional outlet obstruction, the defecography with thickened barium (DTB) and the defecography with barium suspension (DBS)were compared and evaluated objectively. Methods：The comparison of DBS and DTB were carried out on the same day in 25 cases of constipation. Results: The average internal rectal diameter of DTB was 1.16cm larger than that of DBS. The average rectal volume of DTB was 2.2 times as large as of DBS. In 24 abnormal cases detected, the blocking and inlaying of barium were all revealed by the DTB and little barium remained in only 2 cases by the DBS. The average time of defecating with DTB were 9s, and with DBS were 2.5s. The capability of revealing the cause of obstruction at outlet were better in 14 DTB cases and better in 6 DBS cases. Conclusion: For the examination of functional outlet obstruction, the DTB is better than the DBS. The revealing quality can be improved if little concentrated barium suspension is injected before the thickened barium.