REAL-TIME DETECTION OF SURVIVIN mRNA EXPRESSION IN CERVICAL CANCER CELL LINES USING MOLECULAR BEACON IMAGING

Objective To detect the expression of survivin mRNA in cervical cancer cell lines using molecular beacon imaging technology. Methods Human cervical cancer cells （HeLa and SiHa） and human fetal lung fibroblast HFL-I were cultured in vitro. After adding 100 nmol/L survivin mRNA molecular beacon, the fluorescent signals were observed under fluorescent microscope. The expressions of survivin in cervical cancer cells and HFL-I cell were examined by immunocytochemical streptravidin-biothin peroxidase （SP） assay at the same time. Results Two kinds of survivin mRNA molecular beacon, with different color fluorescence, had strong fluorescent signal in cervical cancer cell lines, and the signal in SiHa cell line was stronger, but these signals were not found in HFL-I ; Immunocytochemical staining of positive survivin was located in the cytoplasm of cervical cancer cell lines HeLa and SiHa, whereas, no expression of survivin was detected in HFL-I cell line. Conclusion The technology of molecular beacon imaging can be used to detect the expression of survivin mRNA in viable cells successfully, and may provide a new approach to the diagnosis of early stage cervical cancer and the following-up in the clinic.     

Treatment of Aniline-Contaminated Wastewater by Sequencing Biofilm Batch Reactor System Under Micro-Aerobic Condition

Sequencing biofilm batch reactor(SBBR) under micro-aerobic condition was applied to the treatment of aniline-contaminated wastewater in this study.Hydraulic retention time(HRT) of 12—36 h and dissolved oxygen(DO) concentration of 0.1—0.5 mg/L were selected as the operating variables to model,analyze and optimize the process.Five dependent parameters,aniline(AN),chemical oxygen demand(COD),ammonium,total nitrogen(TN) and total phosphorus(TP) removal efficiencies as the process responses,were studied.From the results,increase in DO concentration could promote the AN,COD and ammonium removal;increase in HRT could also lead to increase of the AN and ammonium removal,but might decrease COD removal due to endogenous respiration and soluble microbial products.In the SBBR system,24 h for HRT and 0.5 mg/L for DO concentration were chosen as the optimum operating condition.The actual removal efficiencies of COD,AN and ammonium under the optimum operating condition were 98.37%,100%and 89.29%,respectively.The experimental findings were in close agreement with the model prediction.The presence of glucose could promote bacterial growth and has positive influence on AN degradation and ammonium removal.     

Design of Micropipette System with High Precision for Small Enzyme Immunoassay Analyzer

A small auto micropipette system is developed to improve the reliability and accuracy of the automatic enzyme immunoassay analyzer's microscale pipetting system. A sophisticated injection mechanism is designed by the means of dislocation parallel distribution of the screw and injector piston rod. It possesses the function of pipetting, taking and removing the pipette tips. In the control system, STM32 controller is used, controlling the single-axis S-type acceleration/deceleration algorithm and multi-threaded coordinated motion. The acceleration/deceleration curves are analyzed and optimized by using the method of segmentation; a minimum injection rate of 1 μL and a step rate of 0.05 μL are realized. The method of digital image processing is used to detect the amount of pipetting in micro-pipetting quantitatively. The liquid area is extracted by background contrast method, and the liquid volume in the tip is obtained by combining the geometric characteristics of the disposable tip, when the pipetting capacity is not qualified to carry out specific guidance on the pipetting system, and avoid the blocking needle, bubble and other abnormal pipetting phenomenon on the impact of pipetting accuracy. The experimental results show that the combination of the automatic sampling system and the image flow detection system can effectively improve the precision and reliability of the micro-pipetting system. Finally, the injection accuracy of the system at the test points with 10, 50 and 100 μL liquid volumes reaches 1.8%, 1.28% and 1.15%respectively.     

Investigation on Zener-Hollomon Parameter in the Warm-Hot Deformation Behavior of CF53

The warm-hot deformation behavior of CF53 steel was studied with hot compression tests at temperature of 1 123 ~1 273 K and strain rate of 0.1~20 s -1 . The activation energy for warm-hot deformation is 274.816 kJ/mol. The influences of Zener-Hollomon parameter, strain and grain size imposing on the flow stress were analyzed in the temperature range of warm-hot forging. Based on the creep theory and mathematic theory of statistics, mathematical models of flow stress were obtained. The results provide a scientific basis for controlling microstructure of forging process through Zener-Hollomon parameter.     

Research on Reduction of Fe_2O_3 by Biomass Sawdust

The research on biomass reduction of Fe_2O_3 was carried out by using sawdust as reductant. The direct reducing agents in the biomass magnetization process were determined by comparing various biomass pyrolysis products with the reduction degree(divalent iron content in total iron), reduction temperature range and valence change of Fe_2O_3 in the reduction process. The microstructure variation of Fe_2O_3 at different stages was also analyzed by scanning electron microscopy(SEM). Nonisothermal thermogravimetric analysis(TGA) was applied to explore the thermal reduction process. The results show that the direct reducing substances in the biomass reaction with Fe_2O_3 are H_2 and bio-oil, and the reduction process can be divided into two steps: biomass pyrolyzing to release H_2 and bio-oil, and reductive volatiles reacting with Fe_2O_3. The two steps are relatively independent.The kinetic of the reduction reaction follows a first-order reaction kinetic model, with 88.99 k J/mol activation energy and 9.55 × 108min~(-1) frequency factor.     

THE HUMORAL AND CELLULAR IMMUNE RESPONSES INDUCED BY HPV18L1-E6/E7 DNA VACCINES IN MICE

Objective To construct eukaryutic expression vector of HPV18 L1- E6, E7 chimeric gene and examine the humorul and cellular immune responses induced by this DNA vaccines in mice. Methods The C-terminal of major rapsid protein L1 gene and mutant zinc finger domains of early E6/7 oncogenes in HPV18 were integrated and inserted into eukaryotic expression vector pVAX1 to generate vaccines pVAX1-L1E6Mxx, ETMxx. CHO cells were transiently transfected with the individual construct. Target protein expressions in the lysate of the transfected cells were measured by ELISA and immunocytochemistry After BALB/c mice were vaccinated with various recombinant plusmids（pVAX1- L1-E6M3 or pVAX1-L1-E7M3 ） and immunie adjuvants （pLXHDmB7-2 or LTB） through different administration routes （intramuscular or intranasal） , the great cellular immune responses were produced us revealed by delayed-type hypersensitivity （DTH） and lymphocyte proliferation, and the expression of IL-4 and IFN- 7 cells in CD4^＋ and CD8^＋ subpopulations. Results The highly efficient expression of pVAX1-L1E6Mxx, E7Mxx vector in host eukaryotic cells were demonstrated both by ELISA and immunocytochemistry. The level of specific serum IgG against HPV in experiment groups mice was much higher than that of control group, and intranuscular immunization group had the highest antibody level. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8^＋ IFN-γ^＋ cells number, but CD4^＋ IL4^＋ cell number was higher in intranasul immunization groups. The immunization groups using pLXHDmB7.-2 as adjuvant were superior to other groups in immunorespouse. Conclusion These DNA vaccines produce remarkable cellular and humorul immune responses in the mouse and may provide us prophylatic and therapeutic candidates for HPV induced cancer treatment.     

PRELIMINARY STUDY OF A NOVEL HUMAN PAPILLOMAVIRUS TYPE 16L1／E6-E7 CHIMERIC RECOMBINANT DNA VACCINE

Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial.     

PROLIFERATION AND MIGRATION OF EPENDYMAL CELLS IN THE BRAIN OF ADULT RATS AFTER PERMANENT FOCAL CEREBRAL ISCHEMIA

Objective Ependymal cells are thought to be the primary source of neural stem cells in the adult central nervous system. The purpose of this study is to examine spatial and temporal profiles of ependymal cell proliferation and migration after focal cerebral ischemia. Methods Eighty male Sprague Dawley rats underwent permanent middle cerebral artery occlusion after injection of 10/μL of 0.2% Dil into the lateral ventricle. Rats were sacrificed and brain sections were acquired for pathological evaluation and laser confocal imaging at day 1,3,7,11,14,21 and 28 after ischemia. Results The density of Dil-labeled cells in the ischemic ipsilateral subventricular zone was significantly higher than that in the control group and these labeled cells dispersed in the ischemic ipsilateral subventricular zone and/or were located in ependyma from day 1 to 11. In the ischemic ipsilateral cortex, some Diilabeled cells occurred in peri-infarction and infarction of parietal region at day14 and peaked at day 21 when some Dil-labeled cell nodules were found in this region. During postischemic day 14--28, a significant decrease in labeled celldensity in the ischemic ipsilateral subventricular zone was coincident with a significant increase in labeled cells density in the cortex (peri-infarction and infarction). Conclusion The results indicate that ependymal cells proliferate and migrate after focal cerebral ischemia in the adult rat brain.     

THE EFFECT OF LIGUSTRAZINE ON NEUROGENESIS IN CORTEX AFTER FOCAL CEREBRAL ISCHEMIA IN RATS

Objective To explore the effect of Ligustrazine on neurogenesis in cortex after focal cerebral ischemia in rats. Methods Focal cerebral ischemia was induced by left middle cerebral artery occlusion with a suture. Two hours later, injection of Ligustrazine （80 mg/kg, 1 time/d） was performed peritoneally. Four hours after the ischemia, 5-bromodeoxyuridine （BrdU） （50 mg/kg, 1 time/d） was injected peritoneally. At 7 d, 14 d and 21 d after ischemia, BrdU positive cells in the cortex were observed by cal staining. Results In ischemic model group, at 7 day, sparsely-distributed BrdU positive cells were observed in the Ⅱ-- Ⅵ layers of the ipsilateral cortex, with a bandlike distribution in ischemic penumbra. With the prolongation of ischemia, the number of BrdU positive cells increased. In Ligustrazine group, BrdU positive cells were also observed in theⅡ-- Ⅵ layers of the cortex, with an intense distribution in ischemic penumbra. The numbers of BrdU positive cells at 7 d, 14 d and 21 d were more than those in ischemic model group respectively. Conclusion Ligustrazine increases the proliferated cells in cortex after focal cerebral ischemia in rats. The results suggest that it may be useful for promoting self-repair after ischemia.     

Role of candesartan against cerebral ischemia and oxidative damage in normotensive rats

Objective Angiotensin Ⅱ (Ang Ⅱ ) contributes to modulating blood pressure by stimulation of Ang Ⅱ AT1 receptors. We devised a rat transient middle cerebral artery occlusion (MCAO) model to assess whether oxidative damage is decreased after pretreatment with Angiotensin Ⅱ AT1 receptor blocker (ARB). Methods After 2 weeks pretreatment with ARB 0. 5 and 1 mg/kg, the male Wister rats were subjected to 2 h middle cerebral artery occlusion (MCAO). At 24 h, the lumen diameter of middle cerebral artery, the plasma level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), and HIF-1 a levels were recorded and compared. Results After pretrcatment with ARB 0.5 and 1 mg/kg, blood pressure did not significantly change compared with that of controls. In the group of candesartan at 1 mg/(kg· day), the lumen diameter was significantly increased compared to that in control group [(86.0±5.0) μm vs. (69.0± 2.1) μm; P<0. 01, n = 6- 8]. The plasma 8-OHdG levels of ARB pretreatment groups were decreased. In immunohistochemical findings, 8-OHdG- and HIF-1α-containing cells in ARB pretreatment groups were decreased. Conclusion Brain ischemia and oxidative damage can be reversed by AT1 receptor blockade in normotensive rats after transient cerebral artery occlusion.     

Research on pharmacokinetics of high-dose tamoxifen in non-small cell lung cancer patients

Objective To study the pharmacokinetics of tamoxifen at a high dosage, which will offer a theoretical support for an appropriate clinical use of the medicine in non-small cell lung cancer （NSCLC） patients. Methods Three qualified NSCLC patients are selected and given tamoxifen （TAM） 160 mg per Os. Blood samples were collected at different times and then analyzed by high-performance liguid chromatography. The PK-GRAPH program was used to obtain the parameters. Results The concentration-time courses of the TAM 160 mg were fitted to one-compartment model. The pharmacokinetic parameters were estimated as follows： Tmax （6. 35±1. 24）h, Cmax （217. 39±7. 71）ng/mL, AUC （12 127.39±636.16）ng · h/mL and T1/2ke（34. 13±2.97）h. Conclusion TAM 160mg one day per Os cannot reach the effective maintenance concentration in vivo required for reversing MDR in vitro. Loading-maintenance dose strategy is recommended to study the pharmacodynamies of tamoxifen at a high dosage in NSCLC patients.     

Reduction of Brain Injury After Stroke in Hyperglycemic Rats via Fasudil Pretreatment

Diabetes is usually associated with cerebrovascular disease, especially stroke. In practice, fasudil is widely accepted to be applied for the treatment of vascular disease. This article demonstrates the study concentrating on the effects of fasudil pretreatment on the prognosis of diabetic stroke. 250—300 g SpragueDawley rats were randomly divided into three groups, non-diabetic stroke group, diabetic stroke group, and fasudil pretreatment group. The rats of diabetes group were treated with intraperitoneal injection of streptozotocin(60 mg/kg), in the meantime the same dose of citrate buffer was injected into those of the control group. The rats of the fasudil group received daily fasudil intraperitoneal injection at 10 mg/kg for three consecutive weeks. After four weeks, all the rats of the experimental group were treated with middle cerebral artery occlusion for 90 min. After sacrifice, the fresh brain samples were collected for following experiments, including infarct volume, edema volume,blood-brain barrier(BBB), which were detected by immunohistochemistry. Inflammatory factors were examined by real-time polymerase chain reaction(RT-PCR) using tissue Ribonucleic Acid(RNA). The concentration of blood glucose is 15 mmol/L or more, which proved that the diabetes model was a success. Fasudil pretreatment decreases the percentage of stroke mortality of diabetes from 43.75% to 31.25%, while the infarction volume decreases from 52.95% ± 12.7% to 45.97% ± 6.7%. Gap formation of tight junction and Immunoglobulin G(Ig G) leakage were reduced(P 0.05), and the expression of inflammatory factors decreases(P 0.05) in fasudil pretreatment after diabetic stroke. Diabetes aggravates the mortality of cerebral ischemic rats. Prolonged fasudil pretreatment can reduce mortality of diabetic stroke, decrease cerebral infarction volume and undermine inflammatory factors expression, and protect the BBB.     

FOLLICULO-STELLATE CELLS OFTHE RAT ANTERIOR PITUITARY RESPONDED TO ANGIOTENSIN Ⅱ BY INCREASING INTRACELLULAR CONCENTRATION

Objective The main purpose of this study was to investigate whether the folliculo-stellate cells (FSC) respond to angiotensin(Ang) Ⅱ by increasing intracellular free concentration ([]i) ,and where the o-rigin of mobilization is if that has occurred. Methods Pituitary cells in primary culture were prepared from male Wister rats(250g) by a conventional method and cultured in MEM supplemented with 4％ normal rat serum. Af-ter 2 days in culutre,cells were loaded with 1 μmol/L fura-PE3/AM for 1 h and subjected to a ment with Quanti Cell 700 system. Excitation wavelengths of 340 and 380 nm were selected by means of a computer-controlled filterwheel. Results The of FSC in the rat anterior pituitary was elevated by Ang Ⅱ. The eleva-tion of of FSC induced by 0. 1,1.0,10 and 100 nmol/L Ang Ⅱ was (56.33±6.18), (117.07± 36.07), (175.59 ± 40.01 ) and (216.02 ±11.52) nmol/L, respectively. The increase of of FSC induced by 100nmol/L Ang Ⅱ was not influenced by the medium without (0Ca),but significantly suppressed by thapsigargin (TG),an inhibitor of ATPase. The rate of responsive FSC to Ang Ⅱ (100 nmol/L) was 61.84％ which was obviously higher than that of pituitary endocrine cells (43.49％). Conclusion The present experiment demonstrates that the FSC in the rat anterior pituitary responds to Ang Ⅱ by increasing [which raises the possibility that Ang Ⅱ re-leased from either lactotrophs or gonadotrophs affects FSC through paracrine mechanism. The elevation of [induced by Ang Ⅱ presents a dosage-dependent relation, and is possibly because of the release of from an intra-cellular pool (s). Fashions of release are relative to the concentration of Ang Ⅱ. The results indicate that Ang Ⅱ functions as a paracrine factor among pituitary cells including FSC.     

Asymmetric PCR method in generation of HBV ssDNA for pyrosequencing

Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction (A-PCR) in producing hepatitis B virus (HBV) single-stranded DNA (ssDNA) for pyrosequencing. Methods A-PCR was carried out to generate HBV ssDNA with forward to reverse primers of different ratios (50 : 1, 100 : 1) and concentrations (13. 0 pmol/25μL and 0.14 pmol/25μL, 19. 5 pmol/25μL and 0. 21 pmol/25μL), and the product yield and quality were compared respectively. Results The forward to reverse primer ratio of 50 : 1 provided better yield and concentration of 19. 5 pmol/25μL and 0. 21 pmol//25μL generated a clearer band. Conclusion A simple and feasible method to produce HBV ssDNA for pyrosequencing in batch is established.     

Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase

Objective To investigate the mechanisms of apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase. Methods Human leukemia cell line K562 were exposed to indole-3-acetic acid （IAA） at 20, 40, 60, 80 or 100 mol/L and horseradish peroxidase（HRP） at 1.2 g/mL for varying times. MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect the arrest of cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay was used to measure apoptosis. 2, 7-dichlorofluorescin diacetate （DCFH-DA） uptake was measured to determine free radical by confocal microscope. Content of malondiadehyde （MDA） and activity of superoxide dismutase （SOD） were measured by biochemical methods. Results IAA/HRP initiated growth inhibition of K562 cells in a dose- and time-dependent manner. Flow cytometry revealed that cell cycle arrested at G1/G0 after 24 hours treatment. After 72 hours treatment, apoptotic rate of 100 mol/L IAA group increased to 43.9 %, which was 5 times that of control（P〈0.01）. Content of MDA and activity of SOD increased respectively in treatments compared with control. Meanwhile, IAA/HRP stimulated the formation of free radical, which was increased by IAA concentration-dependently. Conclusion The combination of IAAand HRP can inhibit the growth of Human leukemia cell line K562 in vitro by inducing apoptosis which is associated with the increase of free radical. The combination of IAA and HRP might be a promising chemopreventive and chemotherapeutic agent against human leukemia.     

Pretreatment with candesartan protects brain against ischemia in normotensive rats

Objective Angiotensin Ⅱ （Ang-Ⅱ ） increases NADPH oxidase activity and stimulates the production of reactive oxygen species （ROS） including superoxide anion through Ang Ⅱ AT1-receptor （AT1-R） activation. ROS is involved in various pathological processes in brain ischemia. We investigated whether the AT1-R blocker （ARB） candesartan can protect normotensive rats against brain ischemia. Methods After 2-week pretreatment with candesartan, rats were subjected to 2 hours middle cerebral artery occlasion-reperfusion （MCAO-R） and 24 hours later, the infarct volume, iNOS, and eNOS mRNA in the internal carotid artery was recorded and compared. Results Candesartan pretreatment reduced cerebral ischemia and oxidative brain damage after MCAO-R in normotensive rats, resulting in a decreased cortical infarct volume [0.5 mg/kg candesartan, （46. 8±13.2）mm^3 ; 1.0 mg/kg candesartan, （ 19.3± 15.3） mm^3 vs. control, （ 111.7 ±14.3 ） mm^3 ; P〈 0.05, P〈 0.01, respectively]. Candesartan pretreatment increased the eNOS mRNA level in the internal carotid artery. Conclusion In normotensive rats exposed to MCAO-R, candesartan protectes against brain ischemia. This effect may represent a significant therapeutic advantage and may induce end-organ protection even at normal blood pressure.