Objective To silence the expression of tissue factor(TF) gene of human umbilical vein endothelial cell(HUVECs) of the newborns with placental abruption(PA) and normal newborns.Methods There were two groups in the experiment,normal group and PA group.Three different treatments were established in each group:(1) the blank; (2) the false-intervention ; (3) the TF gene silencing.There were three samples in each treatments.After these treatments,the changes of mRNA expression of the HUVECs were observed before and aftcr thc gene silencing and the changes of the immunofluorescence of the TF protein level.Results After amplificated,plasmid DNA were sequenced to show that the pENTRTM/U6-TF-shRNA was the positive clone.After the transfected,the levels of the mRNA of TF decreased from 0.657 ± 0.097 to 0.220 ± 0.030 and 1.323 ± 0.323 to 0.207 ± 0.150 in the normal and PA group respectively.Compared with the normal group,there were significant differences for the levels of TF mRNA in PA group with the blank,(1.323 ± 0.323 vs 0.657 ± 0.097,P =0.023) and the same result for the second management (1.057 ±0.178 vs 0.540 ± 0.079,P =0.01).But there was no significant difference between the normal and PA group after RNA interference gene silencing (0.220 ± 0.030 vs 0.207 ± 0.150,P ＞ 0.05).Meanwhile,there were significant differences among the three managements in the themselves groups of normal and pathological ones(F =19.30,P =0.002 ;F =27.66,P =0.001).Conclusion The vectors are transfected into HUVECs and play the biological function.And they silence the expression of TF mRNA.PENTRTM/U6-TF-shRNA could inhibit the expression of TF mRNA of HUVECs in the PA newborn.

Objective To investigate the development strategy of novel T cell based vaccine against HCV infection.Methods BALB/c mice were primed with pSCK-based DNA vaccine and boosted with type 5 adenoviral vector-based vaccine, which expressed the structural proteins ( Core, E1 and E2) de-rived from a Chinese HCV patient (genotype 1b, Hebei strain).Enzyme linked immunospot assay (ELIS-POT) and intracellular cytokine staining ( ICS) were used to analyze the elicited antigen-specific immune re-sponses and the efficacy of cross-protection.Results Immunization of mice with the prime-boost vaccination strategy elicited stronger T cell immune responses against multiple HCV antigens than using the DNA vac-cines alone, especially the IFN-γ-secreting T cell responses against E1 protein as indicated by ELISPOT as-say.ICS data indicated that the prime-boost regimen elicited more TNF-α-producing CD4+and IFN-γ-produ-cing CD8+T cells against E1 protein and high levels of IFN-γ-producing CD4+and CD8+T cells against E2 protein in comparison with immunization with DNA vaccines.Moreover, the prime-boost vaccination was ca-pable of eliciting effective cross-protection in a surrogate challenge model based on a recombinant heterolo-gous HCV (JFH1, 2a) vaccinia virus.Conclusion The prime-boost vaccination using DNA and rAd5-based vaccine expressing HCV structural antigens induced significant cellular immune response and cross-protection in mice, suggesting the possibility of using it as a promising T cell based vaccine against HCV in-fection.

Objective　To compare the Immunoreactivity of various HEV Antigen with Anti-HEV IgM. Methods Solid-phase enzyme immunoassay( EIA ) was developed for detecting anti-HEV IgM by using synthetic peptides E30, E42, E33, and recombinant antigen from HEV ORF-2. Results Of 60 anti-HEV positive sera by using E30, E42, E33 and recombinant antigen as coating antigen, Anti-HEV IgM positive rates were 76.7%, 26.6%, 18.3% and 66.7% respectively. In Acute-phase and convalescence-phase sera of the patients with Hepatitis E, Anti-HEV IgM positive rate was 90% and 3.3% respectively. Conclusions The HEV E30-based EIA will be very useful in the early diagnosis of Hepatitis E.

Objective To study the regulation of microRNA 199a (miR-199a) on adhesion,migration and invasion ability of human eutopic endometrial stromal cells (ESC) from patients with endometriosis.Methods ESC were transfected with miR-199a mimics or negative control (NC) RNA by lipofectamine 2000.The adhesion,migration and invasion ability of ESC were detected by cell adhesion assay,scratch assay,cell migration assay and matrigel invasion assay,respectively.Luciferase reporter assay was used to evaluate whether IKKβ was the target gene of miR-199a.The expression of ikappa B kinase beta (IKKβ),inhibitory kappa B alpha (IκB-α),phospho-IκB-α (p-IκB-α) and nuclear factor-kappa B (NF κB) protein were measured by western blot.Results ( 1 ) Adhesion potential:the adhesion inhibitory rates were ( 14 ± 4 )％ in miR-199a group and 0 in control group,which showed significant difference (P＜0.01 ).(2) Migration and invasion:in the scratch assay,ESC transfected with miR-199a exhibited a lower scratch closure rate than that of controls.In migration and invasion assays,the migration and invasion ability of miR-199a group were significantly decreased compared with those of NC group [ 130 ± 31 vs.247±36 (P＜0.01); 63 ± 15 vs.133 ± 17 (P＜0.01),respectively].(3) The luciferase activity of miR-199a group was significantly lowered than that of control group [ 0.160 ± 0.006 vs.0.383 ± 0.083 ( P ＜0.01 ) ].The protein levels of IKKβ,p-IκB-α,IκB-α and NF-κB of 0.350 ±0.195,0.443 ±0.076,1.970 ±0.486 and 0.454 ± 0.147 in miR-199a group were significantly different compared with the NC group in which the protein levels were set at 1.000 ( P ＜ 0.01 ).Conclusions miR-199a can inhibit the adhesion,migration and invasion of the ESC.IKKβ is the target gene of miR-199a in ESC.One of the mechanisms of the inhibition effect is probably that miR-199a inhibits the activation of NF-κB signaling pathway by targeting IKKβ gene.

Objective To induce bone marrow mesenchymal stem cells (BMSCs) differentiated into hepatocyte-like cells with rebirth liver tissue lixivium of mouse in vitro.Methods Mouse BMSCs were isolated and directionally induced with rebirth liver tissue lixivium of mouse 36 h after partial hepatectomy (PH). The morphology of cells was observed under an invert microscope, and the characteristics of differentiated cells was identified by immunofluorescence test.Results 7 days after induced by liver lixivium, the spindle shaped BMSCs became round and resembled hepatocyte-like cells. 1～2 weeks later, the differentiated cells expressed albumin, CK8 and CK18, which were known as characteristic markers of the hepatocyte.Conclusion BMSCs of mouse can be differentiated into hepatocyte-like cells under induction of liver tissue lixivium of mouse.

AIM:To evaluate the feasibility and advantages of two-port 27-gauge pars plana vitrectomy for treatment of recurrent retinal detachment in silicone oil dwelling eyes.METHODS:A retrospective study.Two-port 27-gauge pars plana vitrectomy was performed in 7 eyes with recurrent retinal detachment without remove of silicone oil.Proliferative membrane peeling, endolaser treatment, and drainage of subretinal fluid were performed.Scleral bulking was performed if necessary.RESULTS:Sucessful retina re-attachment was accomplishied in all eyes.No visually significant intraoperative complication occurred.All eyes expericend quick recoveray of visual acuity and mild postoperative irritation.One eye developed a recurrent inferior retinal detchment in 20d after the surgery using non-remove technique, and recovered after conventional silicone oil removal and retinal reattachment surgery.CONCLUSION:Two-port 27-gauge pars plana vitrectomy is an efficacious (and potentially cost-efficient) method to treat early stage recurrent retinal detachment in silicone oil dwelling eyes.

OBJECTIVETo explore the protection of high intensity microwave radiation on hypothalamo-pituitary-adrenal axis (HPAA) activity and hippocampal CA1 structure in rats and the protectiveeffect of Qindan Granule (QG) on radiation injured rats.

METHODSTotally 48 Wistar rats were randomlydivided into 8 groups, i.e., the normal control group, post-radiation day 1, 7, and 10 groups, 7 and 10days prevention groups, day 7 and 10 treatment groups, 6 in each group. Rats in prevention groups wererespectively administered with QG liquid (1 mL/100 g, 4. 75 g crude drugs) for 7 days and 10 days bygastrogavage and then microwave radiation. Then preventive effect for radiation injury was statisticallycalculated with the normal control group and the post-radiation day 1 group. Rats in treatment groupswere firstly irradiated, and then administered with QG liquid (1 mL/100 g, 4.75 g crude drugs). Finally preventive effect for radiation injury was statistically calculated with the normal control group, post-radiation day 7 and 10 groups. Contents of corticotrophin releasing hormone (CRH), beta endorphin (beta-EP), adrenocorticotropic hormone (ACTH), and heat shock protein 70 (HSP70) were detected. Morphological changes and structure of hippocampal CA1 region were observed under light microscope.

RESULTSCompared with the normal control group, contents of CRH and beta-EP significantly decreased in each radiation group. Serum contents of ACTH and beta-EP significantly increased in post-radiation day 1 and 7 groups (P < 0.05). Compared with radiation groups, beta-EP content in serum and pituitary significantly increased, and serum ACTH content significantly decreased in prevention groups (P < 0.05). Pituitary contents of CRH and beta-EP significantly increased in prevention groups. Serum contents of ACTH, beta-EP, and HSP70 were significantly lower in day 7 treatment group than post-radiation day 7 group (P < 0.05). Morphological results showed that pyramidal neurons in the hippocampal CA1 region arranged in disorder, with swollen cells, shrunken and condensed nucleus, dark dyeing cytoplasm, unclear structure. Vessels in partial regions were dilated with static blood; tissues were swollen and sparse. In prevention and treatment groups pathological damage of hippocampal CA1 region was obviously attenuated; neurons were arranged more regularly; swollen, pycnotic, or deleted neuron number were decreased; vascular dilatation and congestion was lessened.

CONCLUSIONQG could affect HPAA function and activity of high intensity microwave radiated rats, showing certain preventive and therapeutic effects of microwave radiated rats by adjusting synthesis and release of partial bioactive peptides and hormones in HPAA, improving pathological injury in hippocampal CA1 region.

Adrenocorticotropic Hormone ; blood ; Animals ; CA1 Region, Hippocampal ; drug effects ; pathology ; radiation effects ; Corticotropin-Releasing Hormone ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; HSP70 Heat-Shock Proteins ; blood ; Hypothalamo-Hypophyseal System ; drug effects ; radiation effects ; Microwaves ; adverse effects ; Pituitary-Adrenal System ; drug effects ; radiation effects ; Random Allocation ; Rats ; Rats, Wistar ; beta-Endorphin ; blood ; metabolism

Huperzine A is a potent,reversible,and blood-brain barrier permeable acetylcholinesterase irhibitor.The aim of this study was to compare the pharmacokinetics,tolerability,and bioavailability of two formulations with the established reference formulation of huperzine A in a fasting,healthy Chinese male population.This was a randomized,single-dose,3-period,6-sequence crossover study.The plasma concentrations of huperzine A were determined by liquid chromatography tandem mass spectrometry.Tolerability was assessed based on subject interview,vital sign monitoring,physical examination,and routine blood and urine tests.The mean (SD) pharmacokinetic parameters of the reference drug were Cmax,1.550 (0.528) ng/mL;t1/2,12.092 (1.898) h;AUC0-72h,17.550 (3.794) ng.h/mL.Those of the test formulation A and test formulation B were Cmax,1.412 (0.467),1.521 (0.608) ng/mL;t1/2,12.073 (2.068),12.271 (1.678) h;AUC0-72h,15.286 (3.434) ng.h/mL,15.673 (3.586) ng.h/mL.The 90％ confidence intervals for the AUC0-72h and Cmax were between 0.80 and 1.25.No adverse events were reported by the subjects or found with results of clinical laboratory test.The test and reference products met the regulatory criteria for bioequivalence in these fasting,healthy Chinese male volunteers.All three formulations appeared to be well tolerated.

OBJECTIVETo develop an HPLC method for simultaneous determination of gallic acid and hesperidin in Xiaogu capsule, in order to provide a simple, rapid and accurate method for quality control of the compound preparation of traditional Chinese medicine.

METHODXiaogu capsule was extracted with methanol heating reflux method. Synergi 4 mu Hydro-RP 80A (4.6 mm x 250 mm, 5 microm) was adopted as the chromatographic column, with acetonitrile--0.04 mol x L(-1) phosphate monobasic sodium solution (20: 80) as the mobile phase. The flow rate was 1.0 mL x min(-1), the detection wavelength was 283 nm, and the column temperature was 25 degrees C.

RESULTUnder the conditions, gallic acid and hesperidin reached the baseline resolved peak, with a good linearity within the range of 21.6-216.0 mg x L(-1) (r = 0.999 93) for gallic acid, and 4.5-45.0 mg x L(-1) (r = 0.999 95) for hesperidin, respectively. Their average recoveries (n = 9) were 101.5% (RSD 3.7%) and 94.7% (RSD 2.7%), respectively. The average contents of gallic acid and hesperidin contained in Xiaogu capsule were detected to 5.10% and 0.091 1%, respectively.

CONCLUSIONThe method established in this study can determine the content of gallic acid and hesperidin contained in Xiaogu capsule in a rapid and accurate manner, which provided reference for quality evaluation of the medicine.

Capsules ; analysis ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; analysis ; Gallic Acid ; analysis ; Hesperidin ; analysis

OBJECTIVEEstablish the model of mouse with chronic hepatitis B virus (HBV) and nonalcoholic fatty liver disease (NAFLD).

METHODSTake 100 HBV transgenic, BALB/c mice of 4 weeks old, with each gender half. Then pick out 70 mice in one group to feed high-fat feed and the rest to feed normal feed. At the end of week 16, random kill 10 mice of high-fat, then liver tissue and serological detection target identification model is established in this paper. After that, divide the mice into model group and comparison group with 30 mice in each group. Feed model group with high-fat feed, comparison group with normal feed and normal group with normal feed till week 72 (including previous 16 weeks). Kill 10 mice of each group at the end of week 24, 48 and 72 respectively, fully automatic biochemical instrument detection of serum ALT, AST, TC, TG, FBG, fluorescence quantitative PCR method to detect HBV-DNA, chemiluminescence detection of HBsAg, liver biopsy after HE staining to evaluate histology change, observe mice model of dynamic evolution.

RESULTS(1) Feed high fat feed after 16 weeks, mice's weight, serum ALT, AST, TC, TG, FBG and blood biochemical indicators increased, HBV-DNA positive, liver HE staining obviously big blister fatty degeneration of liver cells and within the lobule lymphocytes infiltration, NAFLD activity score (NAS) getting close to NASH, the model of chronic HBV carries with NAFLD mouse built successfully. (2) The TC and TG values of model group in each period were higher than that of comparison group and normal group. (3) In week 24 and 72, HBV-DNA values of each group are obvious different from the other two groups and the difference can be applied to statistical significance (P < 0.05). (4) In week 48 and 72, NAS of each group are obvious different from the other two groups and the difference can be applied to statistical significance (P < 0.05).

CONCLUSIONS(1) Chronic HBV carries with NAFLD mice model can be established by HBV transgenic mice fed by high fat feed. (2) NAFLD accelerates the liver disease of the mice carrying HBV to some extent.

Animals ; Disease Models, Animal ; Fatty Liver ; complications ; pathology ; virology ; Female ; Hepatitis B virus ; genetics ; isolation & purification ; physiology ; Hepatitis B, Chronic ; complications ; pathology ; virology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Non-alcoholic Fatty Liver Disease