BACKGROUND: Apelin/APJ system has a wide range of physiological functions,but its intracellular signal transduction,in particular,apelin receptor desensitization,internalization,resensitization degradation,have still no consistent opinion.OBJECTIVE: To construct eukaryotic expression vector expressing human apelin receptor(APJ)tagged to red fluorescent protein(pDsRED-express-C1),and to determine the expression in human embryo kidney 293 cells.METHODS: The plasmid pcDNA3.1-hAPJ was used as a template for PCR amplification of human APJ.Following PCR amplification the PCR product were removed and enzymatic digestion with EcoR I and BamH I.Same enzymes were used to cut vector pDsRED-express-C1.The digestive product was ligated by conventional methods of connection,then transfected into Competent E.coli TOP10.Single clones were picked plasmid extraction,followed by restriction enzyme digestion and finally DNA sequencing.The recombinant plasmid with correct sequencing was transfected into human embryonic kidney cells,PI staining,followed by the observation under a confocal microscope.RESULTS AND CONCLUSION: PCR amplified a 1.2-kb fragment,which was consistent with the expected size of the human APJ.The pDsRed-hAPJ recombinant plasmid was cut into two fragments,one corresponded to the pDsRED-express-C1 vector size,and the other fragment corresponded to APJ target fragment.Confocal microscopy analysis showed that,APJ was expressed mainly in the membrane of human embryo kidney 293 cells.The pDeRed-hAPJ eukaryotic plasmid expression vector was successfully constructed and effective expression of this fusion protein is achieved,which might be instrumental in the study of displacement and intracellular localization of human APJ.

Purpose:To study the response and toxicity of the regimen of oxaliplatin combined with fluorouracil and calcium folinate in the treatment of consisting of advanced colorectal cancer.Methods:Thirty-seven patients with advanced colorectal cancer received chemotherapy of regimen:oxaliplatin 130 mg/m~2 2 hours iv on day 1,calcium folinate 200 mg/ m~2 iv 2 hours on days 1 to 5,followed by fluorouracil 300 mg/m~2(≤500 mg/d) iv 4 h on days 1 to 5,three or four weeks as one cycle.Results:The total response rate was 29.7%,the main toxicity was bone marrow suppression and neuro-sensory toxicity,leukopenia was observed in 45.9% of the patients,but grade Ⅲ and Ⅳ in only 8.1%,neuro-sensory toxicity was observed in 81.9.%,but grade Ⅲ and Ⅳ in only 5.4%.Conclusions:This study shows that the regimen of oxaliplatin combined fluorouracil and calcium folinate is effective and tolerable in advanced colorectal cancer therapy.

AIM:To observe the effect of fresh amniotic membrane transplantation ( FAMT) in acute ocular chemical burns.
●METHODS:A prospective study of 25 consecutive cases (36 eyes) with acute ocular chemical burns were treated with FAMT. The clinical efficacy was observed such as the time of amniotic membrane absorbed, corneal epithelialization & transparency, visual acuities and complications.
●RESULTS: With follow-up ranged from 3 to 6mo, 31 eyes′ amniotic membrane were dissolved in 2wk (86%). A total of 33 eyes showed corneal epithelialization in 4wk ( 92%) , 3 eyes showed persistent corneal epithelial defects and need secondary limbal stem cell transplantation or corneal transplantation ( 8%) . A total of 10 eyes showed superficial corneal vascularization (28%), 6 eyes′ cornea were opacity in part (17%), and one eye was symblepharon (3%).
●CONCLUSION:Early FAMT is an effective treatment in the management of acute ocular chemical burns to support epithelial healing, restore ocular surface integrity with potential to improve vision and reduce the incidence of complications. Furthermore, FAMT has advantages of easily obtain and convenient usage, which is suitable in local hospital of our country.

OBJECTIVETo observe the effect of Bushen Wenyang Huayu Recipe (BWHR) on hypoxia inducible factor-1α (HIF-1α), proline hydroxylase2 (PHD2), von Hippel Lindau disease (VHL) suppressor gene expressions in endometriosis (EM) rats with Shen yang deficiency blood stasis syndrome (SYDBSS), and to explore the pathogenesis of EM and the mechanism of BWHR for treating EM.

METHODSTotally 50 SD rats were randomly divided into five groups, i.e., the blank control group, the sham-operation group, the model group, the Chinese medicine (CM) group, and the Western medicine (WM) group, 10 in each group. Rats in the blank control group and the sham-operation group were fed routinely. Rats in the rest 3 groups received 30-day "extended refrigerator freezing and ice water immersion" and combined with " autotransplantation" to establish EM rat model with SYDBSS. One Milliliter BWHR at 3.33 g/mL was administered to rats in the CM group by gastrogavage. Gestrinone at the daily dose of 0. 5 mg/kg was administered to rats in the WM group by gastrogavage. Equal volume of normal saline was administered to rats in the model group, the blank control group, and the sham-operation group. The size and morphology of ectopic foci in rats were observed after 4 weeks of medication. Expressions of serum CA125, plasma cyclic adenosine monophosphate (cAMP), and plasma cyclic guanosine monophosphate (cGMP) were detected by radioimmunoassay. Morphological changes of eutopic endometrium and ectopic tissue were observed under the optical microscope by HE staining. Protein expressions and contents of HIF-lα, PHD2, and VHL were detected by immunohistochemical SABC method and Western blot. mRNA expressions of HIF-1α, PHD2, and VHL were detected by RT-PCR.

RESULTSThe ectopic foci grew significantly in the model group. Their volumes were obviously contracted after treated by CM and WM. Compared with the blank control group and the sham-operation group, serum CA125 and plasma cGMP obviously increased, cAMP obviously decreased (P < 0.05); expressions and contents of HIF-1α mRNA and protein all decreased (P < 0.05); mRNA and protein expressions and contents of PHD2 and VHL all decreased in the model group (P < 0.05). Compared with model group, levels of CA125 and cGMP obviously decreased; cAMP levels obviously increased, expressions and contents of HIF-1α mRNA and protein all increased, mRNA and protein expressions and contents of PHD2 and VHL all increased in the WM group and the CM group (P < 0.05). Compared with the CM group, PHD2 protein contents were higher in the WM group (P < 0.05). HIF-1α was negatively correlated with PHD2 (r = -0.799, P = 0.00). HIF-1α was negatively correlated with VHL (r = -0. 625, P = 0.003).

CONCLUSIONSBWHR could effectively treat EM. Its mechanism might be associated with reducing contents of HIF-1α, serum CA125, and plasma cGMP, and up-regulating expressions of PHD2, VHL, and cAMP.

Animals ; Cyclic AMP ; Drugs, Chinese Herbal ; therapeutic use ; Endometriosis ; drug therapy ; metabolism ; Female ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Proline ; metabolism ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Up-Regulation ; Yang Deficiency ; drug therapy ; metabolism

Objective To explore the role of xome apoptosis regulators during the development in rat cochlea.Methods The morphological development process of cochlea was obserred in Wistar rat aged between embryo day 13 to postnatal day 14 in this experiment.Survivin and caspase-3 Were respectively detested at protein and mRNA levels by immunohistochemistry and reverse transcription polymerage chain reaction(RT-PCR).Results The expression of survivin and cagpage-3 located in the bottom wall of the cochlear duct.Not only they widespread in the cell proliferation,but also they gyadually enhanced in the cell differentiation.Both of them had a crest-time.and survivin was prior to caspase-3.In organ of corti during adult time,caspase-3 Wag not present and survivin was only expressed.Conclusions During the development of the rat cochlea,both of them had similar location and trend.But they were derangement.This showed that beth of them participated in the cochlear morphological development.It wag suggested that the interaction between survivin and caspas-3 regulated the apoptosis,promoted the cochlear morphological development.

OBJECTIVETo investigate the correlation between ER-a in the liver and cytokines of T lymphocytes subsets and serum signatures in PBC patients.

METHODSThe research is performed with cross-sectional study. 80 PBC women patients without treatment were enrolled in PBC group, 10 healthy women as baseline-matched in healthy-control group, and 20 patients with non-autoimmune liver disease in non-PBC control group. The expression of IL-6, IL-8, IL-22, TNFa, IFNgamma, AMA-M2, Sp100 and gp210 were analyzed in Peripheral Blood using ELISA in all groups, and ER-a of patients were performed on tissues from liver biopsies in PBC group and non-PBC control group with immunohistochemistry. Spearman correlation test were performed on the indices to identified the association of all Parameters. numerical data were compared with Wilcoxon rank-sum test.

RESULTSCompared with healthy-control group, expression of serum cytokines are significantly higher in PBC and non-PBC groups (P less than 0.01), while no significant difference were observed between PBC and non-PBC groups. The positive rate of ER-a in PBC patients liver tissues in PBC group is higher than that in non-PBC group (Z=4.82, P less than 0.01). Expression of ER-a is positively correlated with positive rates of AMA-M2 antibody, Sp100 and gP210 of tissues of PBC patients ( r=0.898, 0.819, 0.814, P less than 0.01). ER-a is positive correlated with the expression of cytokines, among which the coefficient of correlation of IL-22, TNFa, IFNgamma is more than 0.7 (r=0.71, 0.89, 0.82, P less than 0.01), AMA-M2, Sp100, gp210 is negative in serum of non-PBC control group. No obviously correlations were indicated between the expression of ER-a and cytokines.

CONCLUSIONA high level of expression of cytokines in the serum might be one of the factors of etiopathogenesis of PBC.

Autoantibodies ; blood ; Biomarkers ; blood ; Case-Control Studies ; Estrogen Receptor alpha ; metabolism ; Female ; Humans ; Interleukin-6 ; blood ; Interleukin-8 ; blood ; Interleukins ; blood ; Liver ; metabolism ; Liver Cirrhosis, Biliary ; blood ; immunology ; Middle Aged

BACKGROUNDCentral venous pressure (CVP) and intrathoracic blood volume index (ITBVI) were used to assess the fluid status. It has previously been shown that CVP is not as accurate as ITBVI for all the shock patients. We therefore hypothesized that the change of CVP has the ability to predict fluid responsiveness in some clinical cases of shock.

METHODSFrom September 1st 2009 to September 1st 2011, sixty-three patients with shock from different Intensive Care Unit (ICU) were collected into this retrospective study. All the patients received fluid challenge strategy (infusing 300 ml hydroxyethyl starch in 20 minutes), were monitored with CVP and pulse-indicated continuous cardiac output (PICCO). The correlation between changes in cardiac index (ΔCI), CVP (ΔCVP) and ITBVI (ΔITBVI) were analyzed. Fluid responsiveness was defined as an increase in CI ≥ 10%. Receiver operating characteristic (ROC) curves were generated for ΔCVP and ΔITBVI.

RESULTSFor all the patients, there was no correlation between ΔCI and ΔCVP (P = 0.073), but in the subgroup analysis, the correlation between ΔCI and ΔCVP was significant in those younger than 60 years old (P = 0.018) and those with hypovolemic shock (P = 0.001). The difference of areas under the ROC curves of ΔCVP and ΔITBVI were not statistically significant in the group younger than 60 years old or hypovolemic shock group (P > 0.05, respectively). However, no similar results can be found in the group older than 60 years old and the other two shock type groups from ROC curves of ΔCVP and ΔITBVI.

CONCLUSIONSΔCVP is not suitable for evaluating the volume status of the shock patients with fluid resuscitation regardless of their condition. However, in some ways, ΔCVP have the ability to predict fluid responsiveness in the younger shock patients or in the hypovolemic shock patients.

Adult ; Aged ; Aged, 80 and over ; Central Venous Pressure ; physiology ; Female ; Fluid Therapy ; methods ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Shock ; physiopathology ; therapy

The selective removal of low density lipoprotein (LDL) and fibrinogen (Fib) by degraded carrageenan was studied by the present authors. Degraded carrageenan was prepared by acid with carrageenan as the main material. The effects of acid conditions on the molecular weight were investigated, and the proper reaction conditions were ascertained. The results of infrared spectrometry indicated that the degraded carrageenan is a heparin-like polysaccharide. Then the selective removal of LDL/Fibrinogen by degraded carrageenan was studied. When molecular weight was about 10,000, pH was 5.10 and the concentration of degraded carrageenan was 800 mg/L, the average reduction percentages were 60.0% for total cholesterol(TC), 79.4% for LDL and very low-density lipoprotein (VLDL), and 93.8% for fibrinogen. There were no significant changes with relation to the level of high-density lipoprotein (HDL) and total protein (TP). So, degraded carrageenan was shown to be of good selectivity on plasma LDL/Fibrinogen apheresis.
Carrageenan ; chemistry ; Fibrinogen ; analysis ; isolation & purification ; Humans ; Hyperlipidemias ; blood ; Lipoproteins, LDL ; blood ; isolation & purification

OBJECTIVETo construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization (SSH).

METHODSSbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons (10-5 mol/L for 72 hours). To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein (CREB) mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons (62.85 ± 1.98) compared with normal striatal neurons (28.43 ± 1.46, P < 0.01). Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 (Mtch1; 96.81 ± 2.04 vs. 44.20 ± 1.31, P < 0.01) and thymoma viral proto-oncogene 1 (Akt1; 122.10 ± 2.17 vs. 50.11 ± 2.01, P < 0.01), showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons.

CONCLUSIONSA reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed. Mtch1 and Akt1 might be the candidate genes for the development of morphine tolerance.

Analgesics, Opioid ; pharmacology ; Animals ; Cells, Cultured ; Corpus Striatum ; cytology ; Drug Tolerance ; physiology ; Gene Expression Profiling ; Gene Library ; Molecular Sequence Data ; Morphine ; pharmacology ; Neurons ; cytology ; drug effects ; Nucleic Acid Hybridization ; methods ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; methods

Xingnaojing (XNJ) is an effective clinical drug used to treat acute stroke. Compared with injection administration, its nasal administration has better brain targeting. Therefore, through nasal administration, XNJ microemulsion could help solve the drug load of compound components of different polarities contained in large-dose and high-concentration traditional Chinese medicines, and reduce irritation to nasal mucosa In this study, the modified volume correction method and the improved rat in situ nasal perfusion model were adopted to compare the nasal absorption of geniposide contained in different XNJ preparations. The results showed that the constant absorption rate of geniposide (GE) in XNJ-D was (2.95 +/- 0.25) x 10(-3) min(-1), whereas the constant absorption rate of GE in XNJ-M was (2.16 +/- 0.21) x 10(-3) min(-1). This indicated that the rat nasal absorption of GE in different XNJ preparations complied with the first-order process and could be considered as passive absorption. GE in XNJ-D was absorbed faster than that in XNJ-M, which provided basis for the development of nasal preparations of XNJ.
Absorption ; Administration, Intranasal ; Animals ; Drugs, Chinese Herbal ; pharmacokinetics ; Emulsions ; Iridoids ; pharmacokinetics ; Male ; Nose ; metabolism ; Rats ; Rats, Sprague-Dawley