OBJECTIVETo study the roles of stromal interaction molecule 2 (STIM2) and transient receptor potential canonical 3 (TRPC3) in extracellular Ca(2+)-sensing receptor (CaR)-induced extracellular Ca2+ influx and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVEC).

METHODS(1) The interaction of STIM2 and TRPC3 was determined using the immunofluorescence technique. (2) The expressions of STIM2 and TRPC3 genes were silenced in HUVEC by transfection constructed STIM2 and TRPC3 RNA interference plasmids. The interference efficiency of STIM2, TRPC3 protein and mRNA levels were determined by Western blot and real time RT-PCR, respectively. (3) The second to fifth passage of HUVEC were divided into: STIM2-002 short hairpin RNA (STIM2-002 shRNA ) + spermine + Ca2+ group and TRPC3-004 short hairpin RNA (TRPC3-004 shRNA ) + spermine + Ca2+ group; control group (spermine + Ca2+ group) and vehicle+ spermine + Ca2+ group. The four groups of cells were incubated with CaR agonist spermine, the intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, and the production of NO was determined by DAF-FM (NO fluorescent probe) of each group in HUVEC.

RESULTS(1) Immunofluorescence technique results showed that STIM2 and TRPC3 proteinswere present in the cytoplasm of HUVEC. (2) The results of transfection constructed STIM2 and TRPC3 RNA interference plasmids demonstrated that shRNA targeted to the STIM2 and TRPC3 genes decreased STIM2 and TRPC3 mRNA levels by 88.2% and 74.0%, respectively (P < 0.05), simultaneously, the STIM2 and TRPC3 protein levels were decreased by 79.9% and 71.8%, respectively (P < 0.05). (3) Compared with spermine + Ca2+ group, the [Ca2+]i and the net NO fluorescence intensity of spermine + Ca(2+) + ShSTIM2-002 group, spermine + Ca(2+) + ShTRPC3-004 group and spermine + Ca2+ Vehicle group were not changed (P > 0.05).

CONCLUSIONSTIM2 and TRPC3 do not participate in CaR-mediated Ca2+ influx and NO production individually.

Calcium ; metabolism ; Cell Adhesion Molecules ; physiology ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; physiology ; Humans ; Nitric Oxide ; metabolism ; Stromal Interaction Molecule 2 ; TRPC Cation Channels ; physiology

Objective To explore the effect of different proportions of mixed blood exchange transfusion on blood circulation in neonates with hemolytic disease.Methods Thirty-one newborn infants with hemolytic disease were treated by peripheral arteriovenous synchronization of exchange transfusion with different proportions mixed blood.AB type plasma was mixed with O type red blood cell(RBC) washing.The proportion for the treatment group was 1:1(the O type RBCs 2 U:the AB type plasma 200 mL),by exchange transfusion of haplotypes,in accordance with 80?mL/kg;the proportion for control group was 2:1(the O type RBC 4 U:the AB type plasma 200 mL),by exchange transfusion of double in accordance with 150-180 mL/kg.The indicators were detected,such as the exchange rate of neonatal serum bilirubin,RBC,hemoglobin(Hb),hematocrit(HCT),and the exchange transfusion quantity and days of hospitalization before and after the exchange transfusion were analyzed.Results The exchange rate of serum bilirubin of treatment group and control group was (44.92?3.99)% and (45.69?5.06)%,respectively,there was no significant difference between 2 groups(P=0.639),there was no significant difference of hospitalization days[(8.13?1.13) d vs(8.19?0.91) d]between 2 groups(P=0.884).After exchange transfusion in treatment group,the average level of the RBC,Hb and HCT were increased(P

OBJECTIVETo establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs).

METHODShAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry.

RESULTSThe cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45.

CONCLUSIONSThis study establishes a potential method for isolation of hAMSCs from human amnion,in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.

Amnion ; cytology ; Cell Culture Techniques ; Flow Cytometry ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; cytology

The effects of Zn2 + on GABAA-receptor mediated responses in acutely isolated rat sacral dorsal commissural nucleus (SDCN) were studied using nystatin-perforated whole cell recording techniques.The results demonstrated that ( 1 ) GABA induced inward currents through activation of GABAA-receptor at a holding potential of -40 mV; (2) GABAA-receptor mediated responses were suppressed by Zn2 + in a reversible and voltage-independent manner; and (3) in the presence of Zn2 + , the concentration-response curve of GABA-induced responses was shifted to the right in a parallel manner. The results suggest that Zn2 +allosterically depresses GABAA-receptor mediated currents.

BACKGROUNDIrradiation dose and volume are the major physical factors of radiation-induced lung injury. The study investigated the relationships between the irradiation dose and volume in radiation-induced lung injury by setting up a model of graded volume irradiation of the rat lung.

METHODSAnimals were randomly assigned to three groups. The ELEKTA precise 2.03 treatment plan system was applied to calculate the irradiation dose and volume. The treatment plan for the three groups was: group 1 received a "high dose to a small volume" (25% volume group) with the mean irradiation volume being 1.748 cm(3) (25% lung volume); the total dose and mean lung dose (MLD) were 4610 cGy and 2006 cGy, respectively (bilateral AP-PA fields, source to axis distance (SAD) = 100 cm, 6MVX, single irradiation); Group 2 received a "low dose to a large volume" (100% volume group) with the mean irradiation volume being 6.99 cm(3) (100% lung volume); the total dose was 1153 cGy. MLD was 2006 cGy, which was the same as that of group 1 (bilateral AP-PA fields, SAD = 100 cm, 6MVX, single irradiation); Group 3 was a control group. With the exception of receiving no irradiation, group 3 had rest steps that were the same as those of the experimental groups. After irradiation, functional, histopathological, and CT changes were compared every two weeks till the 16th week.

RESULTSFunctionally, after irradiation breath rate (BR) increases were observed in both group 1 and group 2, especially during the period of 6th - 8th weeks. The changes of BR in the 100% volume group were earlier and faster. For the 25% volume group, although pathology was more severe, hardly any obvious increase in BR was observed. Radiographic changes were observed during the early period (the 4th week) and the most obvious changes manifested during the mediated period (the 8th week). The extensiveness of high density and the decreased lung permeability were presented in the 100% volume group, and ground glass opacity and patchy consolidation were presented in the 25% volume group without pleural effusion, pleural thickening, and lung shrinking. Morphologically, the 100% volume group mainly presented signs of vascular damage, including signs of vascular wall edemas, hypertrophy, and sclerosis. The 25% volume group mainly presented with erythrocyte cell exudation, inflammation, and parenchymal damage.

CONCLUSIONSThe delivery of a small dose of radiation to a large volume is not safe. A low dose smeared out over large volumes, albeit reversible, may lead to fatal respiratory dysfunction. Damage to the lung may be more dependent on the volume of irradiation than on the radiation dose. Clinically, the safest approach is to limit both the volume of the irradiated normal lung and the amount of received radiation.

Animals ; Dose-Response Relationship, Radiation ; Lung ; radiation effects ; Lung Injury ; etiology ; Radiation Injuries, Experimental ; Rats ; Rats, Wistar

OBJECTIVETo observe the effect of the human amniotic membrane (HAM) loaded with human amniotic mesenchymal stem cells (hAMSCs) on the skin wounds of SD rats.

METHODSThe amniotic epithelial cells were removed by trypsin digestion, hAMSCs were loaded onto HAM and then covered on rats' skin defects. The wound healing was observed by HE staining and immunohistochemistry, and the results were compared with the amniotic membrane group and blank control group.

RESULTSThe average wound healing time was (18.3 +/- 0.9) d in the HAM load with hAMSCs group, which was significantly faster than those in the blank control group [(26.4 +/- 0.7) d, P < 0.01] and the amniotic membrane group [(21.5 +/- 1.2) d, P < 0.05]. After 11 d and 14 d, the wound healing rates in the HAM load with hAMSCs group were (81.5 +/- 7.2)% and (94.3 +/- 3.6)%, respectively, which were significantly higher than those in the blank control group [(48.5 +/- 3.2)% and (74.3 +/- 4.3 )%] and the amniotic membrane group [(68.5 +/- 4.5)% and (86.8 +/- 4.8)%] (all P < 0.01). Skin biopsy/HE staining confirmed that the quality of wound healing in the HAM load with hAMSCs group was significantly better than in the amniotic membrane group and the blank control group. Immunohistochemical staining showed that the number of CK19-positive epidermal stem cells in the HAM load with hAMSCs group (48.2 +/- 3.2) was significantly larger than those in the amniotic membrane group (37.7 +/- 3.1) (P < 0.05) and the blank control group (29.6 +/- 2.4) (P < 0.01). Furthermore, the vascular endothelial growth factor expression (64.5 +/- 4.5) in the HAM load with hAMSCs group was also significantly higher than those in the amniotic membrane group (52.6 +/- 3.8) (P < 0.05) and the blank control group (40.7 +/- 3.1) (P < 0.01).

CONCLUSIONHAM loaded with hAMSCs may promote the repair of skin wounds by promoting the regeneration of epidermal stem cells and capillaries.

Amnion ; cytology ; Animals ; Cells, Cultured ; Disease Models, Animal ; Humans ; Male ; Mesenchymal Stem Cell Transplantation ; Rats ; Rats, Sprague-Dawley ; Skin ; injuries ; physiopathology ; Wound Healing

Objective To investigate the effect of a selective muscarinic receptor antagonist (penehyclidine hydrochloride) in three vessel occlusion model of acute global cerebral ischemia-reperfusion in rats.Method One hundred and forty-four male SD rats were randomly divided into four groups:sham operated group,vehicle treated group (saline 1 ml,i.p.),scopolamine treated group (0.01 mg/kg,i.p.) and penehyclidine hydrochloride treated group (0.01 mg/kg,i.p.) with drugs injected 40 minutes before ischemia respectively.The ischemic duration was 10 minutes.The animals were subjected to motor activity tests (open field activity test,beam-walking test and grip test) at 24 hours or on the 3rd and 7th day after reperfusion.HE staining,TUNEL staining and immunohistochemical reactions of bax and bel-2 were carried out at the time points of 2,12,24 hours,3 and 7 days after reperfusion.TTC staining was carried out in some rats for assessment of infarction volume on the 4th day after reperfusion.Results As compared with the vehicle treated group,both penehyclidine hydrochloride treatment and scopolamine treatment decreased the numbers of apeptotie neurons (P

Objective To evaluate the prognosis factors of carcinomatous meningitis (CM).Methods The medical records of 63 patients with CM treated in our hospital from 1998 to 2008 were retrospectively analyzed. The chief prognosis factors evaluated were gender, age, primary tumor type,Karnofsky performance status (KPS) scores, interval between diagnosis of primary tumor and CM,treatment, radiation dose and primary tumor control status. Kaplan-Meier method was employed to calculate the survival time and plot the survival curves. Log-rank test was used to evaluate the differences between the groups. Cox regressive model was used to analyze the prognostic factors. Results All patients died by the end of follow-up. The survival time ranged from 2 to 732 d and the overall one-year survival rate was 7.9% with a median survival time of 67 d. In multivariate analysis, KPS scores, primary tumor control status, interval between diagnosis of primary tumor and CM were independent prognostic factors. Conclusions The main prognostic factors of CM are KPS scores, primary tumor control status, and interval between diagnosis of primary tumor and CM. The most effective treatment modalities still need to be confirmed and individual treatment for each patient with CM should be recommended in clinic.

OBJECTIVETo design and rapidly evaluate a TaqMan assay for detecting influenza A viruses.

METHODSThe probe and the primers of the assay were designed with the software packages of DNA Star and Primer Premier 5.0. Their specificity and conservation were verified through Blast in GenBank and electronic hybridization. The assay's sensitivity was compared with the standard RT-PCR.

RESULTSThe designed primers and probe were confirmed to be very specific and conserved. The assay was 3-27 folds more sensitive than the standard RT-PCR. The RT and PCR steps could be simplified into one step.

CONCLUSIONThe TaqMan Real-time PCR assay is specific, sensitive and easy to perform.

Animals ; Birds ; Humans ; Influenza A virus ; genetics ; isolation & purification ; Influenza in Birds ; diagnosis ; virology ; Influenza, Human ; diagnosis ; virology ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Objective To compare the effects of 20 mg qd and 10 mg bidadministration of iprrazole enteric-coated tablets on the control of gastric acid in healthy subjects.Methods A randomized,single-center,parallel controlled trial was designed to include 8 healthy subjects.Randomly divided into 2 groups,20 mg qd administration group:20 mg enteric-coated tablets of iprrazole in the morning;10 mg bid administration group:10 mg enteric-coated tablets of iprrazole in the morning and 10 mg in the evening.The pH values in the stomach of the subjects before and 24 h after administration were monitored by pH meter.The plasma concentration of iprazole after administration was determined by HPLC-MS/MS.The main pharmacokinetic parameters were calculated by Phoenix WinNonlin(V8.0)software.Results The PK parameters of iprrazole enteric-coated tablets and reference preparations in fasting group were as follows:The Cmax of 20 mg qd group and 10 mg bid group were(595.75±131.15)and(283.50±96.98)ng·mL-1;AUC0-t were(5 531.94±784.35)and(4 686.67±898.23)h·ng·mL-1;AUC0-∞ were(6 003.19±538.59)and(7 361.48±1 816.77)h·ng·mL-1,respectively.The mean time percentage of gastric pH＞3 after 20 mg qd and 10 mg bid were 82.64％and 61.92％,and the median gastric pH within 24 h were 6.25±1.49 and 3.53±2.05,respectively.The mean gastric pH values within 24 h were 5.71±1.36 and 4.23±1.45,respectively.The correlation analysis of pharmacokinetic/pharmacodynamics showed that there was no significant correlation between the peak concentration of drug in plasma and the inhibitory effect of acid.Conclusion Compared with the 20 mg qd group and the 10 mg bid group,the acid inhibition effect is better,the administration times are less,and the safety of the two administration regimes is good.