Aim: To prepare chitosan nanoparticlescarrying gene and study its characteristics in vitro and in vivo.Methods:12-ACSs was dissolved in 0.05mol/L sodium acetate buffer to form a solution of 1mg/ml and a DNA(plasmid-encoding antisense ECE ) solution of 0.1mg/ml was dissolved in 25mmol/L Na2SO4.The charge ratio of components is selected as 2/1 for 12-ACSs /DNA complex.The complex was prepared by mixing 12-ACSs solution with DNA solution prior to observation by using transmission electron microscopy.Using Electrophoretic Retardation of DNA Migration detection,DNA precipitation,Binding Equilibration and DNase Resistivity Assay,the formation of 12-ACSs /DNA complex was determined and its stability was simultaneously evaluated.MTT Cell Proliferation Assays was performed on investigation of the cytotoxicity of 12-ACSs /DNA nanoparticles in bronchial epithelial cells (16HBE).The transfection efficiency of 12-ACSs /DNA nanoparticles in vitro and in vivo was investigated in 16HBE cells and Balb/c mice.Results: 12-ACSs /DNA nanoparticles (100~150nm) were observed by transmission electron microscopy.12-ACSs can protect the plasmid encoding antisence-ECE from degradation by DNase I.12-ACSs can transfer plasmid-encoding antisense ECE into 16HBE cells and into the airway epithelium in mice.As shown by fluorescent microscopy,green fluorescent protein reporter can be observed in the transfected cells as well as in the airway epithelium of the treated mice.And it showed a lower cell cytotocixity in cultured 16HBE cells and in mice treated with12-ACSs /DNA nanoparticles.Conclusion: In summery,the chitosan can be used as an effective protectant for DNA as well as an enhancer for in vitro gene transfection.

Objective To construct an immortalized rat astrocyte strain expressing enkephalin which can be regulated by doxycycline.Methods pRevTet-On and recombinant plasmid pRevTRE/hPPE were transfected into packaging cell PT67 respectively by standard lipofectamine methods.The supernatant containing RevTet-On virus was used to infect immortalized rat astrocyte strain(IAST)and RevTRE/hPPE virus was used to infect the IAST/Tet-On cell.Immortalized rat astrocyte strain that stably expressed Tet-regulated enkephalin was established. hPPE gene expression level of IAST/Tet-On/hPPE cell strain was detected by real time-PCR,immuno- cytochemistry and radio-immunoassay.Results Real time-PCR,immuno-cytochemistry and radio-immunoassay confirmed the level of enkephalin expression was regulated by doxycycline in a dose-dependent manner.Conclusion An immortalized rat astrocyte strain which secrets enkephalin as controlled by doxycycline has been constructed successfully.

Objective To observe the effect of MnCl2 on glutamate-glutamine cycle(Glu-Gln cycle) and apoptosis of dissociated striatum cells and to approach the antagonism of riluzole to the neurotoxicity of MnCl2.Methods Twenty-seven Wistar rats were randomly divided into 3 groups, 9 in each.The first group was the control group and the second group was the single MnCl2 exposure group.Both of the groups were injected with 0.9% NaCl subcutaneously(sc).The third group was the intervention group and was injected with 21.35 ?mol/kg riluzole(sc).Two hours later, the rats in the control group were the given 0.9% NaCl intraperitoneally(ip) injection, the second and third groups were injected with 200 ?mol/kg MnCl2(ip), five times a week, for 4 weeks.Twenty-four hours after the last injection, 6 rats in each group were killed and the contents of Glu and Gln, the activities of glutamine synthetase(GS) and phosphate activated glutaminase(PAG) in the striatum of the rats were determined.The apoptosis of the dissociated striatum cells was detected in the other rats in each group by flow cytometry(FCM) technique.Results Compared with the control group, in MnCl2 group, the content of Glu increased and Gln content decreased significantly(P

Objective To evaluate pharmacodynamics of prepared long-circulating superparamagnetic iron oxide (SPIO) liposomes. Methods Control and experimental groups were established after adding SPIO or long-circulating SPIO liposomes as agents. (1)Macrophages experiment in vitro: the RAW 264.7 macrophage cell strains were recovered, cultured and seeded in the culture plate at a density of 2.5×105 cells/well until they reached 80%-90% confluence. The intracellular Fe uptake of control and experimental group cells were quantified by Ferrozine assay after incubation with different concentrations of drugs. Factorial design analysis of variance was used as statistics method. Prussian blue staining method was used to detect staining of experimental cells.(2)Drug biodistribution in mice: C57BL/6J(n=6) were classified into blank control group (n=2), control group(n=2) and experimental group(n=2).Saline, SPIO and long circulating SPIO were injected via the tail vein in the blank control group, control group and experimental group respectively. Then distribution of drugs in the body was observed by pathological examination.(3) MR imaging of tumor-bearing nude mice: 20 BALB/c nude mice bearing lung cancer models were established and classified into control group and experimental group. After administration of drugs, all animals underwent MR scanning. Signal intensities of livers and tumors were measured, SNR-time dynamic curves were drew. Covariance analysis was used to compare post-enhanced SNR at the 12th hour. (4)Cytotoxicity studies (MTT): cytotoxicity of both drugs on human liver cell line HL-7702 was studied, and statistically analyzed using factorial design analysis of variance. Results (1) Macrophages experiment in vitro: The nanoparticle uptake by macrophage cells evaluated by ferrozine assay showed the uptake of blank SPIO was higher than long-circulating SPIO liposomes. Compared with the blank control group, there was strong blue staining in the macrophages with Prussian staining in the control group and little blue staining in the experimental group. (2) Drug biodistribution in mice: for blue stained cells composed of iron particles, the amounts in the liver, spleen, lung, kidney of the control group were more than those in the experimental group. (3) MR imaging of tumor-bearing nude mice: the non-enhanced SNR of livers and tumors in the control group and experimental group were 31.47 ± 0.56, 30.89 ± 1.41, 58.41 ± 0.61, 58.44 ± 1.08, respectively. After injecting of contrast agents, SNR of livers and tumors in the control group and experimental group were 17.00 ± 0.96, 22.29 ± 0.73, 58.50 ± 0.63, 52.47 ± 1.18, respectively. The covariance analysis showed that SNR of the livers in the control group after 12 hours was significantly lower than the experimental group (F=167.022, P=0.000); while the SNR of the tumors in the experimental group was significantly lower than the control group (F=266.106, P=0.000).(4) Cytotoxicity of nanoparticles by MTT method: the viability of HL-7702 cells tend to decrease with the increase of Fe concentration. Cytotoxicity in the long-circulating SPIO liposomes was lower than the SPIO(F=2256.204,P=0.000). Conclusions Long-circulating SPIO liposomes we prepared reveal suitable sizes, even distribution, and good anti-macrophage ability in vitro and in vivo. They have long circulation characteristic and T2 negative enhancement effect in the transplanted lung cancers, while they still maintain low cytotoxicity.

Objective To investigate the clinical significance of the antenatal detection of four indices of coagulation(PT,APTT, TT,FIB),D-dimer(DD)and platelet(PLT)in the judgment of parturient coagulation function and bleeding risk.Methods 795 pregnant women and childbearing age non-pregnant women with physical examination in this hospital from June 2012 to September 2013 were collected and divided into the normal pregnancy group(early pregnancy,middle pregnancy,late pregnancy,parturient pe-riod),obstetric accident group,pregnancy complicating other disease group and healthy control group.At the same time,PT, APTT,TT,FIB,DD and PLT were detected and the related statistical analysis was conducted.Results DD and FIB in the normal pregnancy group were significantly higher than those in the healthy control group(P<0.05),PT and APTT were significantly low-er than those in the healthy control group(P <0.05),TT had no statistically significant difference between these two groups(P >0.05 );DD and FIB in the obstetric accident group and the pregnancy complicating other disease group were significantly higher than those in the normal pregnancy group(P <0.05),while PT was significantly lower than that in the normal pregnancy group(P <0.05).PLT in the obstetric accident group and the pregnancy complicating other disease group was significantly lower than that in the normal pregnancy group(P <0.05).The sensitivity and specificity of the combined detection of 3 indices in diagnosing the ob-stetrical accidents were greatly superior to those of the single index detection or the combined detection of two indices.Conclusion The prenatal combination detection of four coagulation indices,DD and PLT can preliminarily judge the coagulation status of preg-nancy period in pregnant women,help to treat abnormal bleeding of pregnant women an possess the important guidance significance in formulating the labor scheme and preventing the occurrence of obstetric accidents.

Objective To explore the transfection efficiency of primary lymphocytes from human peripheral blood by different methods to acquire the method with higher transfection efficiency.Methods Mononuclear cells from human peripheral blood were isolated using Ficoll-Hypaque.Cell viability was detected by Trypan blue staining.Suspending lymphocytes were sucked out and were incubated in 24-well plate after cultured in 6-well plate for 2 h.Activated lymphocytes were transfected by electroporation with plasmid(PEGFP-N1).Resting or activated lymphocytes were transfected by lentivirus vector(LVGFP) single infection or repeated infection,respectively.Green fluorescence protein (GFP) was detected under the fluorescence microscopy and percentage of positive cells was checked by flow cytometry at different time points after infection.At the same time,the effectiveness of lentivirus infection was compared under different conditions.Results Purity of mononuclear cells isolated by Ficoll-Hypaque was 95 ％ and its viability was over 95 ％.The percentage of lymphocytes obtained with a uniform shape was 90 ％-95 ％.Scattered fluorescence was observed by electroporation under the conditions of voltage 2 100 V,pulse width 10 ms,pulse number 1 for lymphocyte,while fluorescent became weaker over time and no green fluorescent was observed after transfection for 72 h.After resting lymphocytes were infected once for 48 h by lentivirus vector,green fluorescent was not found and positive cells were less than 1％.1％-5 ％ of activated lymphocytes could express GFP after single lentivirus infection and the expression levels were enhanced with concentration increasing,while 5 ％-10 ％ of activated lymphocytes showed strong green fluorescent by repeated lentivirus infection.In contrast with electroporation,the fluorescent with lentivirus infection was stronger over time.Conclusion Repeated lentivirus infection could efficiently transfect exogenous genes into activated lymphocytes for stable expression.

Purpose To evaluate the breast imaging reporting and data system (BI-RADS) of ultrasound and 18F-lfuorodeoxyglucose (FDG) PET/CT in diagnosis of breast tumor, and to analyze the correlation between them. Materials and Methods 18F-FDG PET/CT images and ultrasound images of 103 patients with suspected breast cancer were retrospectively analyzed to get correlation between the maximum standardized uptake values (SUVmax) and BI-RADS. Sensitivity, speciifcity, positive predictive value, negative predictive value were compared with histology or follow-up results as golden standard. Results Of the 103 lesions, 46 were benign and 57 were malignant. Pearson correlation coefifcient was 0.464 (P<0.01). The sensitivity, speciifcity, positive predictive value, and negative predictive value of PET/CT were 89.47%, 73.91%, 80.95%and 84.99%, respectively;those of BI-RADS were 94.70%, 69.60%, 79.42%and 91.38%, respectively. The sensitivity, speciifcity, positive predictive value, and negative predictive value in patients with BI-RADS 3-4 were 88.90%, 71.40%, 66.65%, and 90.91%, respectively. The sensitivity, speciifcity, positive predictive value, and negative predictive value for BI-RADS grading diagnosis were 88.90%, 46.40%, 51.60%and 86.67%, respectively. Conclusion There is no signiifcant correlation between SUVmax and BI-RADS. BI-RADS has low speciifcity for patients with BI-RADS grade 3-4, while PET/CT can make up this shortcoming. Combined diagnosis in the breast disease can be potentially recommended in clinics.

Objective To explore the clinical characteristics,causes,diagnosis and treatment of pelvic mass in adolescents caused by gynecologic diseases. Methods One hundred and twenty-six adolescents with gynecologic diseases presented as pelvic mass were retrospectively reviewed. Results The average age of all patients was 17. 6 years,ranging from 10. 0 to 19. 0 years. Ectopic pregnancy was found in 38 patients(30. 2%) ,the average age of the patients was 18.6 ±0.5 years, ranging from 17.0 to 19.0 years. Ovarian neoplasm was found in 35 patients (27. 8%) ,the average age of the patients was 17. 1 ± 2. 1 years, ranging from 10. 0 to 19. 0 years. Twenty cases (57. l%)had germ cell tumor, eighteen with mature teratomas, one with endodermal sinus tumor and one with immature teratoma. Thirteen cases(37. 2%)had epithelial ovarian tumors;eight with serous cystadenomas,three with mucinous cystadenoma, one with mucinous cystadenoma complicated with serous cystadenoma and one with borderline mucinous cystadenoma. Two cases had simple cyst of the ovary (5. 7%) . Congenital abnormality of reproductive tract was found in 14 patients (11. 1%), the average age was 14. 7 ±2. 2 years, ranging from 13.0 to 19. 0 years. Seven had imperforate hymens, two had rudimentary horn of uterus,four had atresia of vagina,one had atresia of vagina complicated with rudimentary horn of uterus,and one had oblique septum of vagina. Corpus luteum rupture was found in 11 patients (8. 7%) , the average age was 18. 1 ± 1. 9 years, ranging from 13. 0 to 19. 0 years. 116 patients underwent operative treatment and 10 patients underwent conservative therapy. Conclusions According to the stage of adolescents, main causes of pelvic mass in adolescents are different Main causes of pelvic mass in early adolescents are tumorous disease. Congenital abnormalities of reproductive tract are usually in middle adolescents. The gonads develop closely mature in late adolescent period; main causes of pelvic mass are ectopic pregnancy, ovarian neoplasm and physiologic ovarian cyst Surgery is the main therapy of pelvic mass in adolescents.

Objective To investigate the clinical features of primary retroperitoneal fibrosis (RPF), and to find out better method for diagnosis and therapy. Methods Nine patients with primary RPF in Peking Union Medical College Hospital since 1990 to 2003 were analyzed retrospectively. Results All patients had abnormal findings in CT or MRI, and 8 of them had chronic non-specific inflammation of the retroperitoneum in pathology. Six patients had renal involvement and 2 of them had severe renal failure. Eight of the patients had received therapy with corticosteroid or tamoxifene. Conclusions RPF is an uncommon collagen vascular disease characterized by a chronic non-specific inflammation of the retroperitoneum.Because of the atypical manifestations of RPF, awareness of the disease is important.CT and (or) open biopsy remains the gold standard for diagnosis.Management typically includes surgery and drug treatment.Corticosteroid therapy or more recently,tamoxifen has been used successfully. The clinical and radiographic improvement with drug treatment has been seen in several patients and confirmed the diagnosis,thereby early diagnosis and treatment can bring the patient more excellent renal and general outcome.

Objective To explore the effects of repetitive transcranial magnetic stimulation (rTMS) on cognitive ability in patients suffering from mild cognitive impairment (MCI) after ischemic stroke.Methods Forty five ischemic stroke survivors with MCI but not meeting the criterion for diagnosis as dementia were recruited, and were randomly assigned into an rTMS group (32 patients) and a control group (30 patients) according to a random number table.Both groups received the routine drug therapy of medicine and cognitive function training, and the rTMS group was additionally given rTMS over the left dorsolateral prefrontal cortex at 5 Hz and 80％ motor threshold.The treatments lasted for 4 weeks.The Montreal Cognitive Assessment (MoCA) and auditory event related potential (ERP) were tested for both group before and after the treatment.Results After the treatment, two groups showed significant improvements in the average score of MoCA compared to that before the treatment, and that of the rTMS group was significantly higher than that of the control group.For both groups, the P300 latency shortened and the amplitude increased after the treatment.Moreover, the latency and amplitude of the rTMS group increased to 355.67 ± 16.43 ms and 8.69 ± 1.65 μV, respectively, after the treatment, significantly shortened and lengthened than that of the control group [(372.76 ± 23.35 ms and 7.03 ± 3.04 μV), respectively].Conclusions rTMS can significantly improve the cognitive ability of ischemic stroke survivors in a relatively safe way.