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 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.

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 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 evaluate if 18F-FLT PET imaging could be used as a new clinical method to predict tumor radiosensitivity.Methods MDA-MB-231 and LN229 cells were irradiated with doses of 0,8 and 16 Gy of 6 MV photon energy,then soft agar assay and cellular uptake of 18F-FLT were performed on the 2 cell lines.The t test and one-way analysis of variance were used for the two groups and data before and after irradiation.The MDA-MB-231 and LN229 tumor xenografts were prepared by injecting the tumor cells into the right limbs of female BALB/c nu/nu mice.Once tumors reached a diameter of 10 mm,the two types of mice were divided randomly into 3 groups (20 mice per group) according to the irradiation doses (0,8 and 16 Gy).After irradiation,18F-FLT PET imaging and immunohistochemical staining were conducted.Then correlations between 18F-FLT SUVtumor/SUVmuscle ratio (T/M ratio) and TK1 labeling index percentages (LITK1) were tested using linear correlation analysis.Results The survival fraction of MDA-MB-231 and LN229 cells after irradiated with 8 Gy were (59.73 ± 4.3) ％ and (93.41 ± 3.75) ％,respectively (t =-13.20,P ＜ 0.001).When the dose increased to 16 Gy,the survival fraction decreased to (43.57 ±4.06) ％ and (81.77 ± 4.42) ％,respectively(t =-14.24,P ＜ 0.001).In MDA-MB-231 cells,the cellular uptake of 18F-FLT after irradiation with 8 Gy declined rapidly to (18.32 ± 1.38) kBq/105 cells ((128.22 ± 8.24) kBq/105 cells with the dose of 0 Gy,F =266.41,P ＜ 0.01),and maintained this low level till 72 h.For the LN229 cells,the cellular uptake decreased to (9.87 ± 1.30) kBq/105 cells after 8 Gy irradiation ((134.88 ± 6.59) kBq/105 cells with the dose of 0 Gy,F =346.06,P ＜ 0.01),then increased gradually to (127.17 ± 9.08) kBq/105 cells at 72 h (F =346.06,P ＞ 0.05).The dynamic changes of 18F-FLT cellular uptake in the two cells had the same pattern after being treated with 16 Gy irradiation.In the 18F-FLT PET image of MDA-MB-231 tumor mice after 8 Gy radiotherapy,the T/M ratio decreased to 0.78 ± 0.39 at the first day,but it was 2.84 ± 0.29 before radiotherapy (F =39.78,P ＜0.01).Then the ratio increased slowly,and it was still lower than the baseline at 7 d after radiation (F =39.78,P ＜0.01).The same pattern could be seen in the group of 16 Gy irradiation.In LN229 tumor mice treatment with 8 Gy irradiation,the T/M ratio increased to 2.41 ±0.47 at the first day,and it was 1.58 ±0.29 before radiotherapy (F =34.01,P ＜ 0.05).The ratio decreased steadily to 0.66 ± 0.32 (F =34.01,P＜0.05) at 7 d after radiotherapy.However,in the treatment group with 16 Gy,the T/M ratio decreased gradually and reached 0.44 ± 0.22 at 7 d (F =41.85,P ＜ 0.01).A correlation was found between 18F-FLT T/M ratio and LITK1 (8 Gy:r=0.67,0.73; 16 Gy:r=0.73,0.69; all P＜0.01) in both tumor models.Conclusion 18F-FLT PET imaging may be used as a new assay to predict tumor radiosensitivity,but further investigation is needed before clinical application.

Objective To investigate the effect of oxcarbazepine(OXC) and sodium valproate(VPA) on electroencephalogram(EEG) and peripheral blood levels of homocysteine(Hcy) and asymmetric dimethylarginine(ADMA) in adult patients with partial epilepsy.Methods From May.2014 to May.2015,a total of 100 patients with partial epilepsy were enrolled and randomly divided into treatment group(treated with OXC) and control group(treated with VPA),with 50 cases in each group.After treatment,changes of EEG indices,Hcy,ADMA,cognitive function and adverse reaction were analyzed.Results Before treatment,there was no significant difference of EEG indices between the two groups(P>0.05).After treatment,the incidence rates of α wave decreasing more than 0.5 Hz,increasing of θ wave and increasing of δ wave were significantly different(P< 0.05).Before treatment,there was no significant difference of serum Hcy and ADMA levels between the two groups(P>0.05).After treatment,serum Hcy and ADMA levels were both significantly increased(P<0.05).Before treatment,Mini-mental State Examination(MMSE) scores of the two groups were without significant difference(P>0.05).After treatment,MMSE score of treatment group was higher than that of control group(P<0.05).In treatment group,there were 1 case of skin rash and 2 cases of gastrointestinal discomfort,which were self-improved.In control group,there were 3 cases of dizziness,5 cases of skin rash and 1 case of gastrointestinal discomfort,which were self-improved.Conclusion The effects of OXC and VAP on peripheral blood levels of Hcy and ADMA could be similar,and compared with VAP,OXC could significantly improve cognitive function in patients with epilepsy.

Objective To purify recombinant methioninase and investigate the synergetic inhibitory effect of methioninase and cisplatin on the proliferation of human lung adenocarcinoma cell line GLC. Methods Recombinant methioninase was purified with GST-column from supernatant after ultrasonic disruption of cultured Escherichia coli in the prokaryotic expression system pGEX-4T-1-Met/Dh5a. MTT assay was used to determine the inhibition rate of methioninase in combined with cisplatin on cell proliferation,and their synergistic effect was evaluated by using the q value judge method. Results The concentration of recombinant methionine cleaving enzyme was 0.22 mg/mL, the purity was 95%, and the activity was 0.568 IU/mg. After 72 h culturing, the inhibition rate of cisplatin methionine was 24.80%and 27.49%respectively,while the inhibition rate of the combined drugs was 66.80% ( =1.47>1.15) which showed a significant synergistic effect. Conclusion Both methioninase and cisplatin have the inhibition effect,and the combined drugs display a significant synergistic effect on the proliferation of GLC.

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.