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 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.

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 relationship between the mechanism of ischemic postconditioning-induced activation of nuclear factor-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway during myocardial ischemia-reperfusion (I/R) and reactive oxygen species (ROS).Methods Healthy male Sprague-Dawley rats, aged 16-20 weeks, weighing 250-300 g, were heparinized and anesthetized with intraperitoneal 1％ pentobarbital sodium 40 mg/kg.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution.Thirty-two isolated rat hearts were randomly divided into 4 groups (n=8 each) using a random number table: control group (group C) , group I/R,ischemic postconditioning group (group IPO) , and N-(2-mercaptopropionyl)-glycine (a ROS scavenger) + IPO group (group M + IPO).After 20 min of equilibration, group C was continuously perfused with K-H solution for 100 min, and the isolated hearts received the drugs via the perfusion system in the other groups.Group I/R was perfused with cardioplegic solution 4 ℃ St.Thomas, and then was subjected to 40 min of ischemia at 32 ℃ followed by 60 min of reperfusion.In group IPO, ischemic postconditioning was induced by 6 cycles of 10 s reperfusion followed by 10 s limb ischemia starting from the onset of reperfusion, and the hearts were then perfused for 58 min.In group M + IPO, the hearts were perfused with K-H solution containing N-(2-mercaptopropionyl)-glycine 2 m mol/L for 3 min starting from the onset of reperfusion,underwent 2 min of ischemic postconditioning, and then was perfused for 55 min.Heart rate (HR), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP),and positive maximal pressure of left ventricular increase (+dp/dtmax) were recorded at the end of equilibration and of reperfusion.At 5 min of reperfusion and the end of reperfusion, myocardial specimens were obtained from the left ventricle for determination of ROS content by enzyme-linked immunosorbent assay.At the end of reperfusion, myocardial specimens were obtained from the left ventricle for examination of the ultrastructure of myocardial cells and for determination of Nrf2, heme oxygenase-1 (HO-1) , quinone oxidoreductase 1 (NQO1), and superoxide dismutase 1 (SOD1) mRNA and protein expression (by using Western blot and real-time polymerase chain reaction).The damage to myocardial mitochondria was assessed using Flameng scoring.Results Compared with group C, HR, +dp/dtmax and LVDP were significantly decreased, and LVEDP was increased at the end of reperfusion in I/R and M+IPO groups, HR and LVDP were decreased, LVEDP was increased, and no significant changes were found in +dp/dtmax at the end of reperfusion in IPO group, Flameng score was increased in I/R, IPO and M+IPO groups , the ROS content was increased at the end of reperfusion in I/R, IPO and M+IPO groups, and Nrf2, HO-1,NQO1 and SOD1 mRNA and protein expression was down-regulated at the end of reperfusion in I/R, IPO and M+IPO groups.Compared with group I/R, HR, +dp/dtmax and LVDP were significantly increased, and LVEDP and ROS content were decreased at the end of reperfusion, Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was up-regulated at the end of reperfusion in IPO and M+IPO groups, Flameng score was decreased in IPO group, there was no significant change in Flameng score in M+IPO group.Compared with group IPO, HR, +dp/dtmax and LVDP were significantly decreased, LVEDP and ROS content were increased at the end of reperfusion, Flameng score was increased, and Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was down-regulated in M+IPO group.Conclusion Ischemic postconditioning can regulate ROS level and activate Nrf2-ARE signaling pathway, thus attenuating myocardial I/R injury in rats.

Objective To observe the activation mechanism of Nrf2-ARE pathway in protective effect of ischemia and pinacidil postconditioning on isolated rat hearts.Methods The hearts of adult male Sprague Dawley rats were established ischemia-reperfusion injury model,and devided into six groups(n =8,each group),i.e.Normal group(Group N),ischemiareperfusion group (Group Con,I/R),ischemic postconditioning group (Group IPO),pinacidil postconditioning group (Group P50),N-(2-mercaptopropionyl)-glycine(MPG,2mmol/L) + IPO group(Group M + IPO),MPG + P50 group(Group M + P50).Rat hearts were perfused with Krebs-Henseleit(K-H) buffer for 20 minutes for equilibration.Subsequently,Group N was perfused with K-H buffer for 100 minutes after equilibration,Group Con was perfused with 4℃ ST.Thomas solution to stop the heart beating after equilibration,then the hearts were underwent 40 minutes global ischemia under 32℃,and followed by the K-H solution for 60 minutes.Group IPO after global ischemia period,the hearts were subjected to six 10-seconds cycles of ischemia/reperfusion at the beginning of reperfusion,then were reperfused for 58 minutes.Group P50 after global ischemia,rat hearts were perfused with K-H buffer containing pinacidil(50.μmol/L) for 2 minutes before reperfusion.Group M + IPO after global ischemia,the hearts were subjected to perfuse with K-H buffer containing MPG(2 mmol/L) for 3 minutes,and then underwent six 10-seconds cycles of ischemia/reperfusion before reperfusion.Group M + P50 after global ischemia,the hearts were perfused with K-H buffer containing MPG(2 mmol/L) for 3 minutes,and then subjected to perfuse with K-H buffer containing pinacidil(50 μmol/L) for 2 minutes before reperfusion.Cardiac function indexes(such as HR,LVDP,LVEDP,and the Max dp/dt) at the end point of equilibration and repeffusion were observed and recorded.The ultrastructure of myocardial tissue was observed by electron microscopy and the mitochondrial Flameng score was calculated.RT-PCR and western-blot were applied to detect the gene transcription and protein expression of HO-1,NQO1,SOD1,and Nrf2 in left ventricular myocardial tissue after reperfusion.Results The HR,LVDP and + dp/dtmax at the end of reperfusion:the cardiac function indexes are lower among each group compared with group N,group 1PO and group P50 are better than group Con (P ＜ 0.05).Compared with group IPO,there is no significant difference in group group P50,but group M + IPO is obviously decreased(P ＜ 0.05).Compared with group P50,group M + P50 index is decreased significantly(P ＜ 0.05).The LVEDP at the end of reperfusion is lower than that among each group as compared with group Con,which is significantly increased in group Con (P ＜ 0.05).Compared with group IPO,there is no significant difference in group P50,but group M + IPO is significantly increased(P ＜ 0.05).Compared with group P50,the group M + P50 is obviously decreased(P ＜ 0.05).The ultrastructure of myocardial tissue in group N is mostly normal,group Con presence serious damage.The ultrastructure damage of myocardial tissue is improved in group IPO and group P50 as compared with that in group Con,while group M + IPO is more serious than group IPO,group M + P50 is more serious group P50.The mitochondrial Flameng score is higher among each group as compared with group N (P ＜ 0.05),the score is lower in group IPO and group P50 as compared with group Con and corresponding nonblocking group (M + IPO,M + P50,P ＜0.05).The mRNA and the protein expressions of HO-1,NQO1,SOD1 and Nrf2 among each group are lower as compared with group N(P ＜0.05).Compared with those in group Con,the mRNA and the protein expressions in group IPO and group P50 are obviously increased(P ＜ 0.05),group IPO and group P50 are higher than those in group adding active oxygen scavenger(MPG) (P ＜ 0.05).Conclusion Ischemic postconditioning and pinacidil postconditioning have protective effect of myocardial tissue from ischemia reperfusion injury,while improve the cardiac function index.The cardiac protective effect of Ischemic and Pinacidil postconditioning methods may be involved the ROS in early reperfusion,which activate the Nrf2-ARE pathway,and up-regulate the expression downstream antioxidant protein and phase Ⅱ detoxifying enzyme,ultimately improve the cardiac function index during the reperfusion period.

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 observe the peroxidase body growth activated receptor γ (PPARγ) agonist rosiglitazone on acute pancreatitis in mice with hepatic injury and to investigate the mechanism of hepatic injury .Methods Seventy‐two male Kunming mice were randomly allocated into three groups(24 cases for each group):acute pancreatitis group(AP group) ,rosiglitazone group (AP‐ROS group) ,saline group(NS group) .Mice were killed at 6 ,12 and 24 h after induction of acute pancreatitis .Serum amylase , ALT and AST activities were measured .The expressions of NF‐κB and PPARγmRNA were assessed by RT‐PCR .The expressions of NF‐κB and PPARγ protein were assessed by Western blot .Results Compared with NS group ,serum amylase ,ALT and AST levels at each time point significantly increased in AP group(P< 0 .01);serum amylase ,ALT and AST levels in AP‐ROS group were significantly lower than those in AP group(P<0 .01) .Compared with NS group ,the expressions of liver PPARγ mRNA and protein in AP group were markedly lower at 6 h and 12 h(P<0 .05) ,and the expressions of PPARγmRNA and protein in AP‐ROS group were significantly higher than those in NS group and AP group(P<0 .01) .The expressions of liver NF‐κB mRNA and NF‐κB p65 protein in AP group were significantly higher than those in NS group and AP‐ROS group at all time points(P<0 .01) .Con‐clusion There are clear relationships between NF‐κB and hepatic injury in acute pancreatitis .The expressions of PPARγin injuried hepatic decreased .Rosiglitazone can increase the expressions of PPARγand prevent the expressions of NF‐κB in hepatic during the early phase of acute pancreatitis .

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.