Objective To construct a lentiviral expression vector of murine RelB for RNAi,and then to effectively silence the RelB gene expression of murine bone marrow derived dendritic cells for constructing the bone marrow tolerogenic dendritic cell,in order to provide an experimental foundation for a novel clinical therapy of autoimmune disease.Methods RelB shRNA sequence of mouse was designed by on-line designer software on Corp.Invitrogen,after synthesis and annealing,double strand oligonucleotides(dsoligoes)were cloned into the pENTRTM/U6 plasmid,then after sequencing,a positive clone was further subcloned into pLenti6/BLOCK-iTTM-DEST vector.It was then transformed into stb13 competent cell,and after sequencing,293FT cell line was transfected by above positive recombined plasmid and lentiviral packing materials.It was incubated for 48 to 72 h in a 37℃,5% CO2 incubator.Culture supernatant was harvested and stored at-80℃,then the virus titer was determined by serial dilution assay.Results It was showed from sequencing figures that all the pENTRTM/U6-RelB-shRNA plasmids were positive clone vector,and this positive recombinant vector was recombined with pLenti6/BLOCK-iTTM/U6-DEST vector.It was then transformed into stb13 competent cell and screen positive clone by ampicillin,and resequenced by the use of primer forward U6 primer.The results also showed that recombinant lentiviral vector was positive clone.Viral particle was packaged with other packaging material mediated by lipofectamine 2000 in 293FT cell line.Cultural supernatant was collected and stored at-80℃,and lentiviral particle titer was determined by serial dilution assay with 6?105/transduced unit.Conclusion Lentiviral shRNA expression vector of murine RelB gene for RNAi was successfully constructed.It might be a rational procedure to make tolerogenic DC,and then to develop a DC vaccine and DC-based immunotherapy for autoimmune diseases.

Background Integrated transepithelial photorefractive keratectomy (TransPRK) is a new kind of surface ablation and has a fast reepithelialization and uncorrective visual acuity (UCVA) recovery as well as slighter postoperative pain,and epipolis laser in situ keratomileusis (Epi-LASIK) has been recognized to be an effective method for myopia.But there have been few studies to evaluate the dynamic change of the corneal biomechanical properties and posterior corneal elevation after TransPRK.Objective This study was to assess and compare the effectiveness and safety between TransPRK and Epi-LASIK for myopia with thin cornea.MethodsThis study was approved by Ethic Committee of Jinan Mingshui Eye Hospital,and written informed consent was obtained from each patient.In this prospective non-randomized controlled study,93 right eyes of 93 myopic patients with the central corneal thickness 460 to 500 μm were included in Jinan Mingshui Eye Hospital from June to December 2013 under the informed consent.The eyes were divided into TransPRK group for 46 eyes and Epi-LASIK group for 47 eyes.UCVA,manifest refraction,haze,corneal biomechanical properties,posterior corneal elevation,Qvalue and corneal high order wavefront aberration were analyzed before and 1 week,1 month,3 months and 6 months after operation,respectively,and the examination results were compared between the two groups.Results There was no statistically significant difference in the eyes of postoperative UCVA and manifest refraction between the TransPRK group and the Epi-LASIK group at various time points (all at P＞0.05).Six months after surgery,the percentage of eyes with UCVA of 1.0 or better was 93.9％,and 90.9％ eyes exhibited the targeted refraction in ± 1.00 D in the TransPRK group.Corneal haze was most obvious 1 month after surgery in both groups,with the incidence of 32.6％ (15/46) in the TransPRK group and 17.4％ (8/47) in the Epi-LASIK group,but no significant difference was found in the eye numbers with haze between the two groups (x2 =2.841,P =0.092).No significant differences were seen in the corneal hysteresis(CH) values and corneal resistance factor(CRF) values between the two groups (CH:Fgroup =0.000,P =0.999;CRF:Fgroup =0.110,P =0.741),however,the postoperative CH values and CRF values were significantly declined in comparison with preoperative ones,with significant differences among various time points (CH:Ftime =103.658,P =0.000;CRF:Ftime =132.008,P =0.000),while there were no remarkable differences between any two time points in postoperation (all at P＞0.05).Posterior corneal surface height shifted rearward 1 week,1 month,3 months and 6 months after surgery,showing remarkable differences in comparison with before surgery in both groups (Ftime =12.868,P =0.001),but no significant differences between the two groups (Fgroup =1.923,P=0.169).No significant differences were found in Q-value between the two groups (Fgroup =0.191,P=0.663).Root mean square (RMS) and spherical aberration values elevated in postoperation compared with preoperation,with significant differences between them(all at P＜0.01),but the comparison between intergroup was insignificant (RMS:Fgroup =0.299,P =0.586;Spherical aberration:Fgroup =1.290,P =0.259).Conclusions TransPRK for myopia with thin cornea is safe and stably effective like Epi-LASIK.TransPRK affects corneal biomecbanical properties early after surgery but the effect gradually lessens over time.The posterior corneal elevation shows a tiny backward displacement,while posterior corneal asphericity has no change.

Objective To study the clinical characteristics,pathology,and treatment for papillary carcinoma of the breast.Methods The clinical data of 17 patients of papillary carcinoma of the breast admitted in the First Affiliated Hospital of Wen Zhou Medical College were retrospectively analyzed.Results Papillary carcinoma of the breast accounted for 0.64％ of all breast cancer cases hospitalized during last 10 years.All cases had palpable lumps in the breast.12 cases received modified radical mastectomy,2 cases received simple mastectomy,2 cases underwent breast conservation therapy,1 case underwent simple mastectomy plus sentinel lymph node biopsy.15 patients received postoperative chemotherapy,among those 5 cases also received radiotherapy.During a 32.5-month median follow-up ( 1 month to 8 years),one case with bone metastases died 2 years postoperatively and another one died of multimetastases 7 years later.Conclusions The prognosis of papillary carcinoma of the breast is closely related with its pathology type.For intraductal papillary carcinoma low-traumatic therapy is applicable,while in case of infiltrating papillary carcinoma or invasive micropapillary carcinoma ( IMPC ),more aggressive therapies like that adopted for infiltrating ductal carcinoma are recommended.

Objective To explore the relationship between TLR3 mRNA expression on peripheral blood mononuclear cells(PBMCs)and acute rotavirus(RV)diarrhea.Methods Sixty-one children with acute RV diarrhea served as study subject,the expression of TLR3 mRNA on PBMCs was detected by real-time fluorescence quantitative RT-PCR.the concentrations of IFN-γand TNF-α in serum were measured by the method of Enzyrme-linked immunosorbent assay(EUSA).Results The expression of TLR3 on PBMCs and the serum levels of IFN-γ and TNF-α in the serious diarrhea group were 0. 820±0.051,(33.67±12.88)Pg/ml, (62.21±14.65)pg/ml,respectively,while it were 0.717±0.040,(24.01±10.06)pg/ml,(50.99± 12.18)pg/ml in the slight diarrhea group,and 0.525±0.029,(12.52±5.19)pg/ml,(28.65±7.44)pg/ml in the control group.Compared with the control group.the expression of TLR3 on PBMCs and the serum levels of IFN-γ,TNF-α in the serious and slight diarrhea group were significantly higher(P<0.01).There were significant differences between the serious and slight diarrhea group(P<0.01).There were positive relationship between the expression of TLR3 on PBMCs and tHe serum IFN-γ,TNF-α levels(r=0.431,P< 0.05,r=0.372,P<0.05).Conclusion The expression of TLR3 on PBMCs in children with acute rotavirus dialThea iS up-regulated,TLR3 and its mediated immune response are associated with the development of acute rotavirus diarrhea.

Objective:To study the drug distribution in the local tissues and lymph nodes treated with targeting and continuous interstitial chemotherapy for the oral squamous cell carcinoma before operation. Methods:8 patients with oral squamous cell carcinoma were treated by implanting the sustain-released cisplatin around and into the primary lesion. Drug distribution in tissue were studied by the graphite furnace atomic absorption spectrometry. Results:The drug concentration in the center of the tumor,1 cm and 2 cm from the tumor fringe were (34.877 1?15.720 8),(20.690 0 ?15.688 6) and (6.635 7?3.862 3) ?g/g, respectively. The drug concentration within lymph nodes of the sub-mental and submandibular area, deep cervical lymph nodes for the upper,middle and lower were(2.991 4?3.055 7),(4.026 0 ?3.692 0), (7.192 0 ? 8.958 7) and (5.028 5?3.484 3)?g/g, respectively. Conclusion:Targeting and continuous interstitial chemotherapy for the oral squamous cell carcinoma by implanting sustain-released cisplatin can increase drug concentration in the local tissues and the lymph nodes, to attain good efficacy of targeting chemotherapy.

ObjectiveTo explore the effect and mechanism of Toll-like receptor 4(TLR4) ligand LPS-mediated inhibition hepatitis B virus (HBV) replication in Bewo cells.MethodsFirst of all,2 μg 1.3-fold HBV recombinant vector pcDNA3.1 ( + )-HBV1.3 were transfected into Bewo cells,after 12 h,the cells were treated with LPS for 3 d.To observe the kinetics of IFN-β and TNF-α expression in Bewo cells,the Bewo cells were exposed to TLR4 ligand LPS.And the effect of pyrrolidine dithiocarbamate ( PDTC),an inhibitor of NF-κB,on LPS-induced cytokines was also observed.The HBsAg,HBeAg and HBV DNA level in the culture supernatant were detected by Microparticle Enzyme Immunoassay (MEIA) and fluorescence quantitative PCR,respectively,and the expression of IFN-β,TNF-α,TRIF and MyD88 was detected by ELISA and RT-PCR,respectively.ResultsCompared with control group,LPS could significantly suppress HBV replication in Bewo cells ( P ＜0.01 ),and it could induce the production of TNFα in Bewo cells ( P ＜ 0.05 ),in time-and dose-dependent manners.PDTC strongly inhibited LPS and induced TNF-α production,but had no much effect on IFN-β in Bewo cells ( P ＜ 0.001 ).Compared with control group,the mRNA levels of MyD88 were significantly induced by LPS in the Bewo cells transfected with this recombinant vector( P ＜ 0.001 ).ConclusionsTLR4 ligand LPS could significantly suppress HBV replication by inducing TNF-α production in Bewo cells mainly via the MyD88/ NF-κB signal pathway.

Objective:In this study,a prokaryotic expression of the 3-ketosteroid-Delta (1)-dehydrogenase (KSDD) which came from Arthrobacter simplex was built.Moreover,in order to investigate the catalytic mechanism of KSDD and improve its stability,the structure of KSDD was predicted by computer and the critical sites were confirmed by site-directed mutations.Methods:The recombinant plasmid was constructed by eukaryotic expression vector pET-22b and the recombinant strain was constructed and expressed in Escherichia coli BL21 (DE3).High-performance liquid chromatography was used to determine the transformation rate of 4-AD to ADD.The KSDD structure and key sites were predicted by SWISS-MODEL.Site-directed mutations for the amino acid residues of key sites were constructed and activities of the mutations were detected.Results:The recombinant strain E.coli pET-22-ksdd was successfully constructed.It was induced to express the dehydrogenase by IPTG and the conversion rate of 4-AD to ADD was 27％ at 21 ℃.The structure of 3-ketosteroid-Delta (1)-dehydrogenase and the four key sites was analyzed by SWISS-MODEL.Four mutants,Y120R,Y320L,Y488F and G492Y were constructed.Mutants Y120R and Y488F were inactivated,so they were proved to be the key active sites of KSDD.The conversion rate of mutant Y320L was consistent with that of wild type,but the stability at 37 ℃ was improved.The conversion rate of mutant G492Y was 1.2 times of the wild type and the stability has been improved at 37 ℃.Conclusions:At present,there are few studies about the structure and catalytic mechanism of dehydrogenase.The active sites of the enzyme were verified by this study,which laid the foundation for the further study of the properties of the enzyme KSDD.

Objective To study the protective role of ulinastatin in acute liver failure (ALF) and the effect on the expression of hemeoxygenase-1 (HO-1).Methods Sixty-six S-D rats were divided into three groups:control group,ALF group (model group) and ulinastatin group (intervention group).The rat model of ALF was induced by intraperitoneal injection of D-galactosamine (D-Gal) and lipopolysaccharide (LPS). The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malondialdehyde (MDA) were detected dynamically after 6,12,24,36 and 48 h injection.HO-1 mRNA expression in liver tissue was determined by reverse transcriptionpolymerase chain reaction (RT-PCR), and the expression of HO-1 protein was detected by immunohistochemistry.The differences among multiple groups were compared by univariate ANOVA and pairwise comparison was done by least significant difference (LSD). Results D-Gal/LPS injections successfully induced ALF rat model which presented with elevated levels of serum ALT,AST and liver MDA after 6 h injections (F=23.864,38.446,18.051,respectively,all P＜0.01),and peaked at 12-24 h after injections.Twenty-four hours after D-Gal/LPS treatment,the levels of serum ALT,AST and MDA in model group and intervention group were (8 346.7±1 363.1) U/L vs(4 151.3±970.0) U/L,(9 766.7±1.274.1) U/L vs (4 696.7±1 476.9) U/L,(8.34±1.13)μmol/g vs (4.66±0.91 ) μmol/g,respectively,which were significantly higher than those e (24.0±2.0) U/L,(82.3±16.9) U/L,(2.55±0.22) μmol/g,respectively] in control group (F=55.684,55.501,47.843,respectively,all P＜0.01);while those in intervention group were much lower than those in model group (P＜0.01).The expressions of HO-1 mRNA and protein in model group were significantly increased than those in control group (P＜0.01),while those in intervention group were even higher (P＜0.01).Conclusion Ulinastain could up-regulate the expressions of HO-1 mRNA and protein,which indicates that ulinastain may play anti-oxidant and anti-inflammatory roles in ALF through HO-1 pathway.

Objective To investigate the role of CD4+ CD25+ regulatory T lymphocyte(Tregs)in the immunological pathogenesis of liver fibrosis.Methods Twenty-six rats were divided into two groups:control group(6 rats)and model group(twenty rats).The rat model of liver fibrosis was induced bv subcutaneous injection of carbon tetrachloride(CCl4).The serums were collected for detection of hepatic function and fibrosis parameters.Hepatic tissue samples were used to observe the histopathological changes.The flow cytometry was used to detect the proportions of Tregs in both peripheral blood and spleen.The data were evaluated by t-test.The relationship between two variables was analyzed using Pearson linear correlation.Results The levels of serum alanine aminotransferase (ALT)and aspartate aminotransferase (AST) were significantly increased,but the level of serum albumin (Alb) was obviously decreased.The concentrations of serum hyaluronic acid (HA),procollagen type Ⅲ(PCⅢ),collagen type Ⅳ(CⅣ)of the model group increased to(177.42±61.25)μg/L,(34.86±7.47)μg/L and(7.32±3.71)μg/L,respectively,which were higher than those in the control group(t=-3.670,-5.661,-3.950,respectively;all P<0.01).In model group,hepatic lobules were full of collagen fibers and the hepatic pseudolobule formation was observed.The proportion of peripheral blood Tregs in CD4+ T lymphocyte in liver fibrosis model was(7.41±2.15)%,which was significantly lower than that in control group(12.88±2.93)%(t=3.752,P<0.01).Furthermore,the frequency of Tregs in spleen of the model group was(9.49±1.16)%,which was also significantly lower than that in control group(13.16±2.36)%(t=2.793,P<0.05).In addition,the levels of serum ALT,AST and fibrosis parameters were inverselv correlated with the frequency of Tregs in spleens and peripheral blood(ALT and Tregs in blood:r=-0.727,AST and Tregs in blood:r=-0.698,ALT and Tregs in spleen:r=-0.663,AST and Tregs in spleen:r=-0.535,HA and Tregs in blood:r=-0.719,PCⅢ and Tregs in blood:r=-0.558,CⅣ and Tregs in blood:r=-0.792,HA and Tregs in spleen:,r=-0.424,PCⅢ and Tregs in spleen:r=-0.685,CⅣ and Tregs in spleen:r=-0.506;all P<0.05).However,the linear correlations between serum Alb and Tregs in spleens and peripheral bloods were not observed(r=0.423,0.372,respectively,both P>0.05).Conclusion These findings suggest that the reduction of CD4+ CD25+ Tregs probably play an important role in the immunological pathogenesis of liver fibrosis.

We evaluated the patient in the early,advanced or healing phase of the disease by MRI in the treatment of chronic brucellosis spondylitis and to guide the clinical treatment.MRI images of 40 patients with clinically diagnosed chronic brucellosis spondylitis were analyzed retrospectively.The imaging findings of early,advanced and healing patients were summarized.MRI showed abnormal signals in the vertebral body,intervertebral disc,paraspinaI and psoas muscle.It is early stage if the intervertebral space was normal,and advanced stage if combined with interverbral space stenosis.It demonstrated short T1 and short T2 signal or similar to the normal vertebral body,combined with intervertebral space stenosis,for the healing stage.According to the specific imaging manifestations of chronic brucellar spondylitis in the course of disease development,it is possible to evaluate the clinical stage of the disease and guide the rational selection of clinical treatment.