To optimize Chinese hamster ovary (CHO)expression system and establish a process of screening CHO cell lines with high productivity;neomycin-phosphotransferase (NPT)expressed by the resistance marker gene on the expression vector was mutated with amino acid D at 261 changed to G.After selection by culturing with G418;the survival rate of CHO cells bearing mutant-NPT was significantly lower than that of the cells bear-ing wide type NPT.An enhanced green fluorescent protein (EGFP)was genetically linked to the N terminus of the IgG1 Fc fragment part to generate an EGFP-Fc fusion protein regarded as a report gene;which verified that the resistance of mutant-NPT to G418 was weakened.By comparing fluorescence assay of EGFP intensity in stable transfections after selection with the same concentration of G418 for 3 weeks;mutant-selected pools expressed more exogenous protein than the WT-selected pools.Therefore;the ratio of high producers in a transfected cell population greatly increased.

A novel series of bis-nicotine derivatives (3a-3i) were designed, synthesized and evaluated as bivalent anti-Alzheimer's disease agents. The pharmacological results indicated that compounds 3e-3i inhibited both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the micromolar range (IC50, 2.28-117.86 micromol x L(-1) for AChE and 1.67-125 micromol x L(-1) for BChE), which was at the same potency as rivastigmine. A Lineweaver-Burk plot and molecular modeling study showed that these derivatives targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, these compounds could significantly inhibit the self-induced Abeta aggregation with inhibition activity (11.85%-62.14%) at the concentration of 20 micromol x L(-1).

Objective To establish RT-PCR-RFLP method for studying the genotype of wild mea-sles virus strains isolated from Tianjin area from 2002 to 2008. Methods Isolations of measles virus were carried out by tissue culture method from urine and throat swab specimens collected from suspected cases. RNA were extracted from the virus specimens. The 594 bp fragment of C terminal of the N (nucleoprotein) gene was amplified by one-step RT-PCR, then the PCR products were digested with Bcn I , separated on agarose gel electrophoresis and then analyzed by the method of RFLP (restriction fragment length polymor-phism). In addition, above results were compared with DNA sequencing. Phylogenetic tree was plotted based on the results for the genetic relationship and distance analysis. Results Sixty-nine measles virus strains were isolated from 189 specimens from 2002 to 2008, of which the C terminals of N gene were all de-tected positive. Among the 69 strains of measles virus isolates, 98.55% (68/69) belonged to Hla sub-geno-type which was the predominant sub-genotype, and only one strain (1.45%) belonged to H1b sub-genotype by RFLP analysis which was in accordance with the results by DNA sequencing method. Phylogenetic tree analysis indicated the H1a sub-genotype measles virus strains should be further divided into 2 clades, and the variation fluctuated between 0.2% and 3.8%. There were transmission chains caused by different virus strains co-cireulation. Conclusion A genotype, H1a and H1b sub-genotype can be identified by RT-PCR-RFLP assay specically based on the restriction enzyme Bcn I .The RT-PCR-RFLP assay can be a rapid, simple, accurate and efficient method for large-scale surveillance of measles virus strains in China.

Objective To explore the role of p16 gene methylation in fibroblasts in the occurrence and development of keloid.Methods Skin tissue specimens were resected from the lesions of patients with keloid and normal skin of healthy human controls.Fibroblasts were isolated from these tissue specimens and subjected a primary culture.An immunohistochemical analysis was performed to measure the expression of p16 protein in tissue specimens,real-time fluorescence-based quantitative PCR to determine the mRNA expression level (expressed as 2-△△Ct) of p 16 and DNA methyltransferases (DNMTs) in fibmblasts,and bisulfite sequencing PCR (BSP) to estimate the methylation status of p16 gene in the tissue specimens and primary fibroblasts.Results The keloid fibroblasts (KFbs) showed significandy lower mRNA expression of p16 gene (0.64 ± 0.18 vs.1.92 ± 0.23,t =10.54,P＜ 0.05),but significantly higher mRNA expressions of 3 DNMTs (DNMT1:2.58 ± 0.23 vs.1.13 ± 0.21,t =11.22,P ＜ 0.05; DNMT3A:4.87 ± 0.46 vs.2.38 ± 0.32,t =10.81,P＜ 0.05; DNMT3B:1.57 ± 0.12 vs.0.57 ± 0.16,t =12.45,P＜ 0.05) compared with the normal fibmblasts (NFbs).The DNA methylation rate in the p16 gene promoter region was significantly increased in keloid tissue (1.81％ ± 0.46％) and KFbs (3.15％ ± 0.94％) compared with normal skin tissue (0.90％ ± 0.35％,F =14.23,P＜ 0.01) and NFbs (0.17％ ± 0.29％,F=37.62,P＜ 0.01).Conclusions The methylation and low expression of p16 gene in KFbs may be associated with the uncontrolled growth of keloid,and DNMTs may play a role in the pathogenesis of keloid.

Objective To explore the factors affecting the prognosis of patients with hypertensive cerebral hemorrhage after treatment with minimally invasive operation Methods Clinical data from 50 patients diagnosed as hypertensive cerebral hemorrhage after treatment with minimally invasive debridement were retrospectively analyzed Risk factors affecting the prognosis of patients were analyzed with logistic regression Results The fatality rate of patients was 24 0% and rate of vegetative state and handicap was 36 0% Factors such as the amount of bleeding, bleeding site, preoperative GCS value, changes in pupils and light reaction were associated with the prognosis of patients undergoing minimally invasive debridement for the treatment of hypertensive cerebral hemorrhage The time between bleeding and operation was statistically correlated with the short term prognosis Conclusion The prognosis of patients with hypertensive cerebral hemorrhage is associated with multiple factors The amount of bleeding, bleeding site, preoperative GCS value, changes in pupils and light reaction can be used as the important indexes for the prognosis of patients with hypertensive cerebral hemorrhage after treatment of minimally invasive operation

Background To suppress vascular endothelial growth factor (VEGF) is a researching hot topic for the treatment and prevention of retinal neovascularization.Some detectable efficacy of VEGF small interference RNA (VEGF siRNA) in anti-tumor neovascularization has been well-known.But relevant study on VEGF siRNA on retinal neovascularization is seldom.Objective Present study was to investigate the inhibiting effect of VEGF siRNA on retinal neovascularization.Methods The 48 clean C57BL/6J mice aged 7-day-old were randomly divided into normoxia group,hypoxia control group,vector group and VEGF siRNA group and 12 mice for each.Hypoxia models were established by raising the pups with mother mice in the airtight oxygen-cabin for 5 days.The lipofectamineTM 2000 (LF2000)-mediated vector plasmids or VEGF siRNA recombinant plasmids were then injected intravitreally in 12 12-day-old pup mice respectively.The animals were sacrificed in 1 week after intravitreal injection,and the numbers of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM) were counted by hematoxylin-eosin stain.The expressions of VEGF protein and mRNA in retina were assayed by immunoinfluorescence technique and RT-PCR.Results The numbers of vascular endothelial cell nuclei extending beyond the ILM were 0.19±0.09,24.89±2.03,23.65±2.15 and 8.83±1.12 in normoxia group,model control group,vector group and VEGF siRNA group separately,showing significant decrease in VEGF siRNA group compared with model control group or vector group (q=5.67,q=4.97,P＜0.01).RT-PCR revealed that VEGF mRNA was faintly expressed in mouse retina in normoxia group.However,in model control group and vector group,the level of VEGF mRNA was 52.3 times and 36.7 times more than that of normoxia group respectively and only 3.5 times in VEGF siRNA group,presenting a inhibitory rate of 43.39% of VEGF siRNA on VEGF.Immunofluorescence showed that the expression of VEGF was weaker in normoxia group and strong positive response in model control group and vector group,but the expression intensity of VEGF protein was significantly weaker in VEGF siRNA group.Conclusion VEGF siRNA recombinant plasmids can efficiently inhibit retinal neovascularization in oxygen-induced retinopathy mouse model through intravitreal injection.

Seventy-three patients with type 2 diabetes mellitus were divided into atheroselerosis(AS) group and non-AS group according to the intima-media thickness(IMT)of the carotid artery.The plasma visfatin level in AS group was higher than that in non-As group[(44.95±10.14 vs 34.52±9.08)μg/L,P<0.05],and both of them were higher than that of the control [(24.46±7.18)μg/L,both P<0.05 ].The visfatin level Was positively correlated with IMT,waist-to-hip ratio,visceral fat thickness,fasting insulin,and HOMA insulin resistance index.Age,duration of diabetes,HbA_(1C),and visfatin level were the major risk factors for IMT of the carotid artery.

Objective To summarize the characteristics of common carotid arterial wave intensity (WI) in healthy volunteers and offer the normal reference values of WI. Methods Common carotid arteries (CCA) in 525 healthy volunteers were examined using Aloka Prosound α10 equipped with wave intensity software. The volunteers were divided into six group according to age,group A,＜20 years old;group B,20 - 29 years old;group C,30 - 39 years old;group D,40 - 49 years old;group E,50 - 59 years old and group F,≥60 years old. Carotid arterial wave intensity in normal subjects had two positive peaks, the first peak,W1 ,and the second peak, W2. Between the two positive peaks, a negative area (NA), the time interval between the R-wave of ECG and the first peak(R-W1 ) and that between the first and second peaks (W1-W2) were observed. Results ① 1050 common carotid arteries in 525 volunteers were studied. The mean values of W1,W2,NA,R-W1,and W1-W2 were (8332±4796) mmHg · m-1 · s-3,(1853±1143)mmHg · m-1 · s-3,(38 ± 26) mmHg · m-1 · s-2, (102 ± 19)ms and (266 ± 42)ms,respectively. ②These values were no significant between the left and right CCA in same group. W1 and NA were correlated with age ( r = - 0. 204 and r = -0.301, P ＜0. 001). W1 and NA in group A and B were significant increased than those in other groups( P ＜0. 05 or P ＜0. 001 ). ③There were statistical significances in W1 and W1-W2 of CCA between male and female. ④There were no statistical significances in all values in common carotid arteries of both sides (P ＞0. 05). ConclusionsWI technique is useful for evaluating the dynamic behavior of the heart and the vascular system and their interaction. W1 is very sensitive to the changes in the working condition of the cardiovascular system.

Objective To analyze the risk factors related to survival time of advanced nonsmall cell lung cancer(NSCLC)and to establish a prediction model on survival time. Methods From 2004-2006, 184 patients with advanced NSCLC were enrolled in the Affiliated Hospital to the Nantong Midical College. Related risk factors were analyzed, using the Buckley-James model. Both actual and predicted survival time were compared by log-rank test. Results Through Buckley-James model analysis, data showed that KPS, clinical stage, treatment and pre-treatment hemoglobin were main influencing factors on survival time. Regression equation appeared to be lnMONTH=0.0108 KPS+0.0238 HB+0.4614 Ⅲb+0.8027 Ⅲa+0.3869(radiotherapy+chemotherapy)+0.507 (radiotherapy + operation)+ 0.6082(chemotherapy + operation)- 2.098. There was no statistical difference between the prediction and the actual models of survival time by log-rank test(P=0.575＞0.05). Conclusion KPS, clinical stage, treatment and pre-treatment hemoglobin might be associated. Both the prognosis of patients with advanced NSCLC and the prediction model seemed to have practical significances.

Objective To find the effective therapeutic arrangement through investigating the clinical characteristics of chronic cardiac insufficiency with anaemia. Methods A total of 46 cases of anemia from 315patients who had been admitted to department of cardiology, the First Affiliated Hospital, Harbin Medical University for chronic cardiac insufficiency with anaemia were selected. They were divided into two groups. There were 22 patients in the first group who only accepted treatment to improve cardiac function (normal cardiac, diuretic,vasodilator therapy, etc.), and 24 patients in the second group who accepted treatment to improve cardiac function while receiving anti-anemia therapy treatment, oral ferrous sulfate tablets(0.3 g/tablet), 1 tablet each time, 3 times a day and(or) 2 times per week subcutaneous erythropoietin 3000 U. The hemoglobin(Hb), red blood cell(RBC) ,hematocrit (HCT), left ventricular ejection fraction (LVEF), fractional shortening (FS), stroke volume (SV) , cardiac output(CO) and E peak and A peak ratio(E/A) were observed before and after treatment. By logistic regression, grade grade Ⅱ , Ⅲ , Ⅳ, the incidence of anaemia were 7.9% (10/126), 19.2% (23/120) and 24.6% (17/69),respectively. Grade Ⅱ compared with grades Ⅲ, Ⅳ, the difference was statistically significant (x2 = 4.08, 3.12, all (3.49 ± 0.17) × 1012/L, (0.36 ± 0.08)%, (48.9 ± 3.11)%, (15.6 ± 1.8)%, (38.9 ± 3.7)%, (4.4 ± 1.6)% and (130.7 ±5.75)g/L, (4.12 ± 0.25) × 1012/L, (0.43 ± 0.02)%, (58.5 ± 2.65)%, (18.0 ± 2.5)%, (49.1 ± 7.7)%, (5.1 ± 1.2)%in the first and second groups, respectively. The difference between the two groups was statistically significant(t =value of Hb, RBC, HCT, LVEF, FS,SV, CO were (102.7 ± 6.93)g/L, (3.41 ± 0.12) × 1012/L, (0.35 ± 0.07)%,(47.5 ± 2.86)%, (16.0 ± 2.4)%, (38.2 ± 7.9)%, (3.7 ± 1.4)%, respectively. Compared with those after treatment,the difference was statistically significant (t = 15.632, 13.325, 5.569, 17.182, 3.186, 2.999, 3.074, all P ＜ 0.05);Ⅳ-level relative risk were 1.62, 3.14(P ＜ 0.05 or ＜ 0.01) . Conclusions Based on the standard treatment with treatment of anemia, cardiac contractile function can be improved.