Objective:To investigate the enhancing effect of low dose adenovirus on rAAV2 expression in drug-sensi- tive and drug-resistant tumor cells and to explore the related mechanism.Methods:Human small cell lung cancer NCI- H446 cells,human lung adenocarcinoma A549 cells,human gastric cancer SGC7901 cells,human oral epithelial cancer KB cells,and VCR-resistant KB cells(KB/VCR)were transfected with rAAV2-GFP alone or combined with AdS-RFP or with AdS-TERT-RFP.GFP expression in the infected tumor cells was observed and analyzed by fluorescence microscope and fluorescence activated cell sorting(FACS).GFP protein and phosphorylation level of ERK and AKT in the infected tumor cells were detected by Western blotting.Quantitative analysis of DNA copies of GFP and mRNA expression of GFP, HSPG,?_v integrin,FGFR-1 in the infected tumor cells were performed by Real-time PCR.Results:The results of FACS demonstrated that the mean intensity of GFP fluorescence and the GFP positive rate after infection with rAAV2-GFP com- bined with Ad5-RFP or Ad5-TERT-RFP increased by 0.3-3 and 4-8 folds,respectively.GFP expression in the tumor cells with combined infection showed 4-6 folds increase and the phosphorylation level of ERK and AKT also increased.GFP mRNA expression in the combined infection tumor cells increased by 3.83-7.33 folds;there was no differences in the GFP DNA copies between rAAV2-GFP group and rAAV2-GFP plus Ad5-RFP group.Cellular receptors HSPG,?_v integrin and FGFR-1 mRNA expression was slightly increased in rAAV2-GFP plus Ad5-RFP.Conclusion:Lower dose of recombinant adenovirus can obviously enhance the expression of rAAV2 in drug-sensitive and drug-resistant tumor cells,which might be related to the activation of signal transduction pathway and increase of intracellular transcription.

Objective To study the antiviral and antibacterial effects of the effective site of traditional Chinese medicine(TCM-ES).Methods Chicken embryoes were infected with influenza virus A(FM1 strain) and pre-injected with TCM-ES by means of chicken-embryo inoculation technique,and the antiviral effect of TCM-ES on chicken embryo was assayed by detecting the hemagglutination titers in allantoic fluids.Mice were orally pretreated with various dosages of drugs twice daily for 3 days,then were given drugs continuously for another 4 days following FM1 infection.The protective effects of TCM-ES on mice infected with FM1 were assayed by calculating the weight,index of lung,death-protection rate,and life-prolongation rate,etc.Ribavirin was used as the positive control.In addition,the antibacterial effects of TCM-ES were observed by detecting the minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) by test-tube dilution method.Results In chicken embryo experiments,TCM-ES showed a potential inhibiting effect on influenza virus with the MIC of 10 mg/mL,which was weaker than ribavirin.The results of animal experiment showed that the body-weight(BW) and pulmonary index of infected model group decreased evidently compared with those of the normal group,TCM-ES groups at the dosages of 750 mg/kg and 1500 mg/kg could reverse the decrease of BW and lung index as compared with the infected models,the difference being insignificant as compared with the normal group.Moreover,TCM-ES also increased the death-protection rate and life-prolongation rate of mice in a dose-dependent manner.TCM-ES at dosage of 10 mg/mL(MIC and MBC) had an antibacterial effect on staphylococcus,while had no effect on gram-negative bacilli.Conclusion TCM-ES has obvious antivirus effect on influenza virus FM1 strain,and also has certain antibacterial effect on staphylococcus,which is worth of further development and research.

Objective To observe the effects of training on intrinsic hand muscles after digital flexor tendonrepair. Methods A total of 60 patients with digital flexor tendon repair were randomly assigned into an experimentgroup (n = 30) and a control group (n = 30). Patients in the experiment group received intrinsic hand muscles train-ing, in addition to the passive mobilization of the flexor tendons and active mobilization of the extensor tendons admin-istered to those in the control group. Minnesota manual dexterity test and Purdue pegboard test were adopted to as-sessment the outcome after 3 months of training. Results After 3 months of training, there were significant differ-ences between the two groups in terms of the results of Minnesota manual dexterity test and Purdue pegboard test( P < 0.01 ). Conclusion Intrinsic hand muscles training after digital flexor tendon repair can improve fine move-ment and coordination of hand.

AIM: To observe the short-term curative effect of Suxiao Jiuxin Pill on coronary heart disease (CHD) and angina pectoris. METHODS: 187 patients were divided into two groups at random:the treatment group with 105 cases and the control group with 82 cases. The treatment group was given Suxiao Jiuxin Pill 6 pills tid and the control group was given Compound Danshen Dripping Pill 6 pills tid orally. The treating period was 2 weeks. Before and after treatment angina pectoris was observed and electrocardiogram was determined. RESULTS: Suxiao Jiuxin Pill could significantly alleviate syndrome of CHD in patients, lessen electrocardiogram and decrease dosage of nitroglycerin. The total effective rate on angina pectoris and the effective rate in improving electrocardiogram was higher in treatment group than that in control group (P

Objective To investigate serum total IgE levels of healthy population in two hospitals in Yantai and Weihai areas,to investigate the influencing factors of the levels of serum total IgE,which can provide information for the clinical diagnosis of allergic diseases.Methods The total serum IgE level was measured with chemiluminescence method in 1 200 cases of healthy people and 600 cases of smoking group in different age groups.Results The total IgE level of ≤6 years was (28.53±20.71)IU/mL,7-12 years was (29.74±25.94)IU/mL,13-18 years was (32±22.32)IU/mL,19-44 years was (45.2±36.27)IU/mL,45-60 years was (35.47±27.23)IU/mL,>60 years was (31.2±25.03)IU/mL.There was no effect of serum total IgE in different age groups:≤6 years,7-12 years,13-18 years,45-60 years,>60 years(t=0.610,1.508,0.777,0.160,1.518,all P>0.05),19-44 years was significantly different from other age groups(t=0.075,P<0.01).There was no gender difference of serum total IgE in different age groups (P>0.05).The total IgE level of the smoking group:19-44 years was (55.22±39.16)IU/mL,45-60 years was (42.63±28.46)IU/mL,>60 years was (39.32±26.73)IU/mL.The level of serum IgE in the smoking group was significantly higher than that in the same age group (t=0.142,0.174,0.235,all P<0.05).Conclusion No significant difference existed in healthy people of serum total IgE levels from birth to adulthood.However,the total IgE level rose when reached to 19-44 years,which then slightly declined as the growth of the age.There was no significant difference between male and female of the IgE levels in different age groups.But after the age of 19,smoking can lead to the increase of the total IgE level.

BACKGROUND: Genetically engineered cells have been used in the replacement therapy and gene therapy. However, how to select proper donor cells, target cells, and corresponding viral vectors is the most difficult in the therapy.OBJECTIVE: To compare the transduction efficiency of recombinant adenovirus Ad5 and AdSF35, adeno-associated virus rAAV1/2 and rAAV2, and lentivirus LV in bone marrow mesenchymal stem cells (BMSCs) and exogenous gene expression level so as to select the vectors, which can efficiently transduce BMSCs.DESIGN, TIME AND SETTING: Gene engineering controlled observation, performed in the Central Laboratory, Shanghai First People's Hospital between October 2006 and March 2007.MATERIALS: Ten Sprague Dawley rats of clean grade were used to prepare BMSCs. All recombinant viral vectors carded enhanced green fluorescent protein (EGFP) report gene. Ad5 was prepared by the Central Laboratory, Shanghai First People's Hospital. Ad5F35 was gifted by professor Qian Qi-jun from the Second Military Medical University of Chinese PLA. rAAV2 and rAAVI/2 were the products of Benyuan Zhengyang Gene Technique Co.,Ltd. LV was gifted by professor Cuo Li-be from Shanghai Institute of Biochemistry and Cytobiology, Chinese Academy of Sciences.METHODS: Rat BMSCs were in vitro isolated and cultured by density gradient centrifugation. BMSCs of passage 4 were inoculated into 24-well plate at lxlO5/well. One day later, ceils adhered to the wall and allocated to 5 groups. Ad5-EGFP [10, 100,1 000 multiplicity of infection (MOI)], Ad5F35-EGFP (10,10, 1 000 MOI), rAAVI/2-EGFP (1×104,1x10× vg), rAAV2-EGFP(1×104, 1x105vg), and LV-EGFP (30 TU) were respectively added into the 5 groups. BMSCs were transduced for 2 days with Ad virus and separately for 6 days with rAAV and LV virus.MAIN OUTCOME MEASURES: EGFP-positive expression and fluorescence intensity.RESULTS: After twenty-four hours of Ad5-EGFP transduction, EGFP-positive cells were visible under the microscope. With virus dose increasing, EGFP-positive cells increased and fluorescence intensity strengthened. Twelve days later, EGFP-positive cells gradually reduced and fluorescence intensity weakened. For Ad5F35-EGFP, its transduction was basically similar to Ad5-EGFP, but EGFP-positive cell number and fluorescence intensity were increased. After 6 days of rAAV1/2-EGFP or rAAV2-EGFP transduction, EGFP-posidve cell number and fluorescence intensity were decreased. For LV-EGFP transduction, a small amount of EGFP-positive cells could be visible on the second day, and then EGFP-positive ceils and fluorescence intensity were gradually increased until the sixth day.CONCLUSION: Ad5, Ad5F35 and LV could effectively transduce BMSCs cultured in vitro and express exogenous gene.Furthermore, transduction efficiency was correlated with virus dose in dose-dependent manner, rAAVI/2 and rAAV2 had poor/in vitro transduction efficiency.

Objective: To investigate the effect of cobalt chloride (CoCl2) on transgene expression and viral particle titers in tumor cells infected by conditionally replicating adenovirus expression vector with hypoxia response elements(HRE)-regulated E1AE1B expression (Ad-5HRE-E1AE1B-RFP) and non-HRE regulated replication-deficient adenovirus expression vector (Ad-EGFP, Ad-Luc) in vitro and in vivo. Methods: Ad-5HRE-E1AE1B-RFP had five duplicated HRE and mini CMV acted as a promoter to drive E1AE1B expression and constitutive expression of RFP as reporter. The hypoxia model was optimized by exposing tumor cells to different concentrations of CoCl2. The hypoxia-inducible factor 1 alpha (HIF-1α) protein expression was determined by Western blotting. Under the optimized hypoxia model, the positive expression of exogenous gene and virus replication of Ad-5HRE-E1AE1B-RFP or Ad-EGFP-infected tumor cells were examined by conversed microscopic observation, FACS analysis and plaques formation test. Furthermore, transgene expression induced by combined application of hypoxia-inducible replicative adenovirus and replication deficient adenovirus (Ad-Luc) was also evaluated by examining the lucifererse activity in xenografted tumor models in nude mice by micro PET. Results: Western blotting results showed that CoCl2 at 0.4 and 0.08 μg/mL could stabilize and acumulate HIF-1α protein in gastric cancer SGC7901 cells, which could better mimic hypoxia condition. The microscopic observation and FACS analysis showed that CoCl2 at 0.4 μg/mL could remarkably increase the transduction efficacy of Ad-5HRE-E1AE1B-RFP, which was verified by significant increase in the percentage of positive expression of exogenous gene RFP and fluorescence intensity. But plaques formation test showed that Ad-5HRE-E1AE1B-RFP had no replication. CoCl2 0.4 μg/mL augmented the tranduction efficacy and expression levels of non-HRE regulated replication deficient adenovirus Ad-EGFP and Ad-Luc. Combined intratumoral injection of Ad-5HRE-E1AE1B-RFP and Ad-Luc significantly increased the expression of Ad-Luc in nude mice.Conclusion: CoCl2 markedly enhances transgene expression of recombinant adenovirus. However, the underlying mechanism is not only related to the CoCl2-induced hypoxia, but also probably related to regulation of gene transcription.

Objective:To summarize the experiences in the construction of Clinical Research Unite (CRU) at a hospital level, provide reference for CRU construction in the Chinese hospitals.Methods:The CRU construction should take clinical research project management as working priority, strengthen clinical research team building, improve process management, as well as improve project performance and output.Results:During the CRU construction period, the number and level of clinical research projects have been improved in various disciplines. The sample database and disease specific data have been accumulated. The CRU mode helps the high-quality development of clinical researches in hospital.Conclusions:The way our hospital take in the construction of CRU was a good example, and it may truly improve the quality and capacity of clinical researches.

Objective To explore the value of cMRI and MRS in diagnosis and differential diagnosis of single brain metastases and localized high grade gliomas.Methods The cMRI (T1WI,T2WI and contrast-enhanced MRI)and MRS data of 23 cases with single brain metastases and in 28 cases with high grade gliomas confirmed by pathology were collected and analyzed retrospectively.Results cMRI:①There were no significant differences of T1WI,T2WI and constrast-enhanced MRI between single brain metastases and high-grade gliomas.②Enhanced images of single brain metastases and localized high-grade gliomas showed that there was a significant difference in the peritumoral edema area:the nodular or ring enhancement were found in single brain metastases,the outer outline of ring enhancement was smooth,and there was no enhancement at the peripheral edema area;Irregular patchy or ring enhancement were found in localized high-grade glioma tumor,both of the outer and inner edges of the ring enhancement rough,a little patchy enhancement at the peripheral edema area were found in 1 6 patients,and there was no enhancement of the edema area in the other 1 2 patients.MRS:①There were no significant differences of the tumor parenchyma in the peaks of NAA,Cr,Cho and central Lac between single brain metastases and localized high grade gliomas.②A significant difference of the peripheral edema area were observed between them.The peaks of NAA,Cr,Cho at the edema area in all single brain metastases patients were normal.But in all high-grade glioma patients,the NAA and Cr peaks were decreased,while the Cho peaks were increased.Conclusion The morphological manifestations of single brain metastases are similar to localized high grade gliomas.An obvious difference of cMRI and MRS lines exists in the peritumoral edema area between them,which could be used for differential diagnosis.

OBJECTIVEDesign a convenient and stable eye refractometer based on the theory of blur circle.

METHODSAnalyze the retinal blur circle in both Emsly reduced eye model and Liou & Brennan 1997 eye model by ZEMAX. Design the coefficients including PD (pupil diameter) and NO' (length between node point and fovea) with the purpose of improving the accuracy. At last, compare the clinical optometry data from this refractor with the data obtained from optometry hospital in Wenzhou.

RESULTSThe blur circle diameters are nearly the same in both reduced eye model and the Liou & Brennan 1997 eye model. With the PD = 4 mm and NO' = 20 mm, the refractor shows a fine accuracy in optometry. The paired t test shows that the myopia group and the astigmatism axial direction group have no statistical difference between the data from the blur circle refractor and the hospital (P > 0.05), while the astigmatism degree group has the result of P = 0.41 which may be caused by the poor cooperation of pediatric patients. 80% of the astigmatism degree data differ from the data from the hospital in less than 0.75D.

CONCLUSIONThe blur circle refractor, with the features of convenience and fine accuracy, is promised to be a new style of refractometer in the future.

Astigmatism ; Equipment Design ; Fovea Centralis ; Humans ; Models, Statistical ; Myopia ; Ophthalmoscopes ; Ophthalmoscopy ; Refraction, Ocular ; Visual Acuity