Objective:To clone interleukin 4 cDNA of Tibet pig.Methods:To amplify cDNA,using RT PCR from the total RNA from the lymphocyte stimulated by 10 ?g/ml Con A in vitro,to subclone into pMD T vector,and then to determine it by digesting and sequencing.Results:The cloned Tibet pig interleukin 4 was 98% homologus to the necleotide acids of Chenghua pig a special specie of pig in Sichuan province and 99% homologus to that of hybrid of Landrance with Chanbai pig.Conclusion:IL 4 of Tibet pig was successfully cloned from the lymphocyte after stimulating 70 h with ConA.

The antioxidant activity of lycopene is highest among the current carotenoids.Recently the researches on lycopene are focused on ingredient of functional foods in the world.The fermentation production of lycopene by Streptomyces rimosus was reported first time in China.The determination methods,such as spectrophotometric method and HPLC were constructed.Using a stain Fc of Streptomyces rimosus as a original strain,a high-yield lycopene producing mutant strain Fc' was selected after UV mutation.The lycopene yield of the strain is 2.5 times higher than the original strain Fc.The optimal fermentation conditions were determined by flask experiments,and the lycopene yield of strain Fc' reached 230mg/L in flask under the optimal fermentation condition,meanwhile the Streptomyces rimosus strain could produce purer lycopene without adding any blocking agents in its fermentation process.This results lay a good foundation for lycopene commercial production by fermentation using Streptomyces rimosus.

Objective To construct the lentivirus carrying human ?-catenin-EGFP(enhanced green fluorescent protein)and observe its expression in human follicle stem cells.Methods The ?-catenin gene sequence was amplified by RT-PCR from extraction of total RNA of human vascular endothelial cells.TA cloning technique was utilized to acquire gene subcloned pUCm-T-?-catenin.After transformation reaction,candidate clone was further analyzed by PCR and gene sequencing.Then the plasmid was transfected into FT293 cells.After identification by Western blotting,the plasmid was transfected into FT293 cells again for packaging.Infection titer was monitored by green EGFP expression.The expression of ?-catenin-lentivirus in human follicle stem cells were observed under inverted fluorescence microscope.Results The ?-catenin gene was cloned into the lentivirus successfully.The high expression of green fluorescence protein in FT293 cell line was found under fluorescent microscope.Viral titer checked by real-time PCR was about 2.0?108 TU/mL.When the multiplicity of infection(MOI)was 10,the infection efficiency of ?-catenin-lentivirus in human follicle stem cells was nearly 80% after infection 48 h around.After 3 weeks of continuous observation,we found the infection efficiency still keeping in the range of 80%-90%.Conclusion The lentivirus expression vector for ?-catenin was successfully constructed.It can steadily infect human follicle stem cells and the infection efficiency is considerable high.

Objective To observe liver pathological characteristics of the autoimmune hepatitis (AIH) C57BL/6 mice.Methods We established AIH C57BL/6 mice model with mixed adjuvants of allogenic liver antigens and complete freund's adjuvant by intraperitoneal injection.Then we determined and compared the body weight,alanine aminotransferase (ALT),aspartate aminotransferase (AST) between the normal control group (N group) and AIH model group (M group).And we compared the pathological characteristics of liver,spleen,heart,lung,kidney between two groups.Results We successfully established AIH C57BL/6 mice model.Compared with N group,we found that the average weight increase of mice in M group was decreased,ALT and AST of mice in M group were boosted.In M group,liver of mice presented typical interface of hepatitis,lymphocytic infiltration,even severe hepatitis,and showed spotty necrosis,multi-acinar necrosis.Some showed early sign of liver fibrosis by aprearing fibrous tissue hyperplasia.M group mouse's spleens were enlarged significantly.The spleen had darker color,not neat,and not smooth.Meanwhile,results were analyzed with statistics to confirm whether there was a significant difference between two groups (P＜0.05).About lungs,hearts and kidneys of mice in two groups,the pathological features were not found,and there was no statistic difference (P＞0.05).Conclusions The liver pathological characteristics of AIH C57BL/6 mice are similar to AIH patients.The results provide the pathological basis for the experimental research of autoimmune hepatitis.

Objective To evaluate the impact of three to four cycles of neoadjuvant chemotherapy (NACT) on the survival of patients with N2-N3 nasopharyngeal carcinoma (NPC).Methods The clinical data of 915 patients with T1-4N2-3M0 NPC from 2007 to 2010 were retrospectively analyzed.A total of 179 patients treated with 3-4 cycles of NACT (NACT≥3 group) were matched with 358 patients treated with 2 cycles of NACT (NACT=2 group) and 179 patients treated without NACT (NACT =0 group,concurrent chemoradiotherapy group) for age,N stage,pathological subtype,and NACT regimen.The Kaplan-Meier method was used to calculate overall survival (OS),disease-free survival (DFS),recurrence-free survival (RFS),and distant metastasis-free survival (DMFS) rates,the log-rank test was used for survival difference analysis and univariate prognostic analysis,and the Cox proportional hazards model was used for multivariate prognostic analysis.Results For the NACT≥ 3,NACT =2,and NACT =0 groups,the 5-year OS rates were 89.4％,81.6％,and 73.7％,respectively (P=O.000),the 5-year DFS rates were 83.2％,69.8％,and 64.2％,respectively (P=O.000),the 5-year RFS rates were 86.0％,76.0％,and 69.3％,respectively (P=0.001),and the 5-year DMFS rates were 86.6％,76.0％,and 68.3％,respectively (P=0.000).Three to four cycles of NACT was an independent protective factor for OS,DFS,RFS,and DMFS in patients with N2-N3 NPC.Conclusion Three to four cycles of NACT can significantly improve the survival of patients with N2-N3 NPC.

Objective To describe CT manifestations of intrahepatic spontaneous portosystemic shunts(ISPS) of cirrhosis.Methods Spiral CT(SCT) scans and related clinical findings of 15 cirrhotic patients with ISPS were retrospectively reviewed.Results Three kinds of ISPS were showed :①Right posterior portal vein type was in 12 cases and showed tortuous intrahepatic tubular structures which were all in the posterior segment of the right lobe connecting the right posterior portal vein to the inferior vena cava(IVC) in the suprarenal region in all cases.②Hepatic venous type was in 2 cases,the portal vein communication with the hepatic vein in the liver.③Apex type was in 1 case,the vein arose from the apex of the liver and drains into the internal thoracic vein.Blood ammonia levels were elevated in all cases and symptoms associated with hepatic encephalopahty were present in 2 cases.Conclusion To deeply understand the ISPS,it is of benefit for differential diagnosis of focal hepatic lesions.

This study was aimed to choose the best fixing method of electrocardiogram (ECG) measurement in mice by comparison of ECG index in three different fixing ways. Changes of ST amplitude and heart rate in ECG were measured in the self-made mouse sets, tape bundling or breathing anesthesia state. The amplitudes of the ST seg-ment in C57BL6 mice and β1/β2-AR double knockout mice were elevated in tape bundling or breathing anesthesia state in comparison with that in the self-made mouse sets (all P < 0.01). The heart rate under the tape bundling state in C57BL6 mice was significantly faster than that in the self-made mouse sets (P< 0.05 or P< 0.01). And the heart rate under anesthesia in β1/β2-AR double knockout mice was significantly slower than that in tape bundling state or the self-made mouse sets (both P < 0.01). It was concluded that relative to the binding and anesthesia method, fixing mouse in the self-made mouse sets, which can more truly reflect the physiological or pathological states, is a relatively good fixing method of ECG measurement in experimental study.

Background Though nitric oxide (NO) and NO synthase (NOS) have a critical role in angiogenesis,their effects on corneal neovascularization (CNV) and mechanism need to be further explored.Objective The aim of this study was to explore the effects of NOS and its antagonist,Nw-nitro-L-arginine methyl ester (L-NAME) on experimental CNV in mice,and investigate the influence of NOS and L-NAME on the tube formation of human retinal endothelial cells (RECs) in vitro.Methods The CNV models were established in the left eyes of 36 male BALB/c mice aged 7-8 weeks by application of the filter paper with NaOH in the center of corneas.The mice were randomized into two groups.L-NAME of 10 g/L (0.5 ml) was intraperitoneally injected 1 week before induction of CNV three times a week for three weeks in the mice of the L-NAME injection,and PBS was used in the same way in the control group.CNV was examined under the slit lamp biomicroscope 2,4,7,14 days after NaOH burn.The expression of CD31 in the CNV was assayed to calculate the ratio of CNV area and total corneal area using whole mount technique.The expression of NOS mRNA in the corneal tissue was detected by reverse transcriptase polymerase chain reaction (PCR),and VEGF expression in the human RECs was assayed by Western blot.The vessel formation number of cultured human RECs with or without L-NAME was performed by matrigel in vitro.Grouped t test was used to compare the differences of the parameters between the two groups.Results CNV developed and peaked 2 weeks after the application of NaOH on the mice corneas,and the CNV was obviously less in the L-NAME group compared with the control group.The expression of NOS mRNA in the corneas (NOS mRNA/ GAPDH mRNA)was significantly lower in the L-NAME group than that of the control group 2,4,7 days after CNV induction (t =19.481,t=22.059,t=10.961,all at P＜0.01).The ratio of the CD31 positive area in whole corneal area was 0.59± 0.01 in the L-NAME group,and that of the control group was 0.78±0.10,showing a significant difference between the two groups (t =3.078,P＜0.05).Western blot assay showed that the relative expression of VEGF protein in human RECs was declined in the L-NAME group compared with the control group 0,2,4,7 days,with statistically significant differences in 4 days and 7 days after NaOH burn(t=7.696,t=17.953,both at P＜0.01).The number of vessel network was 46.33±1.86 in the L-NAME group and 64.00±4.51 in the control group,with a significant difference between them (t =3.623,P＜0.05).Conclusions NOS participated in the pathogenesis and aggravation of CNV induced by NaOH.L-NAME arrests CNV formation and human RECs tube formation through down regulating the VEGF expression and NOS activity.

[Introduction] To estimate the prevalence ratios, using a log-binomial model with or without continuous covariates. Prevalence ratios for individuals' attitude towards smoking-ban legislation associated with smoking status, estimated by using a log-binomial model were compared with odds ratios estimated by logistic regression model. In the log-binomial modeling, maximum likelihood method was used when there were no continuous covariates and COPY approach was used if the model did not converge, for example due to the existence of continuous covariates. We examined the association between individuals' attitude towards smoking-ban legislation and smoking status in men and women. Prevalence ratio and odds ratio estimation provided similar results for the association in women since smoking was not common. In men however, the odds ratio estimates were markedly larger than the prevalence ratios due to a higher prevalence of outcome. The log-binomial model did not converge when age was included as a continuous covariate and COPY method was used to deal with the situation. All analysis was performed by SAS. Prevalence ratio seemed to better measure the association than odds ratio when prevalence is high. SAS programs were provided to calculate the prevalence ratios with or without continuous covariates in the log-binomial regression analysis.

Objective To study the effects of semiconductor laser irradiation and high voltage static electric fields on small vessel damage in diabetic rats.Methods Fifty healthy male Wistar rats were randomly divided into two groups:8 rats in a normal group and 42 in a diabetic model group.The diabetic models were created by intrape- ritoneal injection of streptozocin.The diabetic model rats were randomly divided into four subgroups:a diabetes group,a semiconductor laser treatment group,a high voltage static electric field treatment group and a comprehensive treatment group receiving combined semiconductor laser and high voltage static electric field exposure.The rats in each treatment group were subjected to the corresponding intervention.After 20 days of treatment,the venous blood, kidney tissue and myocardium tissue of the rats were collected,and the concentrations of blood glucose,insulin,en- dothelin and tissue were detected.Results Compared with the normal group,a significant increase in blood glu- cose and endothelin was observed in the diabetic model group,along with significantly decreased blood insulin and significant small vessel endothelium proliferation in the kidney tissue.Compared with the diabetes group,endothelin levels were significantly lower in all 3 treatment groups,and blood insulin was also higher in the comprehensive treat- ment group.Conclusion There were severe abnormalities in blood glucose,insulin and endothelin as well as mild impairment of small vessel endothelium proliferation in the diabetic rats.Semiconductor laser and high voltage static electric field exposure have a role in treating and preventing these conditions in diabetic rats.