Objective To investigate the different expressions of adiponectin receptor 2(AdipoR2) mRNA in liver between diet-induced obese rats and controls.Methods The level of AdipoR2 mRNA expression was detected by reverse transcriptase polymerase chain reaction(RT-PCR).Insulin sensitivity was evaluated by insulin sensitivity index(ISI).Results Diet-induced obese rats showed higher expression of AdipoR2 mRNA compare to controls.The ISI of diet-induced obese rats was lower than that of controls.Conclusion High energy diet induceds AdipoR2 expression in liver.

Background Age-related cataract is one of the common causes of blindness.Although the pathophysiology of age-related cataract is far from clearly understood,it is well accepted that DNA damage plays an important role in the disease pathogenesis.Objective The purpose of this study was to quantitatively evaluate the DNA damage in peripheral lymphocytes of age-related cataract.Methods A cross-sectional study was carried out.This study complied Declaration of Helsinki and approved by Ethic Committee of Affiliated Hospital of Nantong University.Written informed consent was obtained from each subject.Two hundred and eleven patients with agerelated cataract and 147 normal subjects were enrolled from a “ Jiangsu Eye Study:Funing 2011 Eye Disease Epidemic Survey”.All the subjects aged from 50 through 80 years with matched age and gender between the two groups.The percentage of tail DNA and Olive tail moment (OTM) were detected by comet assay to assess the extent of DNA damage in peripheral lymphocytes.Statistical analyses were performed with SPSS 17.0 software,and the differences of the percentage of tail DNA and OTM were compared between the age-related cataract group and normal control group by independent sample t test as well as among the 50-59 years group,60-69 years group and ≥70 years group by one-way analysis of variance.Results Comet assay showed a round lymph cell with the clear border in the normal group;while in the age-related cataract group,the cell was bigger with a comet-like tail.The percentage of tail DNA and OTM in peripheral lymphocytes were (21.75 ± 3.51) ％ and 6.54 ± 1.65 in the age-related cataract group,and those in the normal control group were (9.31 ±3.60)％ and 2.18 ± 1.10,respectively,with significant differences between them (t =32.67,P =0.00 ; t =28.02,P =O.00).In the 50-59 years subgroup of the age-related cataract group,the percentage of tail DNA and OTM in peripheral lymphocytes were (20.04±2.86) ％ and 5.92± 1.14,and in the 60-69 years subgroup of the age-related cataract group,the percentage of tail DNA and OTM in peripheral lymphocytes were (20.77 ±2.93) ％ and 6.13 ± 1.14,which were significantly reduced in comparison with (22.79 ± 3.67)％ and 6.95±1.91 of the ≥70years subgroup(TailDNA％:q=2.75,P=0.00; q=2.02,P=0.00;OTM:q=1.03,P =0.02 ; q =0.82,P =0.00).Conclusions The pathogenesis and development of age-related cataract probably is associated with DNA damage.

Objective To observe the protective and anti-apoptosis effects of Rannasangpei (RNSP) on brain ischemia and reperfusion injury in rats. Methods Middle cerebral artery occlusion (MCAO) model was established and the groups were divided as sham group, MCAO group, vehicle + MCAO group and RNSP + MCAO group. Neuronal deficient signs, brain infarct area, the ratio of Bcl-2/Bax and the expression of caspase-3 were evaluated by neuronal deficient score, TTC (2,3,5-Triphenyltetrazolium chloride) staining and Western blot respectively. Results Compared with those parameters in sham group, the neuronal deficient signs, infarct area and caspase-3 expression increased evidently while the ratio of Bcl-2/Bax decreased markedly in MCAO group. But in RNSP+MCAO group, the neuronal deficient signs, infarct area and cas?pase-3 expression decreased greatly while the ratio of Bcl-2/Bax increased markedly compared with those parameters in MCAO group. Conclusion RNSP may have protective effects on brain ischemia and reperfusion, which is related to its an?ti-apoptosis role indicated by upregulation of Bcl-2/Bax ratios and downregulation of caspase-3.

Objective:To prepare monoclonal antibodies specifically against an immunogenic fragment in ectodomain of prostate-specific membrane antigen ( PSMA ).Methods: An polypeptide immunogenic fragment in the ectodomain of PSMA was predicted by biological information technology,and then it was expressed prokaryotically.BALB/c mice were immunized with the prokar-ytically expressed recombinant polypeptide antigen,to prepare the monoclonal antibodies specifically against an immunogenic fragment in ectodomain of PSMA by hybridoma technology,purification of monoclonal antibody by affinity chromatography,characterization of the monoclonal antibodies by Western blot.The radioimmunoimaging in prostate cancer model was performed by using the labeled McAb.Results:Throught the software analysis,we got the antigen fragment in the ectodomain of PSMA containing 310aa sequences higher specificity, artificially synthesized gene sequence of the region, and constructed a prokaryotic expression vector pET-32a-r-ectodomain-PSMA,by prokaryotic expression we obtained the 50 kD target antigen,after hybridization,the three positive hybridoma cell lines (5E6,4A5 and 4D7) were selected by ELISA using target antigen,the isotypes of 5E6 and 4A5 were IgG2a,the isotypes of 4D7 were IgG1,the titer of three monoclonal antibodies was above 1∶256 000.Western blot results showed that the prepared monoclonal anti-bodies could binding specifically to the antigen in the ectodomain of PSMA.Radioimmunoimaging in prostate cancer animal model results further confirm that the prepared monoclonal antibodies could combinate with the antigen in the ectodomain of PSMA in the animal body, and make the tumor imaging.Conclusion: The prepared monoclonal antibodies can specifically recognizes the PSMA antigen,which laid the foundation for the immunodiagnosis and immunotherapy of prostate cancer.

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

Objective To prepare recombinant human adenovirus type 3 expressing Norovirus cap-sid protein gene(Noro-orf2). Methods The cDNA for Noro-orf2 was amplifed by RT-PCR from stool of in-fantile gastroenteritis and cloned into the adenovirus shuttle vector pBSE3CMV-egfp. The vector pBSE3CMV-Nor was linearized with EeoR Ⅴ and Not Ⅰ, and transformed into E. coil BJ5183 with lined edenovirus ge-nomic DNA pLasmid pBRAdv3 by Rsr Ⅱ. The identification of recombinant adenovirus plasmid pBRAdv3E3dNor was performed by PCR, enzyme digestion and DNA sequencing. Then pBRAdv3E3dNor was digested with AsiS Ⅰ and transfeeted into Hep-2 cells with LipofectAMINETM 2000 to package recombi-nant adenovirus particles. Results Noro-orf2 was successfully inserted into the shuttle vector. The recombi-nant adenoviral plasmid pBRAdv3E3dNor was generated by homologous recombination in E. coil BJ5183 and confirmed by PCR and enzyme digestion. The recombinant adenovirus was successfully packaged and puri-fied. Norovirus eapsid protein gene expression was confirmed in Hep-2 cells by immunecytochemistry assay. Conclusion The recombinant type 3 adenovirus expressing Norovirus eapsid protein gene was successfully constructed. This study laid a foundation for developing vaccine against Norovirus.

Canrenone is an important intermediate for the synthesis of eplerenone,a cardiovascular drug.C_ 11 ?-hydroxylation of canrenone is the key reaction,which can be done by microbial transformation.Rhizopus sp.SIPI-0602,kept in our lab,could high selectively transform canrenone to a compound named SIPI-11.By determining and analyzing the MS,UV,NMR etc.spectra of compound SIPI-11,its chemical structure was elucidated to be 11?-hydroxycanrenone.The study on flask transformation technology showed that the transformation ratio exceeded 90% when the substrate concentration was not more than 6g/L.

Objective To evaluate the influence of thermal damage on the cell proliferation,invasive metastasis and epithelial-mesenchymal transition of hepatocellular carcinoma (HCC) through experiments in vitro,and to explore the relationship between thermal ablation and the recurrence,metastasis of HCC.Methods The McA-RH7777 HCC cell thermal damage model was established by using external heating method.The effect of thermal damage on the proliferation of HCC cells was detected by Kit-8 assay (CCK-8),and the cell cycle changes were studied by flow cytometry.The effect of thermal damage on the invasion potential of HCC cells was assessed by using Transwell assay.Fluorescence quantitative polymerase chain reaction (RT-PCR) and Western blot were used to evaluate the influence of thermal damage on HCC cell invasion potential,and on the mRNA and protein expression levels of EMT-related molecular markers,including VEGF,MMP-9,Nm23,E-cadherinand vimentin.Results Heating treatment of McA-RH7777HCC cells was performed by putting the cells in 43.5℃ water basin for 30 min.Two to five days after heating treatment the cell proliferative ability was significantly higher than that of control group (P＜0.05).At 48-72hours after heating treatment the proportion of HCC cells in G1 phase was obviously reduced and the proportion of HCC cells in S+G2 phase was significantly increased,the differences were statistically significant (P＜0.05).Compared with the control group,the difference in HCC cell invasion potential determined at 24 h after heating treatment was not significant,while the HCC cell invasion potential determined at 72 h after heating treatment was strikingly increased (22.3±2.46 vs.14.2±l.82,P＜0.001).Real-time PCR and Western blotting results indicated that at 72 h after heating treatment the expression levels of VEGF,MMP-9 and vimentin were significantly increased,while the expression level of E-cadherin was remarkably decreased,the differences were statistically significant (P＜0.05).Conclusion Thermal damage with sub-lethal heating dose can induce McA-RH7777 HCC cell to develop epithelial-mesenchymal transition and to enhance its proliferation and invasive metastasis potential,and HCC cells show higher malignant potential.

AIM: To compare the pharmacokinetics and relative bioavailability of the domestic and imported sustained-release tablets of gliclazide in healthy volunteers. METHODS:The study was performed by an four-period crossover design with singledose and multiple-dose administration. The plasmadrug concentrations of twenty male healthy volunteers were determined by liquid chromatography with mass spectrum detector method (LC-MS). RESULTS:The pharmacokinetic parameters after a single oral dose of the domestic and imported gliclazide tablets were (7.2+s 1.5) h and (6.9 +1.4) h for tmax, (13.4 ±1.2) h and (13.7 +1.3) h for t1/2, (2.4 +0.8) mg ·L-1and (2.3 ±0.6) mg· L-1 forcmax, (48 ±14)mg · h · L-1 and (48 +14) mg· h · L-1 forAUC0-60,(51+15) mg· h· L-1 and (50±14) mg· h· L-1for AUC0-∞, (22.4 ± 1.9 ) h and (22.8 ± 1.9 ) h for MRT, respectively. The steady state pharmacokinetic parameters after multiple doses of the domestic and imported gliclazide tablets were (6. 1 ± 1.4) h and (6.5+1.4) h for tmax, (4.6±0.9) mg· L-1 and (4.7±1.1) mg· L-1 for cmax, (0.23 ±0.08) mg ·L-1and (0.26±0.08) mg· L-1 forcmin, (1.6±0.3) mg·L-1 and (1.6±0.3) mg · L-1 for mean value of steady plasma-drug concentration (cav),(94±19) mg· h · L-1 and (95 ±20) mg · h · L-1forAUCss, (282 ±33)% and (283 ±43)% for degree of fluctuation DF ), respectively. The relative bioavailability of the domestic gliclazide tablet to the imported gliclazide tablet following a single and multiple dose were ( 102 ± 9) % and (99 ± 10 ) %, respectively. Main pharmacokinetic parameters between the two formulations in both single and multiples dose studies showed no statistical difference ( P >0.05 ). CONCLUSION: The result of two one side t-test shows that the two formulations are bioequivalent.

OBJECTIVETo investigate the method for mandible osteotomy in order to make the mandible of various square face appear harmony.

METHODSAccording to different types of the mandible, mandible angle osteotomy was performed in combination with mandible edge, mandible half ring osteotomy or chin augmentation.

RESULTSA total of 312 cases have been treated since 1996. In this series, mandible angle and mandible edge osteotomy was performed in 200 cases; only mandible edge osteotomy in 23; mandible half ring osteotomy in 15, chin sharpening in 9, chin augmentation with autogenous bone implantation, in 32. Postoperative follow-up of 150 cases for 1-12 months showed that the satisfactory rate was 97%.

CONCLUSIONIntegrated mandible osteotomy can make the square face look natural and nice-looking.