Background The visual evoked potential (VEP) is an important functional index to assess visual cortex plasticity.Monocular form deprivation(MD) of rat is a classic model to study visual cortex plasticity change.Utilizing VEP technique record the shift of ocular dominance in adult rats model is of an important significance for explore the treating opportunities of amblyopia.Objective The present study was to observe the pattern VEP changes in different ages of Long-Evens rats and the adult rats after binocular form deprivation.Methods Thirty-six healthy SPF Long-Evens rats were divided into PW3,PW4,PW5,PW6 groups according to the postnatal weeks,and PW7 rats included 36 SPF Long-Evens rats.The left eye lids of the rats were sutured in PW3,PW6 and PW7 rats for 3,5,7 days respectively,and PVEP in both left and right eye were recorded to assess the rat age that visual cortex plasticity ended.Bilateral PVEP were recorded in PW7 rats,and the bilateral eyelids were sutured to establish the bilateral form deprivation models.The right eyes were opened in 7,10,14 days,and the left eyes were opened in the next 3,5,7 days respectively and the PVEPs were recorded again to find the shift of ocular dominance and whether binocular form deprivation induce the visual cortex plasticity in adult rats.Results The P100amplitudes of the left eyes were gradually declined and those of the right eyes were raised from 3 to 7 days after MD of the left eyes in rats of PW3 group in comparison with before MD ( P＜0.01 ).In PW6 groups,no significant changes in P100amplitudes of both the right and left eyes were found in the third day after MD,but significant raise in the right eyes and lowing in the left eyes were seen in the 5 and 7 days after MD in comparison with before MD(P＜0.01 ).No any P100changes in both eyes were found in 3-7 days after MD compared with before MD in PW7 rats (P＞0.05).The C/I ratio (contralateral VEP amplitude to ipsilateral VEP amplitude in occluded eyes) in the rats of PW3,PW4 and PW5 groups were 1.07±0.15,1.16±0.16 and 1.14±0.15 respectively in 3 days after MD,showing the significant lowing in comparison with before MD (2.69±0.45,2.58±0.4,2.62±0.32) (P＜0.01),but those of PW6 and PW7 were unchanged (2.80±0.48 vs 2.90±0.46,2.59 ±0.36 vs 2.90±0.46,P＞0.05 ),indicating the absence of ocular dominance plasticity in the adult rats.In rats of PW7 with binocular form deprivation for 14 days,a significant decrease was observed in the C/I ratio in rats with next MD for 3 days,demonstrating that the visual cortex plasticity was reactivated after 14 days of binocular form deprivation in PW7 rats( 1.33±0.18 vs 2.70±0.45,P＜0.01 ).Conclusions PVEP can be recorded in Long-Evens rats.It is a major index for identifying the shift of ocular dominance in the Long-Evens rats.Binocular form deprivation can reflect the visual cortex plasticity in the adult rats.

Objective To evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold seeded with rat adipose tissue-derived stromal cells. Methods A dipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by type Ⅰ collagenase and then cultured in vitro. Type Ⅰ collagen was fully mixed with chitosan, freeze-dried and cross-linked with chondroitin sulfate, then freeze-dried again and sterilized by ethylene oxide. The pore diameter, water content, porosity of the scaffold were tested. The adipose tissue-derived stromal cells were digested, seeded into the plates, scaffold, and cen-trifuged into pellet, and then induced into cartilage. MTT detection for cell proliferation was done. After 3 weeks, the cell morphology, and cell proliferation and adhesion were observed, and chondrngenic differenti-ation was also analyzed. Results The pore diameter, water content, porosity tested for the scaffold showed an appropriate form. Cell proliferation showed faster in the scaffold and pellet culture system after 5 day, there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet cul-ture system; ceils on the scaffold proliferated densely showed by histological staining, but there was a scaf-fold structure residues in the inner layer. The finding of type Ⅱ immunohistochemistry stain showed that cells express strong positive for type Ⅱ collagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system; the specific mRNA for cartilage, type Ⅱ collagen, aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR, but type X collagen was expressed continu-ously in the plate culture system and expressed after 21 days in the pellet culture system, whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system. Conclusion The parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study. The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and maintain the phenotype of chondrocytes well; it is the optimal choice for cartilage tissue engineering in the future.

Objective To observe the efficacy and adverse reactions of ECF regimen (epirubicin combined with cisplatinum and fluorouracil) and DOF regimen (docetaxel combined with oxaliplation and fluorouracil ) in the treatment of advanced gastric cancer .Methods 68 cases of advanced gastric cancer were randomly divided into the ECF group (30 cases) and the DOF group (38 cases) .The ECF group was treated with epirubicin 50 mg/m2 on 1 d ,cisplatin 20 mg/m2 on 1-3 d and fluorouracil 500 mg/m2 on 1-5 d .The DOF group was treated with docetaxel 75 mg/m2 on 1 d ,oxaliplatin 130 mg/m2 and fluorouracil 500 mg/m2 on 1-5 d . 21 d were as a cycle of treatment .All cases received two cycles of chemotherapy at least .The efficacy and toxicity were evaluated according to the WHO standard .Results All cases were evaluable for the objective response .The overall response rate was 46 .67% in the ECF group and 42 .11% in the DOF group .The major adverse reactions in the two groups were marrow depression , gastrointestinal reaction ,alopecia and neurotoxicity ,etc .The occurrence rate of neurotoxicity in the DOX group was 39 .47% (26/38) ,which was higher than 13 .33(4/30) in the ECF group ,the occurrence rate of nausea and vomiting in the ECF group was 93 .33% (28/30) ,which was higher than 68 .24(26/38) in the DOF group ,the difference between the two groups was statistically significant (P<0 .05) .Conclusion Both of the two regimens have the similar effect for treating advanced gastric cancer and the ad‐verse reactions are tolerable .

BACKGROUND: The implanted cartilage calls can synthesize cartilage matrix as cartilage in cartilage tissue enginsedng, and the density of implanted cells is the key point.OBJECTIVE: To evaluate the effect of cell seeding concentration on the chondrogenic differentiation of the adipose dadved sromal cells (ADSCs).DESIGN, TIME AND SETTING: The in vitro cellular-scaffold observation was performed at the cytobiological laboratory of China Medical University from November 2007 to July 2008.MATERIALS: Six male SD rata with clean grade were supplied by the Experimental Animal Center of China Medical University.METHODS: Totally 5 g/L type ; collagen solution and 20 g/L chitosan was mixed in a mould with volume ratio of 7:3, after lyophillization, it was cut into pieces with 5 mm ～ 5 mm x 2 mm, followed by crosslinking with ethanol contained of 2% chondroitic acid at room temperature. After washing with double distilled water and freeze drying, the chitosan-collagen-chondroitin sulfate copolymar matrices scaffolds were harvested. ADSCs isolated from rat inguinal fat pads were digested with collagenase and trypsase. The prepared scaffolds were randomly divided into 3 groups, and the third passage cells with density of 2×10 9/L,2×10 109/L, and 2×10 11/L were seeded into chitosan-coflagen-chondroitin sulfate scaffolds, and cultured in chondrogenic medium for 3 weeks.MAIN OUTCOME MEASURES: The expression of cartilage specificity gene was detected by hematoxylin-eosin staining, type Ⅱ collagen immunohistochemical staining and RT-PCR.RESULTS: Hematoxylin-eosin staining showed that after 3 weeks of culture, the cell proliferated and differentiated well, especially in 2x101～/L group, more extrocelluer matrices were produced and cartilage lacuna-structure could be seen. The type Ⅱ collagen was positive expressed in each group, which showed a gradually increasing tendency with the cell seeding concentration increasing. RT-PCR showed that the expression of proteoglycen and type Ⅱ collagen mRNA were slowly increased. However the expression of Ⅹ collagen mRNA was decreased with increasing cell seeding concentration.CONCLUSION: The chitosan-collagen-chondroitin sulfate copolymer matrices can provide an appropdate environment for the generation of cartilage-like tissues and high call seeding concentration of 2×1010/L facilitate ADSCs to differentiate into cartilage.

Objective To investigate the distribution of acquired carbapenemases in carbapenemresistant strains of Klebsiella pneumoniae, and explore its role in epidemiology of nosocomial infection. Methods From November 2008 to March 2009, twenty clinical isolates of carbapenem-resistant Klebsiella pneumoniae were collected from children hospitalized in Wuhan children's hospital. MICs of antibiotics were tested by DNA of Klebsiella pneumoniae. Modified Hodge test was used to screen strains producing carbapenemases,combined imipenem(IPM)-EDTA , meropenem(MEM)- EDTA and ceftazidime(CAZ) - EDTA double-disk synergy test (DDST) were used to detect metallo-β-lactamase-producing. PCR amplification of the carbapenemase and integrase genes, and sequencing were performed. Plasmid conjugation transfer experiments and Southern hybridization were applied to study the mode of drug resistance transmission. Results Four types of Klebsiella pneumoniae were detected by PFGE, type A consisted of 5 strains, including 3 strains of type Al and 2 strains of type A2), type B (2 strains), type C (12strains) and type D (1 strain). Type A and C were the main drug resistant clones. Eight strains of Klebsiella pneumoniae carried both KPC-2 and IMP-4 genes, 10 strains carried IMP-4 gene, 2 strains carried KPC-2 gene. None of NDM-1 ,GIM, SPM, SIM, OXA-23, and VIM carbapenemase genes was detected in 20 isolates. All of 20 isolates carried lntl which were found to be located on bacterial chromosome by Southern blot. Conclusions KPC-2and IMP-4 genes are the major carbapenemase genes in Klebsiella pneumoniae isolated in Wuhan.Transmission of drug resistance is mainly through vertical transmission of type C resistant clone and horizontal transmission of Intl on bacteria chromosome.

Objective To establish a new effective method by using real-time polymerase chain reaction (PCR) to detect single nucleotide polymorphism (SNP) typing for rapid identifying apolipoprotein E alleles.Methods To determine alleles of human apolipoprotein E genetic polymorphism at Cys112Arg locus was detected by PCR melting curve analysis with fluorophore SYBR Green I. In order to increase the speciality of SNP assays, high fidelity Taq polymerase was used. The reliability of SNP typing was validated by comparison with the results of direct DNA sequencing.Results Each sample was determined by double tubes, and two melting curves were analysis. As compared the Tm value of samples with the Tm of standard substance, the apoE genotype of samples was determined. The apoE genotype of 30 samples were E3/3 (27/30) and E3/4 (3/30) respectively, which was accordant with the results of PCR-RFLP and DNA sequencing.Conclusion The presented allelic assay was specific, easy to operate and applicable for discrimination of apolipoprotein E genotyping of human blood.

Objective To explore the effects of chronic fluorosis on neurons in the cerebral cortex of rats,and to provide some morphological evidence of damage in the central nervous system induced by chronic fluorosis.Methods Male Wistar rats 40 days after birth were fed with high fluoride contented water(100 mg/L)for inducing chronic fluorosis.Immunocytochemistry and in situ hybridization were used to detect c-fos and Caspase 8 at cerebral cortical neurons respectively.Results c-fos positive cells rate and gray scale in the cerebral cortex of chronic fluorosis were 35.8%and 0.2756±0.0241,respectively,and that of control group were 32.1%and 0.2774±0.0331with statistical difference(χ2=0.305,t=0.826,P＞0.05).Caspase 8 positive cells rates of fluorosis group and control group were 18.7%and 14.1%,respectively,the difference being statistically significant(χ2=0.419,P＞0.05).The gray scale of fluorosis group and control group were 0.3874±0.0329 and 0.3884±0.0323,respectively,the difference being statistically significant(t=0.641,P＞0.05).Conclusion Chronic fluorosis had no significant influence on apoptosis of cerebral cortical neurons.

Objective To investigate the molecular epidemiology of CRAB isolated from children in wuhan. Methods Forty non-repetitive strains of CRAB were collected from hospitalized children of emergency department, neonatal medicine, cardiothoracic surgery, bone surgery, respiratory medicine and renal medicine in Wuhan children's hospital during December 2008 and May 2009. MIC values were PFGE; KPC, IMP, GIM, SPM, SIM, OXA-23, VIM genes and integrase gene were amplified by PCR and then sequenced to confirm the genotypes.; Plasmid conjugation experiment was used to study the transfer method of bacterial resistance and southern blot hybridization was used to target the resistance genes. Results Susceptible rates of 40 strains to gentamicin, tobramycin, amikacin, ciprofloxacin, levofloxacin, trimoxazole were 20%, 5%, 93%, 93%, 95%, and 23% respectively. Eleven types of clone were detected by PFGE,including 29 strains of type A clone, 2 strains of type B clone, and 1 strain for each type of C to K clone. Eleven isolates produced both IMP-4 and OXA-23 carbapemase. Twenty-six isolates only possessed OXA-23 carbapemase. Thirty-six strains carried class Ⅰ integron. The results of southern blot hybridization showed that Intl, IMP-4 and OXA-23 type were located on chromosome. Conclusions Type A clone of CRAB is the most common. OXA-23 and IMP-4 type are the major acquired carbapemases, especially the OXA-23 is the most common type. The horizontal transmission of OXA-23 and IMP-4 gene mediated by Int1 and the spread of type A resistant clone is the major way of the spread of carbapenem-resistant Acinetobacter baumannii in the region.

Objective To investigate the expression pattern of skeletal muscle specific miR-206,myogenesis related myoD which change with time in dcnervated muscle atrophy rats.Methods From June,2015 to January,2016,40 SPF sprague-dawley rats were equally classified into 5 groups randomly according to standard settled before,5 groups were separately defined as denervated 0d group,denervated 1d group,denervated 7d group,denervated 14d group,and denervated 28d group.Each group contained 8 rats.The rats atrophy models were established by cutting sciatic never on left side.According to the different denervated time,the gastrocnemii on both sides were obtained under anesthesia,respectively.The wet weight ratio of two compared gastrocnemii were measured,and the gastrocnemii transection was observed by HE stain,measured the expression of myoD protein by western blot,obtained the expression of miR-206,myoD mRNA by qPCR.Results According to our study on rats denervated atrophy models,the wet ratio of compared gastrocnemius would decrease rapidly,by HE stain,decease of cross sectional area in muscle fiber was observed as well as degeneration.Collagen fibers hyperplasia appeared and increased with time change.Wet ratio and transaction aera ratio of group Od,1d,7d,14d,28d were 0.99±0.04,0.92±0.07,0.68±0.11,0.39±0.06,0.27±0.07 and 0.99±0.02,0.96±0.04,0.51±0.09,0.34±0.08,0.23±０.03 respectively,difference between experimental groups and control group were statistically significant (P＜ 0.05),the differences between each experimental groups were also statistically significant (P＜ 0.05).After qPCR test of miR-206,myoD mRNA expression,it was found that their expression patterns were similar,miR-206,myoD mRNA increased at first and would reach the expression peak at the 7 th day,after that their contents decreased but still higher at the 14th day when compared with that at the 1 st day.Their expression of group 0d,1 d,7d,14d,28d were 0.24±0.06,0.34±0.04,0.68±0.04,0.49± 0.07,0.25±0.03 and 0.41 ±0.06,0.49±0.09,0.93±0.06,0.66±0.03,0.39±0.04,respectively.All experimental groups were statistically significant different when compared with 0d group except 1d group (P＜ 0.05),the differences between each experimental groups were also statistically significant(P＜ 0.05).The protein expression of myoD was also measured by western blot test,which showed nearly the same expression pattern as the mRNA expression pattern.After injury,the protein expression increased and reached the expression peak at the 7th day.The relative expression of myoD of group 0d,1d,7d,14d,28d measured by grey ratio were 1.03±0.05,1.06±0.06,1.42±0.10,0.66±0.13,0.24±0.07,respectively.The difference between experimental groups and control group were statistically significant (P ＜ 0.05),the differences between each experimental groups were statistically significant (P ＜ 0.05) as well.Conclusion The degree of muscle denervation atrophy was related to the denervated duration in rats.The expression regulation of miR-206 and myoD in gastrocnemius was similar during the muscle denervation atrophy,which suggesting having internal relationship between miR-206 and myoD.

The non-specific accumulation and release of drugs are the main factors affecting the therapeutic effect as well as causing toxic side effects of chemotherapeutic drugs. Nowadays, the application of nanotechnology and responsive drug release is an important strategy to improve the tumor-specific accumulation of drugs and reduce their side effects. In this study, an α-enolase targeted peptide (ETP)-modified polyethylene glycol poly-lysine block copolymer loaded with oxaliplatin prodrug was synthesized first, and then, polymer-coating Fe₃O₄ nanoparticles were prepared by phase transfer dialysis method to improve the blood circulation stability and tumor targeting of oxaliplatin. At the same time, the physicochemical properties, reductant-responsive drug release, cellular uptake, tumor targeting and other biological functions of ETP modified oxaliplatin-loaded Fe₃O₄ nanoparticles were studied in vitro and in vivo. First, the results of reductant-triggered drug release study showed that the drug-loaded nanoparticles could achieve rapid release of more than 80% of the prototype oxaliplatin within 3 h under the reduction conditions simulating the tumor cytoplasmic microenvironment. Secondly, the results of flow cytometry showed that the modification of ETP could increase the ratio of cellular uptake of drug-loaded nanoparticles in tumor cells, and the way that drug-loaded nanoparticles endocytosed by tumor cells were mainly through the energy-dependent and receptor protein and fossin-mediated endocytosis pathway. The animal procedures were approved by the Institutional Animal Care and Use Committee of School of Pharmacy of Fudan University. Moreover, the results of pharmacokinetic experiment showed that the area under the curve (AUC_0-∞) of oxaliplatin could be significantly increased by nano-formulation which was about 5 times than that of free oxaliplatin. Besides, the pharmacokinetic results also showed that the drug-loaded Fe₃O₄ nanoparticles constructed by covalent linkage and chelation had good overall stability in vivo. Finally, the in vivo imaging results showed that ETP modification could increase tumor accumulation of drug-loaded nanoparticles, which would be conducive to the efficacy of oxaliplatin in tumor lesions. In summary, the oxaliplatin-loaded Fe₃O₄ nanoparticles with the capability of reductant-responsive drug release have good drug release characteristics, blood circulation stability and tumor targeting ability, and have the potential to improve the anti-tumor therapeutic effect of oxaliplatin.