Recently,zebrafish,as a new model species,has been used widely in the study of developmental mechanism of the embryo,a model of human disease and the drug screening.Zebrafish has been applied widely in the study of the drug for tumor antiangiogenesis with the development of the advanced technology of the mutagenesis and the confocal microscopy using for observation.Zebrafish applied in the screening of tumor antiangiogenesis drug and the mechanism of tumor angiogenesis are summered.

Multilocus sequence typing (MLST) is a molecular genotyping method based on nucleotide sequencing. The procedure of this method characterizes isolates of bacterial species using the DNA sequencing of multiple housekeeping genes(usually seven). For each housekeeping gene, the different sequences present within a bacterial species are assigned as distinct alleles.For each isolate, the alleles at each of the loci define the allelic profile or sequence type (ST). MLST has the advantages of being robust (based on genetic data) and electronically portable to generate data that allow rapid and global comparisons between different laboratories. In this paper, the principle, method, data analysis, application, advantages and flaws of MLST are introduced.

Objective To analyze the associations of the interleukin-10 (IL-10) promoter-1082G/A and-819C/T single nucleotide polymorphism (SNP) and serum level of IL-10 with intracranial aneurysm (IAs). Methods The polymerase chain reaction (PCR) and DNA direct sequencing methods were used to detect IL-10 gene promoter district two SNP site,-1082G/A and-819C/T genotype frequency and allele frequency in 206 patients with IAs and 187 controls. Chi-square test was used to analyze differences between two groups. The serum level of IL-10 was analyzed by ELISA, and t-test was used to analyze significant differences between two groups. Results There were significant differences in genotypes of GG and GA+AA, as well as the alleles G and A, in-1082G/A locus between IAs group and control group (P<0.01). There were higher frequencies of genotype GA+AA and the allele A in IAs group than those in control group (P<0.01). There was higher risk of suffering IAs in patients with genotype GA+AA (OR=4.137, 95%CI:2.476-6.914) and the allele A (OR=3.368, 95%CI:2.476-4.583). There were higher frequencies of the genotype CT+TT and the allele T in-819C/T locus in IAs group than those of control group (P<0.01). There was higher risk of suffering IAs in patients with genotype CT+TT (OR=3.393, 95%CI:1.952-5.900) and the allele T (OR=3.764, 95%CI:2.730-5.192). The serum level of IL-10 was significantly lower in IAs group than that of control group (P<0.01). Conclusion The IL-10 promoter SNP influences the expression of IL-10. IL-10 promoter-1082G/A and-819C/T polymorphisms are correlated with the formation of IAs.

Highly Active Anti-Retroviral Therapy (HAART) has effectively inhibited the prevalence of HIV-1 and reduced the death rate caused by AIDS. In recent years,the emergence of resistance-conferring RT gene mutations in HIV-1 strains has become the major reason for HAART failure. The detection of drug resistance is important for the HAART regimen choice and novel drug development. A novel assay for the detection of HIV-1 RT drug resistance mutations was developed. HIV-1 drug resistance and wild strains in B subtypes were investigated using Two-Step Mutagenically-Separated PCR (MS-PCR),and point mutations including M41L,K70R,K103N,Y181C,T215F were detected. A longer mutant type primer was designed,using microplates hybridization and ELISA technique to detect several point mutations within a mixed mutant-wild type population. The results indicate that the Two-Step MS-PCR is as sensitive and specific as that in the traditional MS-PCR and MS-PCR combined with ELISA can give a good P/N quotient with better sensitivity,low cost,relatively less time consumption and high-throughput screening. It will be used in clinic usage for the detection of HIV-1 drug resistance mutations as well as other point mutations.

HIV-1 envelope glycoprotein gp41,which is a hopeful target for HIV-1 fusion inhibitors,plays a critical role in the fusion of viral and cellular membranes.In order to build up the screening assay of HIV-1 fusion inhibitors targeting gp41,HIV-1 gp41 5-helix and 6-helix were expressed in prokaryotic cells.Gp41 5-helix and 6-helix recombined plasmids were constructed by using PCR,enzyme digestion and ligation taking the clade B HIV-1 genome as a template.The plasmid was transferred into E.coli BL21(DE3)and then induced by IPTG.The expressed protein was purified by affinity chromatography after denaturation and renaturation.The SDS-PAGE analysis was used during expression and purification.Native-PAGE was used to identify the interaction between gp41 5-helix and T-20.The result will be helpful to build up the screening assay of HIV-1 fusion inhibitors targeting gp41.

Objective To evaluate the clinical feasibility of localization CT enhanced image replacing plain CT scan image for target delineation and dose calculation.Methods Forty cases of NPC were collected and divided into two groups with different concentrations of contrast agents.The contours of planning target volume (PTV) and organs at risk (OARs) of each case were delineated in the plain scan image,and the contours of PTV and OARs were copied to the enhanced image.Two plans based on the plain scan image and the enhanced image were designed in the planning system of Eclipse.The dose distribution and OARs and MU were compared between the groups.Results No statistical differences were found in the dosimetry of PTV,OARs and MU (P＞0.05).Conclusion The image intensifier has little effect on the dose calculation of Eclipse for NPC.In the radiotherapy for NPC,the localization CT enhanced image can be used to replace the plain CT scan image for target delineation and dose calculation.

Objective To clone,express and characterize a tegument protein gene of Schistosoma japonicum(Sj29),and investigate the immune protection of the recombinant protein against S.japonicum in mice.Methods The gene coding for Sj29 protein was amplified by PCR,and the sequence was analyzed by bioinformatics tools.Partial fragment of Sj29 gene was subcloned into the prokaryotic expression vector pET28c(+).The recombinant plasmid was transformed into E.coli BL21(DE3) and induced the recombinant with IPTG.The recombinant protein(rSj29) was purified by His-binding-resin affinity chromatography and characterized by Western blotting.Three groups each with 10 BALB/c mice were immunized subcutaneously three times(two weeks interval) respectively with 100 ?l recombinant rSj29(0.1 mg/ml),adjuvant or PBS.At the 15th day after the final inoculation,each mouse was challenged by 40 ?2 cercariae of S.japonicum.At the 53th day after infection,the mice were sacrificed to obtain the number of adult worms,number of eggs in liver and feces.Serum samples were collected at pre-immunization and certain time after immuniza-tion,and were analyzed for IgG by ELISA.The localization of rSj29 in worms of different developmental stages was demonstrated by immunofluorescent technique.mRNA expression level of Sj29 gene in worms of different developmental stages and three groups after infection was detected by quantitative real-time PCR.Results A 576 bp Sj29 gene fragment was obtained.The recombinant protein rSj29 with Mr 22 900 was expressed in the form of inclusion body.The recombinant rSj29 can be recognized by sera of mice immunized with rSj29 and sera of infected mice.The number of adult worms(15.4?5.9),number of hepatic eggs(40 143.3?2 995.9) and number of fecal eggs(3 803.9?110.9) in re-combinant protein group were significantly higher than those of PBS control group(20?3.4,49 318.1?6 648.3,5 238.1? 303.5,respectively)(P

The pol gene of HIV-1 encodes mainly three enzymes: reverse transcriptase (RT), protease (PR) and integrase (IN). Currently, FDA approved drugs targeting RT and PR are available and administered in various combinations, while no anti-IN drug was approved. HIV-1 integrase is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multi-drug therapy of AIDS. The 288 amino acids of IN (32kDa) recognizes specific sequences in the long terminal repeats (LTRs) of the retroviral DNA. The IN protein catalyzes the 3′-processing step and the 5′-strand transfer step reaction in vivo, which was called integration and this reaction could be analysed by ELISA Assay in vitro. It has been reported that F185K and C280S mutations of HIV-1 integrase would improve the enzyme solubility, and the catalytical activity of the enzyme was the same as that of the wild-type enzyme in vitro. In order to build the platform of screening inhibitor against integrase of HIV-1 virus, the IN enzyme was expressed and the function of integrase protein was assayed. The cDNA of clade B HIV-1 genome was used as a template, overlapped PCR was used to construct site mutagenesis of F185K/C280S and NdeI/Xho I restriction sites were brought in. The PCR product was cloned into the prokaryotic vector pET-28a(+) to form a recombined plasmid, transferred into the host cell E.coli(BL21 DE3). The recombined clones were identified by PCR and Nde I/Xho I digestion .The positive plasmid was sequenced, and the successfully recombined plasmid in the host cell was induced by IPTG. The expressed IN protein was puriied sy the Co+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombined IN protein. The recombinant protein was soluble, and expressed highly and stably in E.coli. The molecular weight of the expression product was identical to the expectation. The IN protein was proved to be functional in 3′ processing and 5′strand transfer by ELISA. It will be helpful to build the platform of screening inhibitors against HIV-1 integrase.

In order to explore if mature neurons derived from neural stem cells have the potentiality to divide, we utilized the chemical digestion method to disperse the adult rat brain tissue into single cells, and culture them in serum-free medium. After being cultured for about eight days in vitro, the neural stem cells were induced to differentiate into neurons. The neurons were further induced to divide. Utilizing the method of serial photograph and NF-160 immunocytochemistry, the processes of division of some neurons were recorded. At the same time, PCNA+NF-160 (or Chat, GABA, GAD) double label were used to investigate if the dividing-neurons were mature ones. After the neural stem cells were induced to differentiate in vitro for eight days, they possessed the shape and character of mature neurons. The differentiated neuron had a big nucleus and one or two distinct nucleolus in the nuclear. Within the perikaryon,there were a large amount of dense and Nissl body-like structure. Several long processes emerged from various locations of the cell body. Then, EGF and bFGF were added into the medium to induce division. After two days of induced-division, neuron-like cells were observed to divide; moreover, the number of neuron-like cells in the region increased continually. Immunocytochemistry demonstrated these cells were NF-160-positive. Serial photographs of dividing-process of neuron-like cells were obtained and their daughter cells were also NF-160-positive. After PCNA+NF-160 (or Chat, GABA, GAD) double label, some cells showed brown cell plasma and black nucleus. The above-mentioned results indicate that neurons, which were previously thought to be end-differentiated, can be re-called into cell cycle under appropriate conditions. Mature neurons still have the potential to divide, proliferate and self-renew.
Animals ; Brain ; cytology ; Cell Differentiation ; Cell Division ; Cell Separation ; Cells, Cultured ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factor 2 ; pharmacology ; Neurons ; cytology ; Photography ; methods ; Proliferating Cell Nuclear Antigen ; pharmacology ; Rats ; Rats, Wistar ; Stem Cells ; cytology

OBJECTIVETo discuss the impact of number of retrieved lymph nodes and lymph node ratio(LNR) on the prognosis in patients with stage II and III colorectal cancer.

METHODSClinicopathological data of 507 patients with stage II and III colorectal cancer were analyzed retrospectively. Follow-up was available in all the patients.

RESULTSThe total number of retrieved lymph nodes was 5801, of which 1122 had metastasis. There was a positive correlation between metastatic lymph nodes and retrieved lymph nodes(r=0.171, P<0.01). In stage II colorectal cancer there was a significant difference in 5-year survival rate between patients with more than 12 lymph nodes retrieved and those with less than 12 lymph nodes retrieved(P<0.01). LNR also affected the 5-year survival rate of patients with stage II and III colorectal cancer(P<0.05). In patients with similar LNR, the 5-year survival rate differed significantly among different regions of lymph node metastasis(P<0.05). LNR influenced the prognosis independent of the number of lymph nodes retrieved.

CONCLUSIONSThe number of retrieved lymph nodes is a prognostic factor for stage II and III colorectal cancer. More than 12 lymph nodes should be retrieved for better staging and prognosis. LNR is also a prognostic factor in stage II and III colorectal cancer. Regions of lymph nodes metastasis should be considered when evaluating the prognosis of patients using LNR.

Colorectal Neoplasms ; diagnosis ; pathology ; surgery ; Female ; Follow-Up Studies ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; diagnosis ; pathology ; Male ; Neoplasm Staging ; Prognosis ; Retrospective Studies