Objective: To clone human interleukin 10(hIL-10) gene full-length cDNA sequence and to construct its eukaryotic expression vector in Chinese hamster ovary(CHO) cells. Methods: cDNA fragment encoding hIL-10 gene was amplified from human peripheral normal white blood cells by RT-PCR and confirmed by DNA sequencing.The ds-DNA was inserted into pcDNA3 vector.This recombinant expressing vector was transfected into CHO cell.The IL-10 molecules expressed were detected by ELISA and the role of inhibition of IL-10 was determined with MTT. Results: The cDNA encoding hIL-10 was cloned by RT-PCR and inserted into T-easy vector and sequence analysis verified that the cloned fragment was hIL-10 cDNA.The IL-10 was expressed in the CHO and it blocked the lymphocyte transformation.Conclusion: The eukaryotic expression plasmid was constructed successfully,which will contribute to further studies on the role of hIL-10 in autoimmunity,transplantation immunity and inflammatory disease.

Objective To investigate the distribution of single nucleotide polymorphisms(SNPs) on retinol binding protein 4(RBP4) genes and forkhead box O1 (FOXO1) gene, and their relationships with the occurrence of type Ⅱ diabetes mellitus (T2DM) in Chinese Han population. Methods Totally ten SNPs on RBP4 and FOXO1 were determined in 384 T2DM patients and 384 normal controls by TaqMan probe genotyping and agarose gel electrophoresis methods. And their serum level of fasting blood glucose (FBG), total cholesterol (TC) and trigly- ceride (TG) were also estimated. Results For RBP4, there was no significance for various genetypes and alleles including - 803 G > A, + 5169 C > T, and + 6969 G > C between two groups (P > 0.05). Each genotype had no relationships with T2DM (using adjusted logistic regression models). No haplotype was associated with T2DM. For FOXO1, among seven SNPs typed, significant variation was found in the frequency distribution of rs7324943 G/T in the two groups(χ~2=4.02, P = 0.044), and further stratification analysis showed that in subjects of aged 40 and non-hypertension, there was a higher risk of T2DM in GT heterozygous carriers than in GG homozygous carriers (OR = 1.47, 1.80), T allele carriers showed higher risk than non-T carriers (OR = 1.42,1.79). For rs17592236 C/T, though no significant frequency variation was found between two groups (χ~2 = 0.39, P = 0.401), but in subjects of aged ≤ 40, stratification analysis showed dramatically increased risk of T2DM in CT and TT carriers than in CC carriers (OR = 6.33,10.15), T allele carriers showed 7. 11-fold higher risk than non-T carriers. A haplotype CT related to T2DM susceptibility was also found, which could decrease the risk of its carriers by 28%. Conclusions For BBP4, the polymorphisms of - 803 G > A, + 5169 C > T, and + 6969 G > C had no relationships with T2DM in Chinese Han population. For FOXO1, the polymorphism of rs7324943 G/T,rs17592236 C/T and a haplotype CT were found related to the susceptibility of T2DM in Chinese Han population. Yet further studies are necessary to explain the impact of these polymorphisms on the disease occurrence.

This is the first report on gene therapy of mice melanoma by HSV - tk/ACV system. The sensitivity to ACV of genetically modified B16 cells (B16LNTK) was much higher than that of the parental cells. The sensitivity to ACV of B16 cells was increased when they were co - cultured with B16LNTK cells with various ratios. which showed the exist of the by stander effects. The tumor volume of B16LNTK (0.25cm3) is 94% less than that of B16 in C57BL/6 mice after 20-days ACV treatment. (P

Background and purpose:Glioblastoma is one of the most common intracranial tumors, the morbidity and mortality are both high, and the molecular biological mechanism of the disease is still unclear. In this study, we detected the gene expression of tyrosine kinase receptor (TKR) pathway in primary glioblastoma(GBM) with low-density array, furthermore we analyzed the significance of the gene expression change. Methods:We detected 26 genes of RTK pathway in 10 primary GBM tissues and 9 normal brain tissues (gained from the decompression operation of brain trauma), and analyzed the different expressions of these two kinds of tissues by statistic method.Results:The Ct values of MAP2K1 and MAP2K4 in normal brain tissues were 1.6?1.7 and 2.2?2.1, the Ct values of MAP2K1 and MAP2K4 in primary GBM tissues were 3.9?1.5 and 5.0?2.0, and the Ct different values between normal brain tissues and primary GBM tissues of both genes were -2.3 and -2.8(P

To describe the fractal feature of CGR (Chaos-game representation) graph of genomes sequences, a multifractal theory is presented in the analysis. By studying the effect of three probability sets on the scale invariance range, the probability set with the best scale invariance is chosen, and then the smooth general dimension spectrum and multifractal spectrum are calculated. The experimental result shows that the probability set composed of the relative probability has the best scale-invariance performance. The scale invariance has three different variance regions, which indicate that genomes sequence segments with different lengths have different distribution rules. It is concluded that the multifractal method is effective for describing the fractal feature of CGR graph of genomes sequences.
Algorithms ; Base Sequence ; Computer Simulation ; Game Theory ; Genome ; Humans ; Mathematical Computing ; Sequence Analysis, DNA ; statistics & numerical data

Biosequence analysis is the primary research field of bioinformatics. In this field, useful information can be extracted by comparison analysis methods. Among them, sequence alignment is the most common comparison method. However the sequence comparison by alignment, which assumes conservation of contiguity between homologous segments, is at odds with genetic recombination. Especially for the multisequence alignment, there exists the difficulty in the complexity of calculation. Therefore, alignment-free sequence comparison methods are required. In this paper, two main categories of alignment-free sequence comparison methods are reviewed. The first one is based on the word (oligomer) frequency and its distribution. The sequences are compared using the distances defined in a Cartesian space by the frequency vectors. In the second category, sequences are compared using Kolmogorov complexity and chaos theory.
Algorithms ; Computational Biology ; Sequence Alignment ; Sequence Analysis ; methods

Objective:To investigate the possible association between Thr241Met polymorphism in the DNA repair gene X-ray repair cross-complementing group 3 (XRCC3) with genetic susceptibility to glioma in a Chinese Han population living in Shanghai and the surrounding provinces in east China. Methods: Genotyping by a TaqMan assay was performed in 771 brain glioma patients living in Shanghai and the surrounding provinces (Jiangsu.Zhejiang, Anhui.etc. )and in 752 control participants matched in age and gender. The genotyping results of TaqMan assay and the association between Thr241Met polymorphism in the DNA repair gene XRCC3 with genetic susceptibility to glioma were statistically analyzed. Results: Genotypes of 1 468 subjects (760 with brain glioma and 708 were cancer-free control) were successfully performed by TaqMan assay, with the successful rate being 96.4%. Statistical analysis result showed that gene(C/T) and genotype(C/CT/CT/T) frequencies of XRCC3 were not significantly different between the glioma and cancer-free groups. Compared with the CC genotype, the variant TC(P = 0. 909; adjusted by age and gender OR = 0. 981; 95%CI = 0. 701-1. 371) or TT(P=0. 642; adjusted by age and gender OR = 0. 7; 95%CI = 0. 156-3. 146) genotypes of XRCC3 Thr241Met were associated with a non-statistically significant increase of glioma risk. Conclusion: The variant TC or TT genotypes of XRCC3 Thr241Met may not be risk factors for brain glioma in Chinese Han population living in Shanghai and the surrounding provinces in east China.

Objective To establish the replicated-deficient recombinant adenovirus-mediated thymidine kinase/acyclovir (Adtk/ACV) system and to evaluate its suicide effect on rat C6 brain gliomas in vitro and in vivo.Methods The plasmid pAdtk and pJM17 were co-infected into 293 cells (adenovector packaging cells) and the results were identified by polymerase chain reaction (PCR) assay. After the glioma C6 cells were transduced by Adtk at different multiplicity of infection (MOI) and exposed to different concentrations of ACV or gancyclovir (GCV), the cell survival curves were studied, and the cell surface was observed with scanning electronic microscopy (SEM). C6 gliomas in vivo at different inoculation days were injected with Adtk intratumorally and ACV intraperitoneally daily, and the survival duration and histologic changes of the rats were observed.Results The infectious Adtk virions had a suicide effect which was enhanced with the increase in MOIs of Adtk and ACV doses along with bystander effect. Under scanning electronic microscope, special pathologic changes were observed. ACV had a similar effect as GCV but a higher dose was used. The survival duration in day 3, day 6 and day 8 groups exceeded 90 days, and the rats in day 10 group survived 28.5±4.6 days, but the survival duration in untreated C6 group and AdLacZ/ACV (adenovirus-mediated LacZ/ACV) treated group were 16.8±3.1 and 14.0±2.2 days respectively. Conclusion Adtk/ACV system can effectively kill the rat brain gliomas in vitro and in vivo.

Objective To establish the polymorphism of DXS102 locus from Xq26.3-27.1 in Chinese population for the gene diagnosis in Hemophilia B family.Methods DNA was extracted from blood samples obtained from Shanghai unrelated volunteer donors with phenol-chloroform method. A total of 23 × chromosomes (154 from females, 80 from males) were studied. A hemophilia B family in which a hemophilia B patient has received gene therapy was analyzed. The polymorphism of DXS102 locus in Chinese population was determined with amplified fragment length polymorphisms assay (Amp-FLP), denaturing polyacrylamide gel electrophoresis, silver stain detection. Short tandem repeats (STRs) linkage analysis was used to conduct gene diagnosis in hemophilia B family.Results Eight alleles were found at DXS102 locus, of which two alleles were first reported. The repeated number of AC dinucleotide ranges from 13 to 21. And the values of the observed heterozygosity, calculated heterozygosity and polymorphism information content(PIC) were 0.87, 0.80, 0.80 respectively. It was also found that the difference of the allele frequencies of DXS102 in Chinese and European populations was significant. By using the linkage analysis of the DXS102 locus, a family with a hemophilia B patient receiving gene therapy in 1994 was analyzed and meanwhile a carrier in that family was then detected.Conclusions The polymorphism of DXS102 locus reveals significant difference between Chinese and European populations. DXS102 locus can be used as a promising marker for gene diagnosis in hemophilia B family.

Retroviral vectors are wildly used as vehicles for gene transfer into hematopoietic cells based on its potency for efficient gene delivery and integration of transgene in host genome. The development of better transduction protocols depends on gene markers that allow a rapid detection and effective selection of genetically transduced cells. In this study, the enhanced green fluorescent protein (EGFP), a gene that is optimized for detection and expression in mammalian cells, was firstly amplified and cloned by high-fidelity PCR. The vector LGSN carrying EGFP gene was then constructed and the retroviral producer cell lines that yield high titers of LGSN vector in supernatants were developed by liposome-mediated transfection in combination with cross infection. Both GP + envAm12 murine fibroblasts and K562 leukemic cells transduced with EGFP virus demonstrated a stable green fluorescence signal readily detectable by flow cytometry or fluorescence microscopy in up to 97% and 86% of examined cells, respectively. The integration of LGSN provirus in transduced cells was confirmed by PCR analysis. These results indicate EGFP is a suitable reporter molecule for gene transfer and expression in hematopoietic cells. Therefore, the bright and long-term expression of EGFP in living cells will advance the study of gene therapy in vitro and in vivo, particularly for human applications.