Objective To investigate the effect of aldehyde-free fixation solution and aldehyde fixation solution on DNA and cytoplasm of nucleus.Methods The DNA quantification (IOD),discrete coefficient (CV),DNA index (DI) and nucleus area (area) were measured by DNA quantification system (ICM) in 8 different fixation solution;combined with cytological staining,the effects of different fixative solution on nuclear DNA and cytoplasmic staining were analyzed.Results Aldehyde fixation solution was better than aldehyde free fixation solution,the formaldehyde in fixtion solution has important influence on Feulgen-eosin staining.Conclusion Aldehyde fixation solution combined with cytological staining can obtained good dyeing effect,which provides the basis for the subsequent multi-technique joint diagnosis.

Objective To study the differentially expressed microRNA (miRNA) between pancreatic ductal adenocarcinoma (PDAC) and para-cancerous tissues,and determine related target genes.Methods Nine fresh PDAC tumor tissues and 3 adjacent normal pancreatic tissues were collected,then Agilent miRNAmicroarray with 713 miRNA loci was used to identify the differentially expressed miRNA.Real-time PCR method was applied to verify the up-regulated miRNA.TargetScan 5.1 and miRandaV5 software were used to analyze the related target genes.Results miRNA microarray identified 11 PDAC related miRNAs,among them,the expressions of miR-194*,miR-192*,miR-602,miR-194 were up-regulated,while the expressions of miR-139-3p,miR-513a-5p,miR-630,miR-30c-1 *,miR-887,miR-508-5p,miR-516a-5p were down-regulated.The expressions of miR-192,miR-194 and their homolog were verified in 31 PDAC tumor tissues.After software analysis,it was found that target genes of miR-192 were ZEB2,CXCL-2,EEF1A1,ERCC3,and target genes of miR-192 * were DCC,SMAD4,FAS,and target genes of miR-194 included DACHI,IGSF11,PTPN2,RBBP4,while target genes of miR-194 * included CD40LG,CIDEB,FHL1.Conclusions Eleven differentially expressed miRNAs are present in PDC,and they may be involved in the occurrence and development of PDC.

Objective To study the aberrant DNA methylation of H19 and MEST in infertile male sperm.Methods Routine semen analysis and morphological analysis were performed on 15 control individuals and 33 oligoastheno-teratozoospermias.Sperms were purified by density gradient centrifugation and DNA was extracted and treated with bisulfite.After PCR,the purified PCR products were cloned into pMD (R)18-T vector and proceed to chemical transformation,restriction enzyme reaction.For each sample,positive clones were selected for sequencing analysis and DNA methylation analysis.Results Five control individuals had high degree of H19 and methylation rate was 100.0％,while the methylation rate of 93.0％ in 10 oligoastheno-teratozoospermias was significantly lower.Significant difference was found in methylation rate between the control individuals and oligoastheno-teratozoospermias (P ＜ 0.01).As to MEST,10 control individuals had a low degree of MEST and the methylation rate was 1.6％,while the methylation rate in 23 oligoastheno-teratozoospermias was 4.8％.No significant difference was found in the methylation rate between control individuals and oligoastheno-teratozoospermias (P ＞ 0.05).Conclusion Male infertility is associated with aberrant methylation of H19 differentially methylated region,and it may be a biomarker of human spermatogenesis defect.

Objective To study the prevalence and genotype of human parvovirus B19 virus among blood products and plasma pools in China. Methods B19 DNA derived from 16 lots of factor Ⅷ concentrate produced by 4 manufactures and 10 lots of plasma pools were detected by nested PCR. Phylogenetic comparison of the partial B19 sequences obtained from positive samples were performed by direct sequencing. Results Twelve of sixteen lots of factor Ⅷ concentrate and all of ten lots of plasma pools were contaminated by B19 DNA. By comparing the partial B19 sequences,all the isolated viruses were genotype Ⅰ and their nucleotides were high conserved with homology of 98. 3%-100%. Conclusion B19 genotype Ⅰ DNA has been detected in high prevalence in factor Ⅷ concentrate and plasma pools. The genetic diversities were shown to be very low.

Objective To study the influence of site-directed deglycosylation of the HIV-1 envelope (Env) on its immunogenicity and assembly of functional pseudovirus. Methods Site-directed deglycosylation were performed using cycling mutagenesis and selection of mutants with DpnⅠ. Single-cycle infection assay was employed to analyze the effect of the mutations on the ability of functional pseudovirus assembly. The influence of deglycosylations on the immunodeficiency of Env was evaluated using pseudovirusbased neutralization assay and ELISPOT assay. Results Mutant N197Q induced higher neutralization activities for both pseudoviruses, but lower Env-specific T-cell response. And N197Q rendered the Env to lose the ability of functional pseudovirus assembly. Mutant G2 induced higher neutralization activities for pseudovirus 74-2 but lower for pseudovirus Wt, and had almost no influence on Env-specific T-cell response and functional pseudovirus forming. Conclusion The site-directed deglycosylation of the HIV-1 Env affects the pseudovirus forming and its immunogenicity.

The ELISPOT assay is increasingly used for assessing cellular immune responses in clinical trials of HIV-1 or cancer vaccines. However, to date, data from clinical trials do not consistently show that immune responses are correlated with clinical endpoints. This is due in part to the lack of assay standardization and validation across laboratories and therefore, a quality control panel is required to establish competency and comparability amongst different laboratories. In this study peripheral blood mononuclear cells (PBMCs) from healthy individuals were screened and frozen in liquid nitrogen. The recovery and viability of the PBMCs and the frequencies of interferon (IFN)-γ-secreting cells after CEF peptide pool stimulation were detected after various intervals in seven different laboratories. The recovery and viability did not differ significantly after different intervals. Although the frequencies of IFN (interferon)-γ-secreting cells among thawed PBMCs (peripheral blood mononuclear cells) fluctuated after CEF peptide pool stimulation at different intervals, they were not significantly decreased compared with those among fresh PBMCs. However, the viabilities, recoveries and frequencies of IFN-γ-secreting cells differed significantly among the seven laboratories. Our results indicate that cryopreserved PBMCs could be used as a quality control panel for ELISPOT. However, the procedures for ELISPOT need to be standardized amongst different laboratories.

Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around Nab epitopes and deletions of variable regions in env.The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γELISPOT.Overall,five mutants(dWt,M2,M5-2,M5-1 and dM7)induced highed neutralization activities for both pseudoviruses than plasmid Wt,while only two of the mutants(dWt and M5-2)showed significant differences(P<0.05).Two mutants(M2 and dM2)induced more Env-specific T cells than plasmid Wt.Statistically however,significance was only reached for mutant M2.Thus,properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.

Data were collected through questionnaire from 505 women enrolled during early pregnancy.Based on soy intake frequency,the subjects were divided into three groups:frequent (three or more times per week),conventional ( more than twice per month but less than three times per week),and occasional ( two or fewer times per month).Serum TSH and FT4 were measured by chemiluminescence immunoassay.The urinary concentrations of two primary isoflavones (daidzein and genistein) and creatinine were further assessed in 95 subjects from the three groups.The percentages of frequent,conventional,and occasional consumers were 18.6％,62.6％,and 18.8％,respectively.No difference was found in age,medical records,family history of thyroid diseases,serum FT4,TSH,and prevalence of thyroid dysfunctions among three groups. Both urinary daidzein and genistein levels were significantly higher in the frequent consumers compared with the other two groups.No correlations were found between urinary isoflavone levels and serum FT4 or TSH.These findings suggest that dietary soy consumption during early pregnancy seems not to be associated with the development of thyroid dysfunction.

Objective To verify the criteria proposed by National Academy of Clinical Biochemistry(NACB)guidelines in investigating the factors that affect serum TSH determination, and to determine the reference range of serum TSH in iodine-sufficient areas of China. Methods In 2007, 5 348 inhabitants were enrolled from 3 iodine-sufficient areas of Liaoning Province, and were asked to fulfill the questionnaire. Serum TSH, thyroid peroxidase antibody(TPOAb), and thyroglobulin antibody(TgAb)were determined, and thyroid ultrasonography was carried out. Results The distribution of TSH levels was skewed in healthy people and closely fit the curve of Gaussian distribution after logarithmic transformation. The levels of TSH in subjects of 12-19 years of age were significantly higher than those of other age groups(P＜0.01), and no significant difference was found among the latter groups. TSH level in females [(1.68±1.90)mIU/L] was higher than in males[(1.45±1.92)mIU/L, P＜0.01]. The reference range of TSH was 0.43-4.74 mIU/L in males, and 0.48-5.39 mIU/L in females. Family history of thyroid disease, abnormal thyroid ultrasonography, and positive thyroid antibodies were the factors that influenced TSH level. Conclusion The reference range of serum TSH in iodine-sufficient areas of China is established.

Objective To investigate the relationship between serum thyrotrophin(TSH)and dyslipidemia in subclinical hypothyroid and euthyroid subjects. Methods An epidemiological study on diabetes and thyroid diseases was performed in Dadong community, Shenyang city, in 2007. 110 subjects with subclinical hypothyroidism(SCH)and 1 240 euthyroid subjects were enrolled in the study. Neither history of thyroid diseases nor administration of thyroid-related and lipid-regulating medicines were reported in these subjects. The levels of serum thyroid hormones, lipids, fasting plasma glucose(FPG), and insulin were determined. Results (1)Patients with SCH had significantly lower HDL-C levels than those who were euthyroid.(2)According to the guideline of treatment of adult dyslipidemia in China, the lipid profiles were each categorized. Mean TSH levels were higher in subjects in the dyslipidemia subclass than subjects in the normal subclass. The differences were significant in high LDL-C subclass in overweight individuals. In euthyroid overweight women, mean TSH levels were significantly higher in high LDL-C subclass. In the euthyroid population, TSH was positively associated with total cholesterol in overweight population. The association was not modified by the homestasis model assessment for insulin resistance(HOMA-IR)values.(3)TSH was associated positively with serum triglycerides and negatively with serum HDL-C in women. TSH was positively associated with total cholesterol in overweight population and positively associated with total cholesterol and LDL-C in overweight women after adjustment for age, sex, and body mass index. Conclusion Raised serum TSH seems to be a risk factor of dyslipidemia in subclinical hypothyroid and euthyroid subjects, which is independent of insulin sensitivity.