MicroRNAs(miRNAs) are endogenous non-coding RNAs,about 20 nucleotides in length.They play a pivotal role in the regulation of genes involved in diverse biology processes such as cell development,proliferation,differentiation and apoptosis by the translation repression or mRNA degradation.Recent evidence has suggested that miRNA alterations are involved in the initiation and progression of various human cancer including hepatocellular carcinoma(HCC),and miRNA-expression profiling of HCC has identified signatures associated with diagnosis,staging,progression and prognosis.As a novel molecular target,miRNAs holds great promise in diagnosis and biotherapy of HCC.

Circulating nucleic acids (CNAs) are extracellular nucleic acids found in cell-free serum,plasma and other body fluids from healthy subjects as well as in patients. The ability to detect and quantitate specific DNA and RNA sequences has opened up the possibility of diagnosis and monitoring of diseases,especially in the field of cancer. Furthermore,in some recent studies it has been suggested a kind of non-coding RNA-microRNA (miRNA),also exist in cell-free serum and plasma,highlighting the field of using CNAs to diagnose cancer. As a novel tumor marker,tumor-specific circulating miRNAs holds great promise in early diagnosis of cancer.

BACKGROUND: Multiple sclerosis(MS) is a chronic autoimmune disease induced by the interaction between genetic and environmental factors. Its pathogen and the mechanism of the relapse and remission m the course of the disease are still unknown. Most of the MS research centers are looking for the pathogenic polypeptide epitope in proteolipid protein(PLP), myelin sheath basic protein (MBP) and oligodendrocyte glycoprotein (MOG) OBJECTIVE: To compare the proliferation of T cell lines(TCL) in MS induced by myelin sheath and delipidated myelin sheath towards 11 components of myelin sheath to mainly search the possible pathogenic polypeptide epitope in PLP, and investigate the possible effects of abnormal dcgrease in myelin sheath.DESIGN: A case-controlled trial.SETTING: Department of neurology in a hospital of a university.PARTICIPANTS: Mononuclear cells(MNC) of 16 MS cases(clinical relapsing-remitting type, patients did not receive any immunosuppresant for at least 3 months when their peripheral blood samples were taken) and 12 HLA-DR15 healthy volunteers were furnished by Dr. Trotter JL of MS Research Center of Washington University from the cell database.INTERVENTIONS: MS-TCL and normal TCL were induced twice by stimulation with myelin sheath and delipidated myelin sheath in vitro by cell culture in vitro. TCL proliferation was tested by 11 antigens including PLP,MBP, M87-106, P30-49, P40-60, P89-106, P95-117, P117-137,P139-151, P178-191, and P185-206.MAIN OUTCOME MEASURES: Difference of scintillation counting in every minute of every well, and the stimulative index of each well were calculated, and the mean wells with positive proliferation of TCL towards each antigen were confirmed as well.RESULTS: The general specific proliferation towards myelin sheath antigens was bigger in MS group than control group 5.49 ±5.31 to 3.10 ± 3. 17, and delipidated myelin sheath-induced TCL was bigger than myelin sheath-induced one 5. 49 ± 5.31 to 3.41 ± 4. 83 . Delipidated myelin sheath significantly changed the immune responses of MS group,especially the changes of responses towards P30-49, P40-60, P89-106,P117-137, P139-161, and P185-206 were significant compared with that the control group only responded to two polypeptides, which indicated that the antigen epitope of MBP, PLP, M87-106, P95-117, P40-60, and P185-206 might have significance in the triggering of MS autoimmune responses.CONCLUSION: TCL induced by MS myelin sheath has different proliferation towards antigen components of myelin sheath from control group. Delipidated myelin sheath significantly increases TCL proliferation in MS group, which suggests that if MS patients developed abnormal degrease in myelin sheath, TCL would produce autoimmune response towards self-myelin sheath, MBP, PLP and its polypeptide segments all can trigger MS or aggravate the state of the illness. Our finding supports the hypothesis of MS autoimmune pathogenic mechanism.

Objective To study the male reproductive ability of male rats with Toxoplasma gondii ( Tg) infection and investigate the variation of Toxoplasma infection in seminal plasma of infertile patients and explore its mechanism. Methods Thirty SD rats were randomly divided into 3 groups. The rats in the Toxoplasma infection group were administrated intraperitoneally with tachyzoites of Tg. in a dosage of 2 × 10~ 5/ml(2ml) , the rats in the treated group were administered with the same dosage of the tachyzoites and from the second day after the infection they were treated with 200 mg/kg azithromycin for 7 days, and the normal group was given physiological saline. Nine weeks after the infection, the serum sex hormone level, number,vitality, activity and quantity of spermatozoa and activities of enzymes in testa's of the testicular tissues were determined in the male rats. The female rats infected with Tg were matched with normal female rats at a ratio of 1: 2 for one week, and on the 21st day of pregnancy, the number of corpora luteum, sex ratio and the weight, body length and tail length of fetus were measured. The ELISA method was used todetermine the seminal plasma's anti-Tg IgG antibody of the 169 patients with infertility and 35 males with normal fertility. Meanwhile the NO levels in their semina were determined by means of nitric acid reducase. Results The number, activity .vitality, serum level of sex hormones were all lower in the infected rats than those in the normal and treated groups. The number of fetus in the pregnant rats matched with the infected male rats was significantly fewer, but the average body weight, body length, tail length of the fetuses and sex proportion showed no significant difference in comparison with those of the control group. The anti-Toxoplasma gondii antibody positive rate in the masculine infertility patients was 18.35% , being significantly higher than 2. 86% in the normal fertility group(P < 0.05 ). The mean NO level in the semina from the infertility group was (146.68 ± 38. 87) μnol/L , which was significantly higher than (84.92 ± 26.72) μnol/L( P < 0.01) in the fertility group. Conclusion Toxoplasma gondii infection can cause certain influences on the male reproductive ability.

Objective To study the pinoresinol diglucoside (PDG) on gene regulation role of ESF-1 cells in collagen secretion, to reveal PDG repair mechanisms on scalded skin.Methods The cells cultured in vitro were divided into the control group, the estradiol group and the three different PDG doses groups. The concentration of the high, medium and low dose groups were 100, 10, 1μmol/L, and that of estradiol group were 10-3μmol/L. The activity of proliferation was detected by MTT. Then collagen type I (Col I), collagen typeⅢ (ColⅢ), tissue inhibitors of metalloproteinase 1 (TIMP-1), tissue inhibitors of metalloproteinase 2 (TIMP-2) and matrix metalloproteinase 1 (MMP-1) expression levels of mRNA after administration of cells were detected by RT-PCR.Results Compared with the control group, the proliferation of ESF-1 cells (0.559 ± 0.027, 0.552 ± 0.034vs. 0.489 ± 0.027,P<0.05) in the estradiol and medium-dose PDG was significantly higher. The expression level of mRNA of ColⅠ(0.958 ± 0.021, 0.929 ± 0.031, 0.916 ± 0.015vs. 0.844 ± 0.022), ColⅢ (0.783 ± 0.038, 0.918 ± 0.021, 0.855 ± 0.017vs. 0.678 ± 0.024), TIMP-1 (0.939 ± 0.025, 0.889 ± 0.036, 0.853 ±  0.015 vs. 0.780 ± 0.023), TIMP-2 (0.507 ± 0.024, 0.655 ± 0.037, 0.572 ± 0.025vs. 0.405 ± 0.062) in the estradiol, low-, medium-dose PDG groups were significantly higher than those in the control group (P<0.05 or P<0.01). Besides, the MMP-1 (0.343 ± 0.038, 0.407 ± 0.046, 0.435 ± 0.037vs.0.519 ± 0.041) mRNA expression level in the middle and low dose PDG groups significantly decrease (P<0.05 orP<0.01). Conclusions The PDG could enhance the activity of ESF-1 cell proliferation, increase the expression of related collagen and tissue inhibitor of metalloproteinases and inhibit that of matrix metalloproteinases to repair scalded skin.

AIM: To clarify the role of eNOS and ET-1 in development of pulmonary hypertension (PH) associated with congenital heart diseases. METHODS: 40 patients were randomly divided into three groups: severe or moderate PH group (group A, 12 cases), slight PH group (group B, 14 cases) and normal group (group C, 14 cases). ET-1 and eNOS were examined by using the technique of immunohistochemistry. RESULTS: ① Plasma ET-1 concentration was significantly higher in group A and B than that in group C (P

Objective To construct,express,purify and identify the gene encodi ng major outer membrane protein of Neisseria gonorrhoeae (Porin I, or PI). Metho ds The gene encoding for PI of N.gonorrhoeae was amplified by PCR and cloned int o expression plasmid pGEX-4T-2 to form pGEX-4T-2/PI recombinants. A high lev el expression of GST-PI fusion protein was obtained in GST gene fusion system (GST:glutathione S transferase). The analysis indicated that the expressed pr otein was present predominantly in the insoluble form. Therefore, the induced pr otein was purified by SDS-PAGE, and bands corresponding to polypeptides of GST-PI fusion protein were excised and subjected to electroelution. A dot immunoch romatographic assay was employed to demonstrate whether the purified protein was gonococcal PI specific. Results The pGEX-4T-2/PI expression recombinants were constructed,expressed,purified and identified successfully. SDS-PAGE analysis and dot immunochromatographic assay suggested that the recombinant GST-PI fusio n protein was a 60 000 molecular weight protein andidentical in size to native PI and reacted with anti-PI monoclonal antibody. Conclusion Our results may lead to a potentiality for further study of diagnosti c kits and vaccine for Neisseria gonorrhoeae.

Objective To establish a method based on repetitive protein sequences and linear B cell epitope to predict and screen specific peptides of Plasmodium vivax. Methods A P. vivax protein sequence database was reconstructed based on Plas-moDB data,and a customized software for searching of repetitive sequences was used to count the repetition times of each 16 aa peptide in the whole database,and the highly repetitive peptides were chosen to predict the potential linear B cell epitopes. The re-petitive peptides with P. vivax specificity were selected as candidate antigen peptides to synthesize and to couple with KLH carrier protein for immunizing BALB/c mice. After the immunization,the antibody titers of the immunized mice were detected. Results The repetitive information of 16 aa peptides was analyzed by screening of the total 5 432 peptide sequences in the P. vivax data-base. A total of 22 peptides were identified as candidate peptides from the top 1 000 repetitive peptides by linear B cell epitope pre-diction on the BcePred website. Through clustering analysis and similarity comparison,five potential P. vivax specific peptides were selected,synthesized and then coupled with KLH to immunize the mice. The antibody titers of the immunized mice induced by the 5 peptides were all above 1:9 000. Conclusion The method for predicting and screening of specific peptides of P. vivax based on repetitive protein sequences and linear B cell epitope has been established successfully,and all the 5 peptides obtained by the method can induce the high titer antibody in mice.

Aim To develop the in vitro and in vivo models induced by shrimp tropomyosin( ST) and mono-clonal tropomyosin-specific murine IgE antibody ( anti-ST-IgE mAb) . Methods ST was purified from Metap-enaeusensis by an isoelectric precipitation method. The anti-ST-IgE mAb was obtained from hybridomas. After RBL-2 H3 cells were sensitized with anti-ST-IgE mAb and challenged with ST,β-hexosaminidase release was determined. Passive systemic anaphylaxis ( PSA ) was induced in mice and the rectal temperature was recor-ded after ST challenge within 30 min by a thermal probe. Results A significant increase ofβ-hexosamin-idase was observed in sensitized cells after ST chal-lenge. The average temperature drop after ST challenge was 1. 44℃ in PSA mice within 30 min. Conclusion The in vitro and in vivo models induced by ST and anti-ST-IgE mAb are established as an improvement of pres-ent models of type Ⅰ allergy.

Objective:To develop murine models of Th2 response induced by shrimp tropomyosin (ST). Methods:Mice were sensitized with ST for 6 weeks. The serum antigen-special IgE (sIgE),total IgE and sIgG level,Th1/Th2 cytokines production were measured by ELISA. The basophil activation in mice was measured by flow cytometry. Results:The intraperitoneal sensitization with ST for 6 weeks induced significant increase of serum sIgE,total IgE and sIgG (sIgG1,sIgG2a and sIgG2b) level in mice. Th2 cell response was induced and cytokines (IL-4,IL-5,IL-10 and IL-13) production increased in splenocytes stimulated by ST,while Th1 cytokine (IFN-γ) production decreased. As the markers of basophil activation,CD200R and CD41 expression also increased in response to ST. Conclusion:The Th2 response is dominant in ST-induced anaphylaxis in mice.