Abstract:Clinical proteomics is the application of proteomics in clinical medicine, which focuses on the prevention, diagnosis and therapy of disease. Cancer has been the primary research object in clinical proteomics. Because biomarker is invaluable in early diagnosis of cancer, one of the central goals in clinical proteomics is to discover appropriate biomarker and the interest shifts to looking for multi-biomarkers combined. Study design, sample collection and pre-process in clinical proteomics and the application and progress of clinical proteomics technology have been introduced.

Abstract:The most familiar action of mode performed by RNA regulation is the posttranscriptional gene silencing in the cytoplasm. The discovery of siRNA-directed DNA methylation in nucleus has proved that siRNA can induce transcriptional gene silencing by siRNA-guided genome modifications. DNA methylation was once predicted to be an epigenetic mechanism of tumor biogenesis. It is well known that the abnormal inhibition of tumor-suppressor genes is associated with DNA de novo methylation in the gene promoter region. Potential molecular mechanisms of tumour-suppressor gene silencing was analized, the principle of siRNA-directed transcriptional suppression was explored, and the possible relationship between them was clarified. The detailed study on the relationship between DNA methylation and RNA interference will be beneficial to better understand tumor biology and epigenetics, and also provide new therapeutic strategies for the prevention and treatment of cancer.

Abstract:Peripheral nerve injury of a limb usually causes functional reorganization of the contralateral somatosensory cortex. However, the patients with an operation of the contralateral seventh cervical nerve (C7) transfer to an injured arm with brachial plexus root avulsions usually have the sole tactile sensibility of the healthy hand when the injured hand is touched at the early stage after the operation. Then, at later stage they gradually get normal sense from the injured and the normal hands independently. Mimicked the process in a rat model based on the above operation, representations of the injured forepaw and the healthy forepaw in the somatosensory cortex were studied by means of somatosensory evoked potential (SEP) recording. Somatosensory function shown in SEP response amplitude and peak latency of the injured forepaw gradually recovered with time after the operation due to the contralateral C7 regeneration toward the injured limb, accompanied with the recovery process of limb movement. The somatosensory representation of the injured forepaw was observed exclusively in the ipsilateral somatosensory cortex since the 5th month after the operation. Accordingly, the overlapped representation of the injured and healthy forepaws emerged in the ipsilateral somatosensory cortex of 13 rats studied except one with separated representation though the SEP latency and response amplitude were different in responding to stimuli on the two forepaws. It is concluded that the contralateral peripheral nerve transfer to the injured arm can cause dynamically functional reorganization in the ipsilateral somatosensory cortex suggesting a remarkable plasticity of the brain function induced by an alteration of sensory input between two sides of the body in adult rats.

Abstract:A mAb T2-2 was generated using hybridoma techniques, and its target was identified as a 46 ku-cytokeratin (CK), based on biochemical study and a completely overlapped binding pattern of mAb T2-2 with anti-pan-CKs antibodies. An epithelia-specificity of the mAb T2-2 was determined by screening 68 human normal and 65 tumor tissues using immunohistochemistry. Unlike most of anti-CKs antibodies, the mAb T2-2 recognized a mono-specific epitope only expressed on the 46 ku CK, suggesting that mAb T2-2 is superior to most anti-CKs antibodies that cross-reacted with many different kinds of CKs. In addition, it was found that the mAb T2-2 was multipurpose with a broad applicability to ELISA, immunohistochemistry, immunofluorescence, Western blotting, and was also compatible with various fixation reagents. These results strongly indicate that the mAb T2-2 has potential applications for studying CKs function and for diagnosis of tumor and other disorders.

Abstract:New strategies are needed for prevention of Pseudomonas aeruginosa (P. aeruginosa) infections, a widespread disease caused by P. aeruginosa with strong drug resistance. The immunoprotective capacity of the receptor of autoinducers protein LasR/RhlR was examined in the BALB/c mice. At first, specialized expression plasmids were developed to facilitate expression of LasR/RhlR proteins in Escherichia coli (E.coli). Then, biofilms were grown from clinical isolated P. aeruginosa PA0305 to investigate the relative contributions of cell signaling for biofilm formation. Morphological characters of biofilm were quantified using Image-Pro Plus software. Fluorescence analysis demonstrated that cell signal molecule LasR/RhlR significantly (P < 0.05) influenced development of P. aeruginosa biofilm. Active immunization of mice with LasR/RhlR was found to provide significant protection against homologous challenge with P. aeruginosa in mice lungs. In 10 days after lungs inoculation, the bacterial clearance rate of the immunized mice was clearly higher than that of non-immunized groups on the basis of microbiological and histological assays. The protective effects of immunization with LasR and RhlR together were the same as the result of LasR or RhlR immunized mice alone. These data indicate that the manner of LasR, RhlR or both is an important determinant of immunoprotection in mice lungs infection.

Abstract:In an attampt to establish the functional expression of STGC3 with doxycycline (Dox) induced Tet-on regulating system in human nasopharyngeal carcinoma cell line CNE2, an ideal experimental platform was provided for further studies of STGC3. pTet-on regulating plasmid was transfected into CNE2，and stable expression of Tet-on was established in CNE2 through G418 select. Then the response plasmid of recombinant pTRE-STGC3 was steadily transfected into positive CNE2/Tet-on cells with hygromycin screen. Dox was used to induce the expression of STGC3 and a cell clone sensitive to Dox was selected. The best-induced concentration was determined with different concentration of Dox induction. Growth curves, clone formation rate and cell cycle distribution were detected after STGC3 gene up-regulated expression with Dox induction. The growth capacity and clone formation potential of CNE2/Tet /pTRE-STGC3 was significantly suppressed, compared with the controls (P＜0.05). FCM analysis indicated that G0/G1 phrase cell rate of CNE2/Tet /pTRE-STGC3 was markedly higher than that of the controls and CNE2/Tet/pTRE-STGC3 cells were arrested in G0/G1 phase of cell cycle. Functional expression of STGC3 under Dox induced Tet-on regulation system was successfully established in CNE2, which provided an ideal experimental platform for further functional study of STGC3.

Abstract:NGX6, a candidate of tumor suppressor genes, can inhibit the proliferation of colon cancer in vivo and in vitro. The uncontrolled proliferation of tumor cell closely related with the dysfunction of the cell cycle. Flow cytometry and Western blot were used to explore effects of NGX6 on cell cycle progression and the expression of cyclins, CKI(p27,p16) in colon cancer cell line HT-29. Flow cytometry (FCM) showed that the overexpression of NGX6 increased the proportion of HT-29 cells at G0/G1 phase and decreased that at S, G2 and M phase. Western blot and FCM indicated that NGX6 could down-regulate the expression of cyclin E, cyclin D1 and increase the p27 expression,while NGX6 has no significant effect on expression of cyclin A and cyclin B. The expression of p16 was not detected in all groups. These results demonstrated that NGX6 arrested cell cycle progression at G0/G1 phase by down-regulating the expression of cyclin E, cyclin D1 and up-regulating that of p27 to inhibit the proliferation of HT-29 cells.

Abstract:In clinic, the low tumour-targeted ability of SOD is a critical shortage in its application, which is a difficulty for scientist at all time. For solving this problem, Nostoc commune CHEN iron-superoxide dismutase (Fe-SOD) and anti-lung adenocarcinoma LC-1 single chain Fv (ScFv) were fused, and the fusion protein named SOD-ScFv was expressed. After purification, fusion protein demonstrated both SOD and LC-1 antibody activities. The result of tracing of SOD-ScFv by FITC dyeing and quantification of ROS(reactive oxygan species) in SPC-A-1(lung adenocarcinoma) cells showed that the fusion protein possessed the ability to recognize SPC-A-1 cells and eliminate the cellular ROS. The tumour-targeted theory put up in this research will overcome two applied disadvantages of SOD in clinic: it neither target the tumour cell nor permeates through the cell membrane. Also, the research provides a feasible idea that ScFv can be used to target the anticancer drug to tumour.

Abstract:Glycosylation is one of the most important forms of post-translational modification of proteins. Glycan influences folding and stability of proteins and their biological functions. A berrant glycosylation has been associated with many malignant tumor. Proteomic analysis methods combined with Pro-Q Emerald glycoprotein staining technology was applied to investigate the glycosylation difference of glycoprotein between normal human liver cell lines and hepatocellular carcinoma cell lines. Total proteins were extracted by using cell-splitting methods, then subjected to two-dimension electophoresis(2-DE). After that, 2-DE gel were stained with Pro-Q Emerald glycoprotein stain. Glycoprotein patterns of both cell lines were obtained. Glycosylation status of glycoproteins were quantificationally compared by using Dymention software and glycoproteins with altered glycosylation were identified with mass spectrometry. Results showed that (74±2) glycoproteins (n＝3) were detected in normal human liver cell lines whereas (78±3) glycoproteins (n＝3) were detected in human hepatocellular carcinoma cell lines. There were 31 matched glycoproteins between both cell lines. Glycoprotein patterns had distinct difference between normal human liver cells and human hepatocellular carcinoma cells. 25 glycoproteins presented glycoslytion changes in human hepatocellular carcinoma cells comparing with normal human liver cells. Among such glycoproteins, glycosylation level of 10 glycoproteins were up-regulated whereas glycosylation level of 15 glycoproteins were down-regulated and 12 of these glycoproteins had been identified by mass spectrometry. These results imply that glycosylation changes may play key roles in occurrence and development of liver cancer.

Abstract:In order to identify candidate tumor suppressor genes (TSGs) in childhood acute lymphoblastic leukemia (ALL), firstly, loss of heterozygosity (LOH) of 6q16.3～21 in 139 primary ALL samples was analyzed by using polymerase chain reaction (PCR) and 11 microsatellite markers. The frequency of LOH on 6q16.3～21 was 32%. A 2-cM high frequency deletion region was flanked by D6S1709 and D6S301 loci at 6q16.3～21. Clinical data showed that patient with 6q16.3～21 LOH had higher WBC counts and blast cells (P < 0.05). The statistics about age, sex, classification of morphology and immunology were indistinct (P > 0.05). Then, positional cloning strategy, bioinformatics technology and reverse transcription-polymerase chain reaction (RT-PCR) were used to identify candidate TSGs and its cDNA fragments at 6q16.3～21, especially at the high frequency deletion region. Comparing with expression of normal peripheral blood mononuclear cell, EST screened in D6S1709-D6S301(GenBank Accession No.AA403058) was down-regulation in ten of fifteen childhood ALL (P < 0.05). Digital differential display showed that the expression levels of AMD1, PPIL6 and WASF1 were lower in cancer tissues than in normal tissues(P < 0.05). These findings may provide new clues in cloning of childhood ALL TSGs at 6q16.3～21.

Abstract:Several studies have demonstrated that heparin can significantly inhibit the P-selectin-mediated interaction of platelets and tumor cells during metastasis as a P-selectin ligand. However, little information is available about the specific oligosaccharide structures of heparin in recognition by P-selectin. Two chemically modified heparins, CR-heparin and SCR-heparin were prepared, to explore if such heparin derivatives can reduce the P-selectin-mediated A375 tumor cell adhesion. The results indicated that CR-heparin with low anticoagulant activity could significantly inhibit the P-selectin-mediated A375 tumor cell adhesion, demonstrating that 6-carboxyl group of the glucuronic acid in heparin may not be crucial for recognizing by P-selectin. In contrast, SCR-heparin reduced the inhibiting activity dramatically, suggesting that the recognition of P-selectin to heparin depend on not only densities of negative charge. These results provide valuable experimental evidence for clarifying the molecular mechanism of P-selectin-mediated tumor cell adhesion.

Abstract:In the cells infected by HSV, various viral proteins are expressed in a cascade style. The expression of immediate-early (IE) gene occurred in early phase of infection is under the control of regulatory elements in upstream sequence of promoter. A series of pCAT expression vectors containing different upstream sequence fragment of α4 - gene, in which relative element is deleted orderly, is constructed and detected in CAT assay. The transcriptional ability of these DNA fragments on α4 - gene are evaluated to investigate their efficacy in early phase infection. The data collected indicated that the expression of IE gene in cells is controlled by various elements in its upstream. Topological distribution of these elements provides probably useful information for further understanding about the mechanism of virus replication.

Abstract:In order to develop a new method for screening and identification of dominant B cell epitopes using UreB protein as a target antigen 11 amino acid fragments from UreB protein of Helicobacter pylori (Hp) were synthesized by Fmoc solid phase peptide synthesis strategy, and fluorescein FITC was labeled to the N-terminals of all peptides respectively. The antigenicity of synthetic peptides is determined by analyzing the recognition and combination between peptides and standard antibody samples by fluorescence polarization (FP) immunoassay. In order to screen the dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against 10 UreB synthetic peptides respectively in 159 UreB antibody-positive antiserum samples. There are 10 of 11 UreB synthetic peptides have distinct antigenicity by FP assay. The results showed that 3 out of the 10 antigenic peptides may be immunodominant, for the antibodies against them existed more widely among the samples and the antibody titers were higher than those of other peptides’s. The methods for predicting and identifying epitopes are useful for epitope mapping, and the fluorescence polarized method for antibody immunoassay can be widely used in the diagnosis, typing and therapy of diseases in clinic in the future.

Abstract:The design of DNA-based alphavirus vectors significantly improves the utility of these replicon vectors. The DNA-based replicon vectors can be used in expressing foreign genes and preparing RVP in virto efficiently, also in developing replicon vaccines and gene therapy vectors in vivo. The approach involved the conversion a RNA-based replicon vector into a layered DNA-based replicon vector by the RNA polymerase Ⅱ promoter and transcription termination/polyadenylation signal transcribed replicon RNA from DNA. When DNA-based alphavirus vector tranfected into cells, the first layer includes a eukaryotic RNA polymerase Ⅱ expression cassette that initiates transcription of RNA in nucleus. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the RNA vector corresponds to virus RNA replication cycle and results in high level expression of foreign gene. DNA and RNA-based bifunctional replicon expression vector pSCTA and helper vector pSHCTA were successfully constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with CMV promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3′-untranslated region (UTR). In order to obtain DNA-based highly efficient replicon vectors, they were further modified to construct additional three DNA-based SFV replicon expression vectors and corresponding helper vectors. To investigate the efficiency of foreign gene expression level by the four different DNA-based SFV expression vectors and recombinant virus particle (RVP) prepared by cotranfecting with corresponding helper vectors, improved DNA-based replicon vectors pSCAR and pSHCAR derived from SFV were developed. high level protein could be generated using the new vector system by transfecting DNA into BHK21 cells and High titer of RVP produced by cotranfecting with helper vector. Antigen genes were also expressed in cells by the replicon expression vector. Additionally, reporter gene expression was observed in mice muscle following injection with SFV DNA vector. Anti-β-Gal antibody response and cell-mediated immune response were induced after intramuscular inoculation of the β-Gal-encoding SFV replicon DNA. The results suggested that highly efficient DNA-based replicon vectors pSCAR and pSHCAR were constructed by modifying the SFV vectors. The improved DNA-based replicon vectors enhance the utility of them, and can be developed as potentially replicon vaccines and gene therapy vectors.

Abstract:Extraction of high-quality RNA from Panax ginseng tissues is particularly difficult due to high levels of polyphenolics and polysaccharides, or other compounds that bind and/or coprecipitate with RNA. The methods such as guanidinum thiocyanate, CTAB and modified Trizol were applied to isolate high purity and integrity RNA from Panax ginseng. By all of these methods total RNA can be obtained, but only by the modified Trizol method, it extracts integrity and undegraded RNA from mature leaf tissue. Following this efficient procedure, 90～120 μg/g total RNA from leaf tissues can be obtained. The ratio of intensities of the 28 S and 18 S rRNA bands was 2∶1. The absorbance ratios (A260/A280) were 1.8 to 2.0. The RNA is suitable for RT-PCR and cDNA library construction.