NEK2 promotes the progression of liver cancer by resisting the cellular senescence / 中南大学学报(医学版)

OBJECTIVES@#Liver cancer is the sixth most common malignant tumor in the world. Hepatocellular carcinoma (HCC) accounts for 85%-90% of all patients with liver cancer. It possesses the characteristics of insidious onset, rapid progression, early recurrence, easy drug resistance, and poor prognosis. NIMA related kinase 2 (NEK2) is a cell cycle regulating kinases, which regulates cell cycle in mitosis. Cellular senescence is a complex heterogeneous process, and is a stable form of cell cycle arrest that limits the proliferative potential of cells. This study aims to investigate the relationship between the expression level of NEK2 and the senescence in hepatoma cells, and to explore the effect of NEK2 expression on hepatoma cell senescence and the underlying molecular mechanism.@*METHODS@#A total of 581 senescence-relevant genes were obtained from the GenAge website. The gene expression data of tumor tissues of 370 HCC patients were downloaded from the Cancer Genome Atlas database. The co-expression of NEK2 and aging-related genes was analyzed by R-package. KEGG was used to analyze the significant gene enrichment pathway of differentially expressed genes in NEK2 overexpression HEK293. The stable transfected cell lines with overexpression and knockdown of NEK2 were constructed in hepatoma cell line SMMC-7721 and HepG2, and senescence-associated β-galactosidase (SA-β-gal) staining was used to detect senescence, the cell proliferation was detected by CCK-8 method and clone formation experiment, the cell cycle was analyzed by flow cytometry, and the expression of proteins related to p53/p21, p16/Rb, and phosphatase and tensin homolog deleted on chromosome ten (PTEN)/Akt signal transduction pathway was detected by Western blotting.@*RESULTS@#There were 320 senescence related genes co-expressed with NEK2. KEGG analysis showed that the senescence signaling pathway was significantly enriched in HEK293 cells with overexpression of NEK2.Compared with SMMC-7721 or HepG2 without knockdown of NEK2, the senescent cells of SMMC-7721 and HepG2 with knockdown of NEK2 were increased, cell proliferation and clone formation were decreased significantly, the percentage of cells in G0/G1 phase was increased, the expression levels of phospho-Akt (p-Akt) and phospho-Rb (p-Rb) protein were decreased significantly, and the expression level of p16 protein was increased significantly (all P<0.05). Compared with SMMC-7721 or HepG2 transfected with blank plasmid, the senescent cells of SMMC-7721 and HepG2 overexpressing NEK2 were decreased, the cell proliferation and clone formation were increased significantly, the percentage of cells in G0/G1 phase were decreased, the expression levels of p-Akt and p-Rb protein were increased significantly, and the expression level of p16 protein was decreased significantly (all P<0.05).@*CONCLUSIONS@#NEK2 may mediate the anti-aging effect of hepatoma cells through p16/Rb and PTEN/Akt signal transduction pathways, which provides a new theoretical basis for NEK2 to promote the progress of liver cancer and a new idea for the targeting treatment for liver cancer.

Protocatechuic Aldehyde Represses Proliferation and Migration of Breast Cancer Cells through Targeting C-terminal Binding Protein 1 / 한국유방암학회지

PURPOSE: C-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor that is overexpressed in many cancers. CtBP1 transcriptionally represses a broad array of tumor suppressors, which promotes cancer cell proliferation, migration, invasion, and resistance to apoptosis. Recent studies have demonstrated that CtBP1 is a potential target for cancer therapy. This study was designed to screen for compounds that potentially target CtBP1.METHODS: Using a structure-based virtual screening for CtBP1 inhibitors, we found protocatechuic aldehyde (PA), a natural compound found in the root of a traditional Chinese herb, Salvia miltiorrhiza, that directly binds to CtBP1. Microscale thermophoresis assay was performed to determine whether PA and CtBP1 directly bind to each other. Further, clustered regularly interspaced short palindromic repeats associated Cas9 nuclease-mediated CtBP1 knockout in breast cancer cells was used to validate the CtBP1 targeting specificity of PA.RESULTS: Functional studies showed that PA repressed the proliferation and migration of breast cancer cells. Furthermore, PA elevated the expression of the downstream targets of CtBP1, p21 and E-cadherin, and decreased CtBP1 binding affinity for the promoter regions of p21 and E-cadherin in breast cancer cells. However, PA did not affect the expression of p21 and E-cadherin in the CtBP1 knockout breast cancer cells. In addition, the CtBP1 knockout breast cancer cells showed resistance to PA-induced repression of proliferation and migration.CONCLUSION: Our findings demonstrated that PA directly bound to CtBP1 and inhibited the growth and migration of breast cancer cells through CtBP1 inhibition. Structural modifications of PA are further required to enhance its binding affinity and selectivity for CtBP1.

MicroRNA-33a regulates the invasion of cervical cancer cells via targeting Twist1 / 中南大学学报(医学版)

OBJECTIVE@#To examine the expression of Twist1 in cervical cancer and to explore its biological function in the progression of cervical cancer.
@*METHODS@#The expressions of Twist1 in 32 cervical cancers and matched normal tissues were examined by immunohistochemistry (IHC). Cell invasive ability and the expression of invasion-related genes were determined in RNAi-based Twist1-silencing HeLa cells. The relationship between Twist1 and microRNA-33a (miR-33a) in cervical cancer was studied by Pearson correlation analysis, and the roles of miR-33a in regulation of Twist1 and cell invasiveness were studied.
@*RESULTS@#The positive expression rate of Twist1 was 75.0% (24/32) and 21.9% (7/32) in the cervical cancer and the matched normal tissues, respectively, with significant difference between them (P<0.05). Twist1 shRNA significantly decreased the invasiveness of HeLa cells (P<0.05). Compared with the matched normal tissues, the expression of miR-33a was increased in the cervical cancer tissues, which was negatively correlated with Twist1 (r=-0.661, P<0.05). Overexpression of miR-33a could significantly suppress Twist1 expression as well as cell invasiveness (P<0.05).
@*CONCLUSION@#Twist1 is critical for the invasiveness of cervical cancer cells; miR-33a, as a tumor suppressor gene, functions as an upstream regulator of Twist1 and is involved in the invasiveness of cervical cancer cell.

Expression of Toll-like receptor 4 in cervical cell lines and cervical lesions and its relation to HPV16 infection / 中南大学学报(医学版)

OBJECTIVE@#To explore the relation between human papillomavirus (HPV16) infection and expression of Toll-like receptor 4 (TLR4) in cervical cell lines and cervical lesion tissues and to investigate the effect of TLR4 on cervical cancer progression.@*METHODS@#Expression of HPV16 E6 mRNA was detected by RT-PCR. Western blot and immunohistochemistry were used to detect the expression of TLR4 in H8, SiHa, Caski cell lines and formalin-fixed and paraffin-embedded cervical tissue specimens with cervicitis, cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinama (CSCC). DNA was extracted from paraffin-embedded cervical cancer tissues and HPV16 genes were detected.@*RESULTS@#The differentiation expression of HPV16 E6 mRNA and TLR4 in SiHa and Caski was significantly higher than that of normal cervical cell H8 (P0.05). The expression of TLR4 was significantly correlated with HPV16 infection in CIN and CSCC (r=0.303, P<0.05, r=0.633, P<0.05).@*CONCLUSION@#High expression of TLR4 may play important roles in the development and progression of CIN and CSCC, and the expression of TLR4 can be up-regulated by HPV16 infection.

Molecular cloning and alternative splicing analysis of hepatoma associated gene HTA / 中南大学学报(医学版)

Objective:To obtain the full length cDNA sequences of hepatoma associated gene HTA, analyze its alternative splicing, detect the expression pattern of 2 HTA gene transcripts in different hepatic cell lines, and to establish a base for further study of HTA gene function in hepatocellular carcinoma (HCC) occurence and development. Methods:The full length cDNA of HTA gene was cloned by rapid amplification of cDNA 3' ends (3'-RACE), rapid ampliifcation of cDNA 5' ends (5'-RACE) and DNA sequencing. The gene structure and alternative splicing were analysed. Northern blot assay was performed to detect the expression pattern of 2 HTA gene transcripts in different hepatic cell lines. Results:The full length of HTA gene was 1414 bp, composed of 3 exons and 2 introns, and the second intron could be retained in mRNA. Northern blot assay showed that 2 transcripts of HTA mRNA(1.4 kb and 1.7 kb) could express in the HCC cell lines HepG2 and QGY-7703, but not in the non-malignant cell line L-02 and HUVEC. The expression level of 1.4 kb transcript was much higher than 1.7 kb one. Conclusion:This study successfully has obtained the full length cDNA of HTA gene, and analysed the gene sequence and alternative splicing, 2 transcripts of HTA mRNA specifically expressed in HCC cell lines. As a hepatoma associated gene, HTA deserves further investigation.

Recombinant expression of hepatoma associated gene and its protein function / 中南大学学报(医学版)

OBJECTIVE@#To recombinant express hepatoma associated gene(HTA) and pre-test the function of HTA to determine the role of HTA in the development of liver cancer.@*METHODS@#HTA338-616 was amplified from HepG2 cells and cloned into the prokaryotic expression vector pET21a(+)-MBP. The proteins MBP and MBP-HTA were induced, purified by His-tag magnetic bead purification kit and identified by Western blot and ELISA. HepG2 cells were stimulated with MBP or MBP-HTA proteins. MTT assay and colony formation assay were employed to examine the proliferation of these cells and the changes of cell cycle distribution were determined by flow cytometry.@*RESULTS@#The prokaryotic expression plasmid pET21a(+)-MBP-HTA was successfully constructed. We got a 52 kD purified purpose protein.The proliferation of HepG2 cells stimulated with MBP-HTA was significantly higher than those stimulated with MBP and negative controls. HepG2 cells stimulated with MBP-HTA showed significant decrease fraction in G1 phase and increase fraction in S phase, and the cell proliferation was enhanced.@*CONCLUSION@#HTA protein can significantly promote the proliferation of HepG2 cells, which may be related to the promotion of G1 phase to S phase.

Expression of scFv SA3 against hepatoma fused with enhanced green fluorecsent protein and its targeted ability in vivo / 中南大学学报(医学版)

Objective To express and purify the human scFv antibody,SA3,against the hepatoma fused to enhanced green fluorecsent protein,and to observe the targeted capacity of fusion protein EGFP-SA3 in vivo.Methods SA3 and EGFP genes were cloned into plasmid pET-25b( + )to construct the recombinant plasmid EGFP-SA3/pET-25b ( + ),followed by DNA sequencing.Then it was transformed into E.coli BL21 ( DE3 ) and induced for fusion expression of EGFP-SA3with IPTG.The expressed fusion protein EGFP-SA3 was purified and detected with SDS-PAGE.HepG2 cells were incubated with the fusion protein EGFP-SA3 in vitro,and the binding bioactivity was observed under the fluorecsent microscope.Further more,we injected the EGFP-SA3 by caudal vein into nude mice planted by hepatoma and observed the whole body fluorescence image of EGFP.Results SA3 and EGFP genes were successfully cloned into pET-25b( + ),which was confirmed by restriction enzyme Nco I-Xho I or Nco I-Eco RI.A band migrated at the position 750 bp,same to EGFP gene,emerged when recombinant plasmid was digested by restriction enzyme Nco I-Eco RI.Similarly,a band,about 1 500 bp,emerged when digested by Nco I-Xho I.The open-reading frame was confirmed by DNA sequencing.Fusion protein EGFP-SA3 was expressed as inclusion body.After purification and refolding,the result of immunofluorecsence detection verified that EGFP-SA3could specifically bind to HepG2 cells and maximum tumor penetration was at 24 h after the injection.Conclusion The purified fusion protein EGFP-SA3 has strong binding capacity to HepG2cells,indicating the scFv SA3 has a potential value as a targeting molecule for diagnosis and targeted therapy for liver cancer.

Expression and activity identification of a human nasopharyngeal carcinoma I50 anti-idiotype antibody / 中南大学学报(医学版)

Objective To obtain I50 anti-idiotype antibody and identify its activity in vitro.Methods I50 anti-idiotype (Id) antibody gene was amplified from the template of fuse 5-I50 by PCR to construct a prokaryotic expression vector pET25b-I50. The expression of pET25b-I50 in E. coli BL21(DE3) was induced by isopropylthio-β-D-galactopyranoside (IPTG) and was confirmed by SDS-PAGE and Western blot with Ab1(FC2) monoclonal antibody and an anti-hexahistidine tag antibody. The method of dialysis refolding was used to restore the activity of I50 anti-Id antibody, which was measured by Dot-ELISA and lymphocyte proliferation assay. Results The recombinant vector was successfully constructed and the recombinant protein was successfully expressed and purified with 90% purity. The relative molecular weight of the expressed protein was 15 kD, which was in accordance with expectation. The activity of I50 anti-Id antibody could be restored and could promote the proliferation of lymphocyte in a dose-dependent manner. Conclusion These results suggested that I50 anti-Id protein vaccine is likely an option in the therapy against nasopharyngeal carcinoma in vivo.

In vitro anti-tumor immune mechanism of nasopharyngeal carcinoma bispecific anti-idiotype antibody / 中南大学学报(医学版)

Objective To compare the in vitro antitumor immune responses induced by bivalent bispecific anti-idiotype antibody G22-I50 and monovalent anti-idiotype antibody G22 and I50, and explore its possible mechanism. Methods Proteins G22-I50, G22, and I50 were induced and identified by Western blot and ELISA. Peripheral blood monoclear cells (PBMC) were isolated and stimulated with G22-I50, G22, and I50 anti-idiotype antibodies, respectively. MTT assay and LDH release test were employed to examine the proliferation and cytotoxicity of the PBMC. The levels of IFN-γ, IL-2, and IL-4 in the supernatant were detected by ELISA and changes of T lymphocyte subsets were determined by flow cytometry. Results Western blot showed that G22-I50, G22, and I50 had specific binding capabilities to FC2 (Ab1). The activities of G22-I50, G22, and I50 had recovered and these proteins could be used in the in vitro study. The proliferation and cytotoxicity of the PBMC stimulated with G22-I50 were significantly higher than those stimulated with G22 or I50, The level of IFN-γ and IL-2 in the culture supernatant of the PBMC stimulated with G22-I50 was higher than that in the G22 or I50 group, but the level of IL-4 did not increase.Compared with the G22 or I50 group, the proportion of CD4+ and CD8+ T cells and CD4+/CD8+ ratio significantly increased, and the proportion of CD4+CD25+ T cells significantly decreased in the PBMC stimulated with G22-I50. Conclusion G22-I50 has more potent immunogenicity and would enhance specific antitumor effect which might relate to improving PBMC proliferation, inducing the secretion of Th1 type cytokines, activating CD8+T cells, and suppressing the expression of CD4+CD25+ T cells.

Survey of Prevalence and Need of Visual Disability in Shanxi Province / 中国康复理论与实践

@#ObjectiveTo investigate the situation characteristic, cause and needs of visual disability in Shanxi Province and to provide prevention measures corresponding to different causes.MethodsBy cluster random sampling, 88 townships were selected and 75016 persons received the eye examination in 22 countries in Shanxi from July 1 to May 31 in 2006. Visual acuity and eye examination were performed by ophthalmologist and judged the causes.ResultsThe total prevalence rate of visual impairment in Shanxi was estimated to be 6.21‰.The was a significant increase in the prevalence rate of visual impairment as the age grew older(χ2＝415.54, P<0.05), those in women was higher than that in men(χ2＝40.62, P<0.05). The prevalence rate of visual impairment in countryside was significantly higher than that in city(χ2＝25.37, P<0.05).The chief causes of visual impairment were cataract(39.34%), there are 43.91% of visual impairment who had not received any rehabilitation services, and most of those were medical services (35.26%).ConclusionVisual impairment prevention should establish a social security system and security system. Increase the investment of medical services and relief. To take different preventive measures corresponding to different ages. The preventive of visual impairment of shanxi should be focus on senile eye diseases. The major work should be put in remote rural area.

Screening and characterization of human scFv antibodies against nasopharyngeal carcinoma / 中国医师杂志

Objective To screen the anti-nasopharyngeal carcinoma scFv from a human anti-nasopharyngeal carcinoma single-chain phage antibody library, and identify its characteristics. Methods The single-chain phage antibody library was subjected to three rounds of positive and negative cell panning and enrichment, and then it was selected by ELISA. The binding specificity of phage antibodies with naso-pharyngeal carcinoma cells was confirmed by immunohistochemistry. Results After panning, enrichment and testing by ELISA, 3 phage an-tibody clones reacting with CNE2 more strongly than HUVEC and NP69 were picked out from 4212 clones. One clone, HNSAO33, was fur-ther analyzed after DNA sequencing. The results of immunohistochemistry with cultured cells were similar to those of ELISA. HNSAO33 spe-cifically reacted to nasopharyngeal carcinoma cells in most human nasopharyngeal carcinoma tissue sections except a few human normal naso-pharyngeal tissue sections. The distinction of positive rates was of a great statistical significance. Conclusion ELISA and immunohisto-chemistry results confirmed HNSAO33 specifically bind with nasopharyngeal carcinoma cells. The seFv fragment against nasopharyngeal carci-noma may be further developed and applied in clinical diagnosis and therapy of nasopharyngeal carcinoma.

Construct a subtractive library of Caski cell lines induced by exposing to the space environment by suppression subtractive hybridization / 中国医师杂志

Objectives Construct a subtractive library of Caski cell line induced by exposing to the space environment by suppression subtractive hybridization and pave the way to explain the molecular mechanisms of the changes at the gene level. Methods Super SMART cDNA synthesis and suppression subtractive hybridization (SSH) were performed to isolate differentially expressed cDNA fragments from strains subclonal 48A9 cell line. cDNA from the 48A9 cell line were used as " tester" , and the other from the control Caski cell line as "driver". Subtractive products were directly inserted into T/A cloning vector, and then transformed into host bacteria to set up a subtractive cDNA library of specially or highly expressed genes in strains subclonal 48A9 cell line. Results mRNA were directly extracted and purified with good quality. Double strand cDNA were reverse transcripted integratedly, and then cut by Rsa I into even length short segments. Liga-tion was identified as high effective. After two hybridizations, a subtractive library of differentially expressed genes in strains subclonal 48A9 cell line was successfully constructed by SSH. Conclusion SSH is an effective approach to isolate differentially expressed genes.

Effect of gamma-aminobutyric acid on malignant phenotypes of hepatocellular carcinoma cell line HepG-2 / 中南大学学报(医学版)

OBJECTIVE@#To determine the effect of gamma-aminobutyric acid(GABA) on proliferation and malignant phenotypes of hepatocellular carcinoma cell line HepG-2.@*METHODS@#HepG-2 cells were cultured by routine method, and then treated with different concentrations of GABA. The proliferation of HepG-2 cells was measured through MTT, doubling time and cell cycles by flow cytometry. The malignant phenotypes were investigated by soft agar colony formation assay.@*RESULTS@#Compared with the control group, GABA efficiently stimulated the proliferation of HepG-2 cells in a dose-dependent manner and affected the distribution of cell cycles of HepG-2 cells. The doubling time of the control group and the GABA-treated group were 39.0, 30.6, 30.0, 27.3, 26.6, and 38.2 h, respectively. The colony formation rates were 3.2%, 4.2%, 5.4%, 6.6%,6.5%, and 3.5%, respectively. Tumorigenicity testing showed that the average weights of tumor was 1.382 g, and 0.285 g for the 2 groups. The difference between the control group and the GABA-treated groups was significant (P<0.01).@*CONCLUSION@#GABA can enhance the proliferation and malignant phenotypes of HepG-2 cells.

In silico identification of cancer genes by high throughput analyses / 西安交通大学学报(医学版)

Objective To identify potential candidate genes related to the cancerous phenotype by analyzing databases publicly available. Methods Using a data-mining tool called Digital Differential Display (DDD) from the Cancer Gene Anatomy Project database, ESTs from 17 different tumor types were analyzed for differential expression. Results We obtained 130 up-regulated and 159 down-regulated genes, most of which are related to cytoskeleton, ribosomal subunit, substance metabolism, cell cycle, signal conduction, transcription and translation. These genes appear most frequently on chromosome 12 but rarely on chromosome 21 and Y. Conclusion In silico identification is a high-throughput screening strategy. Our study may lay a foundation for identification of future caner markers and provide a new thought for screening strategy of cancer markers.

A clinical trial of active immunotherapy with anti-idiotypic vaccine in nasopharyngeal carcinoma patients / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the effect of active immunotherapy with anti-idiotypic vaccine in patients with nasopharyngeal carcinoma (NPC).</p><p><b>METHODS</b>Anti-idiotypic antibodies (2H4/5D3) bearing the internal image of the NPC antigen were used in active immunotherapy in NPC patients receiving radiotherapy. Antibodies and cytokine levels in patient sera were determined using ELISA before and after active immunotherapy. IL-2 mRNA expression in the peripheral blood mononuclear cells (PBMC) was measured by in situ hybridization.</p><p><b>RESULTS</b>Nineteen patients with NPC at stage IV were treated with alum-precipitated 2H4 or 5D3. Neither hypersensitivity nor adverse side effects were observed. The levels of anti-anti-idiotypic antibodies (Ab3) and anti-NPC antibodies (Ab1') were increased. Human anti-mouse antibodies (HAMA) were seen in 19 patients of the experimental group; the levels of Ab1' did not increase in the control group. Serum IL-2, IFN-gamma and TNF-alpha levels were increased in most patients in the experimental group, while no differences were observed in Ab1' and cytokine levels between pre- and post-therapy in the control group. In addition, IL-2 mRNA expression in PBMCs from NPC patients was closely related to serum IL-2 (r = + 0.8829) levels by in situ hybridization.</p><p><b>CONCLUSIONS</b>Anti-idiotype vaccine is safe for clinical active immunotherapy. Anti-idiotypic vaccine might be able to enhance humoral and/or cellular immunity in NPC patients receiving radiotherapy.</p>

Purification and Identification of human Myocardial Troponin T / 中国医师杂志

Objective To obtain high pure myocardiac Troponin T from myocardiac tissue. Methods After the myocardiac tissues were homogenized and purified with high salt solution, the Sephacryl S-300HR column chromatography was employed to separate the cTnT from cardiac proteins, and identified them with SDS-PAGE and Western blot. Results The crude cTn was purified by column chromatography, and there appeared three peaks. The second peak was single component, which could react with mouse anti-human Troponin T antibody, and its molecular weight was about 34kD. Conclusions In this experiment, high pure cTnT was obtained, which could pave a way for further research.

Construction of cDNA Library From Human Nasopharyngeal Carcinoma Cell HNE_2 / 中国医师杂志

Objective To construct a cDNA library from human nasopharyngeal carcinoma cell HNE 2.Methods The total RNA was separated from human NPC(nasopharyngeal carcinoma)cell HNE 2 and the mRNA was isolated from the total RNA by MagneSphere technique,then the first-strand cDNA was synthesized with oligo(dT) primer containing sfiI site while the double-strand cDNA was amplified through LD-PCR(Long-distance PCR) by SMART technique.The double-strand cDNA was digested by sfiI(IA &IB)restriction enzyme before cDNA size fractionation ,the double-strand cDNA fractionated was ligated into the ?TripIEx2 vector and then was packaged in vitro.Results The unamplified human NPC cell HNE 2 cDNA library consists of 0 78?10 6 independent clones,and the percentage of recombinant clones was more than 96%.The titer of the amplified cDNA library was 1 02?10 9 pfu/ml and the average insert of the recombinants was 1 2kb.Conclusions The quality of the constructed human NPC cell HNE 2 cDNA library is excellent and helpful to screen NPC specific-antigen.

Active Immunotherapy with Anti-Idiotypic Antibody for Nasopharyngeal Cancer / 中国肿瘤生物治疗杂志

According to the immune network hypothesis proposed by Jeme, certain anti-idiotypic antibodies (Ab2) express three dimensional shapes which resemble the structure of natural antigens. Here we propose a study of active immunotherapy by Ab2 for patients with Nasopharyngeal Cancer (NPC) . Two Ab2,designated 2H4 and 5D3,against two Abl (FC_2 and HNL5)that recognize NPC associated antigen were generated. They could substitute NPC antigen to induce humoral and cellular immune response against NPC cells in syngeneic mice. Nineteen patients with NPC at stage IV were chosen for active immunotherapy. They were treated with Alum-precipitated 2H4 or 5D3 accompanying radiotherapy. Nine patients with radiotherapy alone were as control. Both anti-anti-idiotypic antibodies (Ab3) and anti-NPC antibodies (Abl') were increased and human anti-mouse antibodies (HAMA) occurred in nineteen patients of the experimental group; whereas the levels of Abl' did not rise in control group.Serum IL-2, IFN-? and TNF-? level were increased in most patients in experimental group. While in the control group, there was no difference of cytokine level between pretherapy and post- therapy.

Autoantibodies:the early diagnostic biomarkers for tumors / 西安交通大学学报(医学版)

Tumor markers refer to the specific substances that exist in tumor cells themselves or are secreted by tumor cells. They can reflect the existence and growth of the tumor. The serum tumor biomarkers have been widely applied in tumor detection,but the detection of these markers is based on the tumor antigens,and thus has many inadequacies in tumor screening and diagnosis. In this paper,we reviewed autoantibodies as tumor biomarkers against self-antigens in vivo. This method is to examine the tumor autoantibodies by using the tumor antigens,and its specificity and sensitivity are superior to the traditional examination methods. Using autoantibodies to detect tumors would provide a new method for tumor screening and diagnosis.

Construction of phage antibody library using sensitized in vitro B-lymphocytes of liver cancer patients / 西安交通大学学报(医学版)

Objective To construct human phage antibody library against hepatoma carcinoma.Methods Peripheral blood mononuclear cells(PBMCs) of patients with liver cancer were sensitized in vitro and transformed by Epstein-Barr virus(EBV).The genes of light chain and Fd of antibodies were amplified by RT-PCR.Fab genes were cloned into vector pComb3 and transformed into E.coli XLI-Blue by electroporation to construct the Fab-displaying phage antibody library.Results ELISA detection showed that 4 liver cancer patients' B cells transformed by EBV could produce specific antibodies to hepatoma carcinoma cell.Totally 13 types of light chain genes and 28 types of Fd genes were obtained by RT-PCR.The capacity of the primary phage library was 1.7?107pfu/mL.The percentage of recombinant clones was about 100%.Conclusion A human phage antibody library has been constructed successfully by means of EBV transformation technique.