Objective To establish a method to detect the glutathione stransferase(GST) M1 gene polymorphisms rapidly and to apply preliminary for detection.Methods GSTM1 gene and internal control gene CYP1A1 were amplified by multi-PCR with appropriate fluorescent dye SYBR green I in the reaction system,The changes of fluorescence values were recorded from 65 ℃ to 95 ℃ by a rate of(0.1 ℃/s) when PCR was just completed.Results There were two peaks in the melting curve of GSTM1~+ genotype,but only a single peak occurred for the GSTM1-genotype.The temperatures of two peaks corresponded to the expected Tm.agarose gel electrophoresis analysis demonstrated that these peaks correspond to the bands of the predicted molecular size.The detection can be completed within an hour after DNA was extracted.Conclusion Melting curve analysis combined with SYBR green I is a easy,rapid,accurate methodfor detection of GSTM1 gene polymorphisms.

Objective To investigate the expression and clinical significance of early B-cell factor 3 (EBF3) mRNA and protein in hepatocellular carcinoma(HCC) and the effect of EBF3 overexpression on HepG2 cell proliferation. Methods The expression levels of EBF3 mRNA in 20 pairs surgical specimens of HCC and their distant noncancerous tissues were detected by fluorescence quantitative real-time polymerase chain reaction(FQ-RT-PCR). Western blot was used to detect the expression levels of EBF3 protein in 5 pairs surgical specimens of HCC and distant noncancerous tissues. The fusion protein EBF3-EGFP was ex-pressed in HepG2 cells by transfection of pEGFP/EBF3 into the synchronized HepG2 cells using lipo-fectAMINE 2000 regent. Expression of EBF3-EGFP fusion protein was observed under the inverted fluores-cence microscope. S-phase fraction(SPF) and proliferating index(PI) were analyzed with flow cytometry (FCM). Results The ratio of EBF3 mRNA to β_2 mRNA in HCC tissues was significantly higher than that in distant liver non-cancerous tissues(0.55 ±0.12 versus 0. 22 ± 0.23, t = 5.69, P < 0.001 ). EBF3 protein in nuclear extracts of HCC tissues was about 4 fold that in distant non-cancerous tissues (26.35 ±14.06 versus 7.86 ± 8.47, t = 2.52, P = 0.036). Fluorescence microscopy revealed that 24 h after trans-fection of pEGFP/EBF3 into hepatoma HepG2, the fluorescence of EBF3-EGFP fusion protein was mainly observed in the nucleus. After transfection for 24 h and 48 h, SPF and PI were markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfeeted cells. Conclusion The expression of EBF3 at both mRNA and protein levels was up-regulated in HCC tissues. EBF3 promotes HepG2 cells proliferation through DNA replication, effect of EBF3 in ttCC needs to be further investigated.

Objective:To clone the encoding sequence of human EBF3 gene,construct recombinant eukaryotic expression plasmid vector pEGFP/EBF3,and study the effect of EBF3 on HepG2 cell cycling.Methods:Total RNA was isolated from placental tissue.Full-length human EBF3 cDNA was amplified by RT-PCR,cloned into eukaryotic expression plasmid vector pEGFP-N1 and sequenced.The expression and sub-cellular localization of the fusion protein EBF3-EGFP in HepG2 cells were analyzed by Western blot.Cell cycles were analyzed with flow cytometry analysis.Results:Obtained full encoding sequence of early B cell factor 3 was identical with that included in GeneBank,and the eukaryotic expression plasmid vector pEGFP/EBF3 was constructed correctly.24 h after transfected by pEGFP/EBF3,the fusion protein EBF3-EGFP was observed mainly in the cellular nucleus under the inverted fluorescence microscope.Western blot analysis confirmed that the EBF3-EGFP fusion proteins of Mr 87 000 were detected in both cytoplasmic and nuclear protein of the HepG2 transfected by pEGFP/EBF3 for 24 h or 48 h.Flow cytometry analysis revealed that the percentage of cells in the S phase was markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfected cells.These findings suggested that transfection of EBF3 gene into HepG2 induced cell proliferation by increasing the number of cells from G1 phase to G2 phase.Conclusion:The recombinant eukaryotic expression plasmid vector pEGFP/EBF3 is successfully established.The percentage of cells in the S phase is markedly increased in pEGFP/EBF3 transfected cells as opposed to pEGFP-N1 transfected cells.It is likely that EBF3 promotes HepG2 cells proliferation through DNA replication.

Objective To investigate the expression differences of mRNA and protein of early B-cell factor 3(EBF3) in the tissues between hepatocellular carcinoma(HCC) and distant noncancerous tissues and the clinical significance.Methods The expression levels of EBF3 mRNA in 18 pairs surgical specimens of HCC and their distant noncancerous tissues were detected by real-time fluorescence quantitative polymerase chain reaction(FQ-RT-PCR).Western blotting was used to detect the expression levels of EBF3 protein in 5 pairs surgical specimens of HCC and distant noncancerous tissues.Results The ratios of EBF3 mRNA tobeta2 microglobulin mRNA in eighteen liver cancerous tissues(0.52?0.17) were significantly higher than those in distant liver noncancerous tissues(0.28?0.23,t=3.56,P=0.0011).The average gray scale level of EBF3 in five liver cancerous tissues(26.35?14.06)were significantly higher than those in distant noncancerous tissues(7.86?8.47,t=2.52,P=0.036).Conclusion Theexpression levels of EBF3 mRNA and protein up-regulated in primary hepatocellular carcinoma,suggestting that EBF3 may contribute to occurrence of primary hepatocellular carcinoma.

Objective To evaluate the diagnosis value of plasma Dickkopf-1(DKK1)in hepatocellular carcinoma (HCC)pa-tients.Methods Selected 48 patients with HCC,20 patients with liver cirrhosis (LC),20 patients with chronic hepatitis B (CHB),and all of them were clinically diagnosed in the Third People’s Hospital of Nantong City,chose 20 cases of health examination as healthy controls (HC),that were ruled out other chronic disease.Enzyme-linked immunosorbent assay (ELISA)and Abbott i2000 microparticle chemiluminescence immunoassay analyzer were used to determine the plasma DKK1 and alpha-fetoprotein (AFP)levels.At the same time,analysesed and compared the receiver operating characteristic (ROC)curve and correlation of the DKK1 and AFP results.Results The plasma level of DKK1 in patients with HCC was significantly higher than that in patients with chronic hepatitis B,cirrhosis and healthy controls (Z=-4.132~-5.828,P<0.001).The area under ROC curve (AUC)of plasma DKK1 in the diagnosis of HCC was 0.889 and 95% confidence in-terval was 0.831~0.947,when the Cut-off value of DKK1 was 565 ng/L,the sensitivity was 93.8% and specificity was 70% in diagnosing HCC.The areaunder ROC curve of AFP in the diagnosis of HCC was 0.759 and 95% confidence interval was 0.667~0.850.The AUC of DKK1 was significantly higher than that of AFP (Z=2.28,P=0.022).DKK1 and AFP in diagnosing HCC were no significantly correlated (r=0.148,P=0.316).21 patients with AFP under 20μg/L showed higer DKK1 above 565 ng/L in 48 patients with HCC.Conclusion Detection of DKK1 in plasma can be used as a complement of AFP in the diagnosis of HCC,Especially DKK1 early diagnostic value of AFP negative HCC patients is remarkable.

Objective To estimate the role of the posterior corneal parameters measured by Pentacam in screening subclinical keratoconus.Design Retrospective case-controlled study.Participants Forty five healthy subjects(73 eyes),14 keratoconus patients (20 eyes),43 subclinical keratoconus patients(58 eyes).Methods Based TOPOLYZER and biomicroscope signs,eyes were diagnosed as subclinical keratoconus and keratoconus to compare the differnces of related posterior corneal parameters provided by Pentacam in normal eyes and subclinical keratoconus or keratoconus eyes.Main Outcome Measures Back-Diff(6 mm) max,Back-Diff(6 mm )min, Back-Difference(6 mm),Back-Rmin,Back-Astig.Results The parameters which were statistically different between normal corneas and clinical keratoconus,normal corneas and subclinical keratoconus include Back-Diff(6 mm)max(P

Objective To construct and screen siRNA targeting a proliferation-inducing ligand (APRIL) gene in a mouse colorectal cancer celline, CT-26. To investigate the effects to the cell growth and migrant capacity of CT-26 after knockdown APRIL gene, lay the foundation for molecular targeted therapy to colorectal cancer. Methods Four pairs of APRIL siRNA were designed and chemically synthesized. And disorder sequences were synthesized as a negative control. These sequences were transfected with LipofectAMINE 2000 into CT-26 cells, which high-expressed APRIL gene. The transfection efficency rate of 6-FAM labelled control siRNA was detected by fluorescence microscope. The inhibition effectiveness of APRIL mRNA and protein was analyzed by FQ-RT-PCR and Western blot, respectively. Cell proliferation activity was analyzed by cell counting kit-8, cell migration capacity was detected by the repair of cell damage, and MMP-2 together with TIMP-1, two important regulatory genes in cell metastasis, were measured by RT-PCR.Results The different kinds of APRIL siRNA effectively suppressed the level of APRIL mRNA and the protein expression in CT-26 (P ＜ 0.05 ). Cell proliferation and metastasis ability were repressed after APRIL siRNA transfection( P ＜ 0.05 ), compared with random siRNA control and nontransfected control. The mRNA levels of MMP-2 and TIMP-1 genes wre significantly altered among APRIL siRNA groups and two control groups ( P ＜ 0.05). Conclusion We have constructed and screened a kind of siRNA (APsi737) targeting APRIL gene in a mouse colorectal cancer cell line, CT-26. APRIL siRNA can effectively inhibit the cell growth and migration capacity, maybe be regulated by MMP-2 and TIMP-1.

Objective To investigate the regulation of B-lymphocyte stimulator(BLyS) levels in response to IFN-γand IL-6. Methods Flow cytometry, quantitative polymerase chain reaction, ELISA and Western blot were applied to examine the expression level of BLyS in response to IFN-γ and IL-6 . Results IFN-γand IL-6 induced BLyS expression in KM3 cells. After treated with BAY11-7082, an IkB-α phospho- rylation inhibitor, the up regulation of BI,yS induced by IFN-γ was completely inhibited. Inhibiting the nu-clear faetor-kB (NF-kB) and mitogen activated protein kinase(MAPK) activation in KM3 cells reduced BLyS protein and gene expression. Conclusion MAPK and NF-kB pathways are involved in the regulation of BLyS expression, which suggests that MAPK and NF-kB might be used for the treatment of multiple mye- loma.

Objective To investigate the action and mechanism of NF-κB pathway in up-regulating B cell-activating factor receptor (BAFF-R) expression in multiple myeloma cells induced by IFN-γ.Methods Activated NF-κB were detected with Western blot, while the expression of BAFF-R were measured with RT-PCR and ELISA, and investigated the effect of BAY11-7082 on transcription of BAFF-R mRNA and translation of protein in multiple myeloma cells stimulated by IFN-γ. Results IFN-γ can induce the degradation of IκB-α in time-dependent and dosage-dependent manner, and up-regulated BAFF-R expression in multiple myeloma cells. BAY11-7082, an NF-κB inhibitor, inhibited not only the transcription of BAFF-R mRNA but also the protein of regulated by IFN-γin dosage-dependent manner. Conclusion NFκB may play an important role in high expression of BAFF-R in multiple myeloma cells induced by IFN-γ.

Objective To investigate the effects of a proliferation-inducing ligand(APRIL)gene silencing by small interfering RNA(siRNA)on cell cycle and proliferation of colon carcinoma SW480 cells.Methods The siRNA plasmid vector targeting APRIL gene,named as siRNA-APRIL,was transfected into SW480 cells,transfected with scrambled vector as a nontargeting control and nontransfected group as another control.APRIL mRNA and protein expression were examined by real-time PCR and Western blot,respectively.Cell proliferation activity was analyzed by cell counting kit-8(CK-8),cell cycle was detected by flow cytometry,and p21 together with p27,two important regulatory genes in cell cycle,were measured by RTPCR.Results Compared with nontargeting control and nontransfected control,APRIL expression was inhibited significantly at both mRNA and protein level by siRNA-APRIL being transfected in SW480 cells(P ＜0.05).Cell proliferation ability was drastically repressed after siRNA-APRIL being transfected at 48 h,72 h and 96 h(P ＜ 0.05).After transfected 48 h,the percent of Go/G1 phase cell was significantly increased,S and G2/M phase cell were significantly decreased,the number of cell in apoptosis was increased and the expression of p21 and p27 mRNA were up-regulated(P ＜ 0.05).There was no significant difference when compared the two control groups each other(P ＞ 0.05).Conclusion siRNA-APRIL can effectively knockdown the expression of APRIL gene in SW480 cells,moreover,it can inhibit the cell proliferation and induce G0/G1 phase cell cycle arrest,which occurrence may involve in upregulation the mRNA expression of p21 and p27.