2.Establishment and preliminary application of a gene chip for detection of hepatitis B virus "a" determinant hotpoint mutation.
Rui ZHANG ; Rong-cheng LI ; Yan-ping LI ; Sheng-qi WANG ; Zheng-lun LIANG ; He-min LI ; Hui ZHUANG
Chinese Journal of Hepatology 2007;15(2):103-106
OBJECTIVETo develop a gene chip for rapid detection of the "a" determinant hotpoint mutation of hepatitis B virus (HBV).
METHODSPrimers were designed in the HBV conservative region, and probes for detecting 126A, 126S, 144A, 145R, 145E, 144A+145R, and 144A+145E mutants were developed for that gene chip. PCR amplification and gene chip technology were optimized. The performance of the gene chip was evaluated by detecting the reference plasmids. Forty five samples of serum obtained from patients with chronic hepatitis B were used to compare the sensitivity of the gene chip and the direct sequencing of PCR products.
RESULTSThe oligonucleotide microarray was specific for mutant and native plasmids. The sensitivity of the gene chip was 5 x 10(3)copies/micro l with a high reproducibility. The gene chip could detect minor variants when they were more than 10% among the HBV strains. The positive rates of 126A, 126S-1, 126S-2 detected in the 45 specimens by the gene chip (46.67%, 35.56% and 24.44%, respectively) were higher than those detected by direct sequencing of PCR products (9.00%, 4.44% and 2.22%; P=0.000, P=0.000 and P=0.002, respectively). The sequencing of cloned PCR products demonstrated that the gene chip was specific for the "a" determinant hotpoint mutation detection.
CONCLUSIONHBV "a" determinant hotpoint mutations can be detected by oligonucleotide microarray with high sensitivity and specificity, providing a method for large scale screening of the mutants.
Hepatitis B ; blood ; diagnosis ; Hepatitis B virus ; genetics ; Humans ; Oligonucleotide Array Sequence Analysis ; methods ; Point Mutation
3.Suppression of invasion and angiogenesis in human prostate cancer PC-3 cells by adenovirus-mediated co-transfer of PTEN and P27.
Zhen QIU ; Fei-lun CUI ; Chuan-Liang XU ; Zheng-qin GU ; Ying-hao SUN
National Journal of Andrology 2007;13(3):201-205
OBJECTIVETo investigate the effects of adenovirus-mediated PTEN and P27 on the invasion of PC-3 in vitro and angiogenesis, along with their synergy in the treatment of prostate cancer.
METHODSRecombinant adenovirus vectors of the human tumor suppressor genes PTEN and P27 were constructed. The replication-incompetent recombinant adenovirus was packaged and propagated in HEK293 cells. The viral titer was examined by plaque assay and the mRNA and protein expressions of PTEN and P27 in human prostate cancer cell line PC-3 infected with Ad-PTEN and Ad-P27 were determined by RT-PCR and Western blot respectively. The invasion of PC-3 cells in vitro was examined by Boyden chamber assay. MTT assay was used to testify the effect of supernatant from PC-3 infected with Ad-PTEN and Ad-P27 on the proliferation of endothelial cells ECV-304 and the CAM test was used to testify the effect of PTEN and P27 on angiogenesis. The difference between the combined therapy group and the single gene therapy group was also examined.
RESULTSThe viral titers of Ad-PTEN and Ad-P27 were 1.8 x 10(7) pfu/ml and 1.2 x 10(9) pfu/ml respectively. Adenovirus infection verified that the mRNA and protein expression of PTEN and P27 were steady in human PC-3 cells. The invasion in vitro of PC-3 cells was significantly inhibited by infection with Ad-PTEN or/and Ad-P27. CAM and MTT assays of ECV-304 confirmed that the supernatant from PC-3 cells infected with Ad-PTEN or/and Ad-P27 could inhibit the angiogenesis effectively. There was a significant difference between the combined therapy group and the single gene therapy group.
CONCLUSIONThe combined gene therapy of Ad-PTEN and Ad-P27 plays a synergistic role in inhibiting the invasiveness of PC-3 cells and angiogenesis.
Adenoviridae ; genetics ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; Humans ; Male ; Neoplasm Invasiveness ; PTEN Phosphohydrolase ; genetics ; Prostatic Neoplasms ; blood supply ; pathology ; Transfection
4.Interruption failure of heptitis B virus vaccination in mother-to-infant transmission and heptitis B virus genotypes and preC/BCP mutations.
Jia WANG ; Jie LI ; Hui ZHUANG ; She-lan LIU ; Rong-cheng LI ; Yan-ping LI ; Zheng-lun LIANG
Chinese Journal of Epidemiology 2007;28(4):331-333
OBJECTIVETo investigate the association of heptitis B virus (HBV) genotypes and precore(PreC)/basal core promoter(BCP) mutation with interruption failure of HBV vaccination in mother-to-infant transmission.
METHODSA total number of 208 serum samples were collected from infants and mothers,including 16 infants who had become HBsAg-positive despite a complete and timely course of immunization and another 88 infants successfully protected from mother-to infant HBV transmission. HBV genotypes were determined by type-specific primers PCR method. PreC/BCP mutations were detected by direct sequencing of PCR products, and Clustal W 1.8 software was applied to analyzing the sequences.
RESULTSOf 16 mothers who were having vaccine failure infants, 15 (93.8%) were HBeAg positive and infected with genotype C (15/15, 100%). Among 88 mothers of having children being protected by vaccine, 51 (58.0%) were HBeAg positive, with 45.1% (23/51) of genotype C. The proportion of genotype C in HBeAg mothers of infants with vaccine failure, was significantly higher than that of mothers with vaccine protected infants (chi2 = 14.3, P = 0.003). However, the frequencies of T1762/A1764 mutations had no significant differences between genotype C HBeAg positive mothers with vaccine failure or protected infants (33.3% and 13.3%, respectively, P = 0.4). No A1896 mutation was found in these two groups.
CONCLUSIONHBV genotype C might contribute to the immune failure of HBV vaccination in mother-to-infant transmission, while PreC/BCP mutation might not have correlation with it.
Adult ; Female ; Genes, Viral ; Genotype ; Hepatitis B ; immunology ; prevention & control ; transmission ; Hepatitis B Vaccines ; immunology ; Hepatitis B virus ; genetics ; immunology ; Humans ; Infant, Newborn ; Infectious Disease Transmission, Vertical ; prevention & control ; Mutation ; Polymerase Chain Reaction ; Pregnancy ; Pregnancy Complications, Infectious ; immunology ; virology ; Promoter Regions, Genetic
5.Sensitivity and specificity of 4 domestic ELISA kits for detection of hepatitis B virus markers
Xing WU ; Cheng ZHOU ; Wei-Jin HUANG ; Zibai QI ; Zheng-Lun LIANG ; He-Min LI ; Hui ZHUANG
Chinese Journal of Epidemiology 2008;29(9):915-918
Objective To compare and analyze the sensitivity,specificity of 4 domestic ELISA kits for detection of hepatitis B virus (HBV) markers (HBsAg,anti-HBs,HBeAg,anti-HBe,and anti-HBc).Methods Five hundred and ninety four serum samples collected from patients with chronic hepatitis B and abnormal blood donors were detected for HBV markers and by 4 domestic ELISA kits.Samples with conflicting results by different diagnostic kits were retested.Samples with the HBsAg values close to the cut-off point were detected by Abbott HBsAg confirmation kit (Architect HBsAg confirm).Sensitivity of the kits was determined,using the national sensitivity reference panels for HBsAg,anti-HBs,HBeAg,anti-HBe and anti-HBc.Results The rates of sensitivity on 4 domestic kits for detection of HBsAg were 4 to 10 times lower,and on the 4 domestic kits for detection of anti-HBs,HBeAg,anti-HBc and anti-HBc were 4 to 16 times lower,as compared to Abbott Architect kits.In addition,the domestic HBV ELISA kits had some false positive results.The total coincidence rates of HBsAg,anti-HBs,HBeAg,anti-HBe,anti-HBc were 96.46%-98.15%,94.28%-98.15%,98.15%-99.49%,90.07%-96.30%,92.09%-96.80%,respectively.Conclusion Both sensitivity and specificity of the domestically produced HBV ELISA kits should be improved.
6.Structure-based design, synthesis and evaluation of bioactivity of anti-P-gp peptide mimetic.
Jing QI ; Hui PENG ; Ying-dai GAO ; Chen XU ; Zhong-qin LIANG ; Zhen-lun GU ; Chun-zheng YANG
Acta Pharmaceutica Sinica 2003;38(11):826-830
AIMTo design and evaluate the small peptide mimetic of anti-P-glycoprotein (P-gp) antibody (PHMA02).
METHODSFrom the three dementional structure analysis of computer modeling of PHMA02 CDR loops, a small peptide mimetic was designed and determined by flow cytometry.
RESULTSAnti-P-gp peptide mimetic functionally similar to PHMA02 was developed. The peptide mimetic competitively inhibits PHMA02 binding to P-gp and partially block the P-gp function as a drug efflux pump in K562/A02 cells.
CONCLUSIONSome special conformational properties of CDR loops of antibody might serve as lead structures for develop new biological peptide mimetics. Antibody-structure-based design would develop new drug in the future.
ATP-Binding Cassette, Sub-Family B, Member 1 ; chemistry ; immunology ; Antibodies, Monoclonal ; chemistry ; Binding, Competitive ; Complementarity Determining Regions ; chemistry ; Drug Design ; Drug Resistance, Multiple ; Humans ; K562 Cells ; Molecular Mimicry ; Peptides ; chemical synthesis ; chemistry ; metabolism ; Protein Conformation
7.Epidemiology of hepatitis E in clinical patients in Dongtai, Jiangsu province.
Xin YAO ; Cheng ZHOU ; Feng-cai ZHU ; Xing WU ; Xue-feng ZHANG ; Zhong-ze WANG ; Chuan JI ; Zheng-lun LIANG
Chinese Journal of Epidemiology 2010;31(5):594-594
China
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epidemiology
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Female
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Hepatitis E
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blood
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epidemiology
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Hepatitis E virus
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immunology
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Humans
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Immunoglobulin M
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blood
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Male
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Middle Aged
8.Establishment of an imatinib resistant cell line K562/G01 and its characterization.
Jing QI ; Hui PENG ; Zhen-Lun GU ; Zhong-Qin LIANG ; Chun-Zheng YANG
Chinese Journal of Hematology 2004;25(6):337-341
OBJECTIVETo establish a BCR/ABL+ cell line with resistance to imatinib, and investigate the possible mechanisms of the acquired resistance.
METHODSK562 cells were cultured in gradually increased concentrations of imatinib over a period of several months to generate their resistance line. MTT assay, RT-PCR, Western blotting, and FISH were used to study the possible molecular mechanisms of the resistance.
RESULTSA resistant cell line, K562/G01, was established with 15.2 +/- 3.0-fold resistant to imatinib as compared with that of the parental sensitive cell line. The resistant cell line also had the cross-resistance to a broad spectrum of other anticancer agents excepting for DOX. There was no difference between the two cell lines in terms of the cell morphology, proliferation doubling time, and fraction distribution of cell cycle. K562/G01 cells showed increased levels of BCR/ABL, mdr1 mRNA and their coding proteins and the increased tyrosine kinase activity. No point mutation in the BCR/ABL ATP-binding site was detected while the copies of BCR/ABL fusion gene were increased in K562/G01 cells.
CONCLUSIONAn imatinib-resistant human leukemia cell line, K562/G01, was established. The mechanisms of resistance of K562/G01 cells to imatinib involved increased expression of BCR/ABL and mdr1/P-gp, amplification of BCR/ABL fusion gene, and increased activity of BCR/ABL.
ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Benzamides ; Drug Resistance, Neoplasm ; Drug Screening Assays, Antitumor ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; Imatinib Mesylate ; K562 Cells ; drug effects ; metabolism ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology
9.Clinical research of improved plastic and reconstruction of anus in situ.
Ben-Shou ZHANG ; Zi-hao LUN ; Zeng-nan LI ; Xiang ZHENG ; Liang-biao XU ; Zhi-jiu XU ; Xiang-bing SHUI ; Ling JIA ; Hui CHU ; Fu-tang WEI ; Yun-fei WU
Chinese Journal of Plastic Surgery 2006;22(6):455-457
OBJECTIVETo explore the effect of the improved plastic and reconstruction of the anus in situ.
METHODSImproved plastic and reconstruction of anus in situ was performed in 38 cases of low rectal cancers operated while Miles radical operation. Improvement includes: (1) The internal sphincter was rebuilt with 4 layers of muscle layer of the endmost of colon. (2) The last of gracilis was divided into 2 parts to reconstruct the superficial part and deep part of external sphincter muscle. (3) The rectum cape improvement is to firmly stitch the levator ani outside the external sphincter muscle in front of the colon. (4) The rectum valve is improved into three artificial rectum valves.
RESULTSThe form and function and their long term survival rate were good, the rate of superior anus function was 94.73%.
CONCLUSIONIt mains the results of improved plastic and reconstruction of anus in situ is near that of normal persons.
Adult ; Aged ; Anal Canal ; surgery ; Female ; Humans ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; methods ; Rectum ; surgery
10.Ursolic acid induces human hepatoma cell line SMMC-7721 apoptosis via p53-dependent pathway.
Yan-Xia YU ; Zhen-Lun GU ; Jiang-Lin YIN ; Wen-Hsien CHOU ; Chi-Yi KWOK ; Zheng-Hong QIN ; Zhong-Qin LIANG
Chinese Medical Journal 2010;123(14):1915-1923
BACKGROUNDUrsolic acid (UA) is a ubiquitous molecule in the plant kingdom with specific anticancer effects that have been shown in vitro and in vivo. Although UA can inhibit the proliferation of liver cancer cells and induce apoptosis of many types of tumor cells, the molecular mechanism of its anti-hepatoma activity is still not well defined. The objective of this study was to investigate the inhibitory effect and mechanisms of UA on the human hepatoma cell line SMMC-7721.
METHODSAfter treatment with UA, the growth inhibition of SMMC-7721 cells was assessed by MTT assay. Cells were also evaluated by flow cytometric analysis, Wright-Giemasa staining, Hoechst 33258 staining and transmission electron microscope after they were induced by UA. DNA microarray technology was used to investigate the gene expression pattern of SMMC-7721 cells exposed to UA 40 micromol/L. The molecular mechanism of cells death was analyzed by real-time RT-PCR and Western blotting.
RESULTSThe proliferation of SMMC-7721 cells was significantly inhibited in a dose- and time-dependent manner after UA treatment. UA induced cell cycle arrest and apoptosis. The DNA microarray analysis indicated that 64 genes were found to be markedly up- or down-expressed, including GDF15, SOD2, ATF3, and fos. The result of Western blotting showed the apoptotic proteins p53 and Bax were up-regulated while the anti-apoptotic protein Bcl-2 was down-regulated. Real-time RT-PCR confirmed UA could up-regulate the mRNA expressions of GDF15, SOD2, ATF3 and down-regulate the mRAN expression of fos. Meanwhile these effects were partly blocked by pretreatment with the p53 inhibitor Pft-alpha.
CONCLUSIONActivation of the p53 pathway is involved in UA inhibition of SMMC-7721 human hepatocellular carcinoma cell growth and induction of apoptosis.
Apoptosis ; drug effects ; Blotting, Western ; Carcinoma, Hepatocellular ; drug therapy ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Flow Cytometry ; Humans ; Liver Neoplasms ; drug therapy ; metabolism ; Microscopy, Electron, Transmission ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; Triterpenes ; therapeutic use ; Tumor Suppressor Protein p53 ; metabolism