D Lu

Fudan University, Shanghai, Shanghai Shi, China

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Publications (13)6.09 Total impact

  • H Wang · F Wang · L Chen · J Xue · D Lu
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    ABSTRACT: To probe into the feasibility of increasing hFIX cDNA transfer and expression in muscle. The high-frequency electric field was used to promote both Lac-Z-encoding plasmid pCMV beta and hFIX-expressing plasmid G1NaMCIX to transfer and express in muscle. The effects of frequency and length of square pulse, as well as eletroporation time on hFIX expression were investigated. Electric stimulation could increase the transfer and expression of pCMV beta in muscle, the number of X-gal positive myofiber cells in electroporation-treated mice is 2.1 times larger than that of mice not treated by electroporation (P<0.01). The most optimal electric simulation condition for hFIX cDNA transfer and expression was obtained, under this condition, the highest level of hFIX antigen in plasma is (40+/- 5.4) ng/ml and 7 times higher than that of mice without electroporation P < 0.001). Electroporation is able to enhance hFIX cDNA transfer and expression in muscle efficiently.
    No preview · Article · Dec 2001 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
  • Z Jiang · J Shi · S Yang · C Zhang · H Jiang · Z Chen · L Jin · D Lu · W Huang
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    ABSTRACT: To establish a new method for single nucleotide polymorphism(SNP) typing based on allele specific PCR: single-tube bi-directional amplification (SB-ASA), and study the influence on specific extension by introducing a mismatch at the third 3'terminal base of allele specific primers. Two allele specific primers, with a mismatch introduced at the third 3'terminal base, were both included in PCR system; they extended in opposite directions and amplified two allele specific fragments different in size. The genotype was determined by observing the length of amplified fragments after agarose electrophoresis. The proper ranges of annealing temperature (Ta) under which primers can specifically extend were achieved by observing the amplification status at different temperatures. SB-ASA was successfully used to type 36 samples for four different kinds of SNPs. Typing results were completely consistent with those by directional sequencing. Proper Ta ranges of two primers were expanded respectively from 64-69 degrees centigrade to 46-66 degrees centigrade and from 60-62 degrees centigrade to 56-61 degrees centigrade by introducing a mismatch at the third 3'terminal base. SB-ASA is a simple, rapid and efficient new method for SNP typing. During allele specific PCR reaction, specific primers with a mismatch at the third 3'terminal base have more power to identify two alleles.
    No preview · Article · Sep 2001 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
  • W U Hong · G Cai · H Xu · H Chen · J Xiao · D Lu · J Xue · X Qiu · L Jin
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    ABSTRACT: The study was conducted to investigate single nucleotide polymorphism(SNP) in beta2-adrenoceptor(beta2-AR) gene and the distribution of these identified SNPs in Chinese Han ethnic group. beta2-AR gene was sequenced to detect SNPs by fluorescent labeling automatic sequencing method in 80 unrelated samples from territory of Dabie Mountain in Anhui province. A total of 8 SNPs were identified in length of 3.8 kb, including 5 SNPs in code region, 3 SNPs in regulatory region. Although the variations, -468C to G, -367T to C, -47C to T,-20T to C, +79C to G, +100G to A, +491C to T, +1098T to C have been identified in other ethnic groups, they have not been found in our study. The allele distribution of SNPs is in good unity with the Hardy-Weinberg equilibrium. The distribution of SNPs in beta2-AR gene is not equable and the SNPs in different ethnic groups differ greatly. The allele distribution of SNPs conforms well to the Hardy-Weinberg equilibrium.
    No preview · Article · Mar 2001 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
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    JS Wang · ZX Chen · X Xia · DR Lu · JL Xue · CG Ruan
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    ABSTRACT: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.
    Full-text · Article · Feb 2001 · Chinese medical journal
  • H Wu · G Cai · H Chen · J Xiao · W Huang · D Lu · J Xue · L Jin
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    ABSTRACT: To detect single nucleotide polymorphisms(SNPs) existing in code region of beta(2)-adrenoceptor(2-AR) gene and to investigate association of the identified SNPs with essential hypertension in Chinese Han population. Beta(2)-AR gene was sequenced with fluorescent labelling automatic sequencing method in unrelated Chinese Han population from Dabie Mountain in Anhui Province. Genotype of the SNPs were typed with PCR-RFLP method. Two SNPs were identified in length of 774bp, at position + 1053 with G-->C substitution and + 1239 with A-->G substitution respectively. The frequency of genotype of the two SNPs complied well with the Hardy-Weinberg equilibrium in normal group. Distribution of genotype AA, GA, GG of the SNPs at locus + 1239 in hypertension group was significantly different from that in normal group (chi(2) = 6.70, df = 2, P < 0.05). No significant difference was observed in distribution of genotypes of the SNPs at locus + 1053 between the two groups. These results indicate that the SNPs at locus + 1239 of beta(2)-AR gene is associated with EH. The SNPs at position + 1053 was not linked to hypertension.
    No preview · Article · Jan 2001 · Zhonghua nei ke za zhi [Chinese journal of internal medicine]
  • J Luo · Y Ji · Y Peng · J Xiao · Y Yao · H Xu · M Yang · J Zhen · D Lu · L Jin
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    ABSTRACT: To investigate the linkage between asthma and 5q31-33 in a Chinese population. The linkage between microsatellite markers in 5q and asthma and allergy was tested by lod score analysis. The linkage between asthma and 5q31-33 was not confirmed. The genes at 5q31-33 are not likely to contribute to inheritance of asthma in this Chinese population.
    No preview · Article · Nov 1999 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
  • Q Wang · DR Lu · YN Xing · JL Xue · XF Qiu
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    ABSTRACT: To establish the replicated-deficient recombinant adenovirus-mediated thymidine kinase/acyclovir (Adtk/ACV) system and to evaluate its suicide effect on rat C6 brain gliomas in vitro and in vivo. The plasmid pAdtk and pJM17 were co-infected into 293 cells (adenovector packaging cells) and the results were identified by polymerase chain reaction (PCR) assay. After the glioma C6 cells were transduced by Adtk at different multiplicity of infection (MOI) and exposed to different concentrations of ACV or gancyclovir (GCV), the cell survival curves were studied, and the cell surface was observed with scanning electronic microscopy (SEM). C6 gliomas in vivo at different inoculation days were injected with Adtk intratumorally and ACV intraperitoneally daily, and the survival duration and histologic changes of the rats were observed. The infectious Adtk virions had a suicide effect which was enhanced with the increase in MOIs of Adtk and ACV doses along with bystander effect. Under scanning electronic microscope, special pathologic changes were observed. ACV had a similar effect as GCV but a higher dose was used. The survival duration in day 3, day 6 and day 8 groups exceeded 90 days, and the rats in day 10 group survived 28.5 +/- 4.6 days, but the survival duration in untreated C6 group and AdLacZ/ACV (adenovirus-mediated LacZ/ACV) treated group were 16.8 +/- 3.1 and 14.0 +/- 2.2 days respectively. Adtk/ACV system can effectively kill the rat brain gliomas in vitro and in vivo.
    No preview · Article · Jul 1998 · Chinese medical journal
  • Y Bao · D Lu · H Xu · Q Shi · X Qiu · J Xue
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    ABSTRACT: To establish the polymorphism of DXS102 locus from Xq26.3-27.1 in Chinese population for the gene diagnosis in Hemophilia B family. DNA was extracted from blood samples obtained from Shanghai unrelated volunteer donors with phenol-chloroform method. A total of 23x chromosomes (154 from females, 80 from males) were studied. A hemophilia B family in which a hemophilia B patient has received gene therapy was analyzed. The polymorphism of DXS102 locus in Chinese population was determined with amplified fragment length polymorphisms assay (Amp-FLP), denaturing polyacrylamide gel electrophoresis, silver stain detection. Short tandem repeats (STRs) linkage analysis was used to conduct gene diagnosis in hemophilia B family. Eight alleles were found at DXS102 locus, of which two alleles were first reported. The repeated number of AC dinucleotide ranges from 13 to 21. And the values of the observed heterozygosity, calculated heterozygosity and polymorphism information content(PIC) were 0.87, 0.80, 0.80 respectively. It was also found that the difference of the allele frequencies of DXS102 in Chinese and European populations was significant. By using the linkage analysis of the DXS102 locus, a family with a hemophilia B patient receiving gene therapy in 1994 was analyzed and meanwhile a carrier in that family was then detected. The polymorphism of DXS102 locus reveals significant difference between Chinese and European populations. DXS102 locus can be used as a promising marker for gene diagnosis in hemophilia B family.
    No preview · Article · Jul 1998 · Chinese medical journal
  • Y Bao · D Lu · Q Shi · H Xu · X Qiu · J Xue
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    ABSTRACT: To determine the polymorphism of DXS102 and use it in gene diagnosis in hemophilia B. Amp-FLP and linkage analysis on 234 chromosomes. Eight alleles were found at DXS102 locus. The number of AC dinucleotide repeats ranged from 13 to 21. And the observed heterozygosity, calculated heterozygosity and polymorphism information content(PIC) were 0.87, 0.80 and 0.80, respectively. It was noted that the difference of the allele frequencies of DXS102 in Chinese and European populations was significant. By using the linkage analysis of the DXS102 locus, a family with a hemophilia B patient receiving gene therapy in 1994 was analyzed and a carrier in that family was then detected. The polymorphism of DXS102 locus reveals significant difference between Chinese and European populations. DXS102 locus can be used as a promising marker for gene diagnosis in hemophilia B family.
    No preview · Article · Mar 1998 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
  • K Zhang · D Lu · J Xue · Y Huang · S Huang
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    ABSTRACT: Two expression vectors comprised of mouse matrix attachment regions (MARs), bovine beta-casein gene sequence, human factor IX (hFIX) minigene, and cDNA, pMCIXm and pMCIX, were constructed for the purpose of a mammary gland bioreactor. A secretory expression system of hFIX protein in milk was made using stearylamine (SA) liposome to transfect plasmid DNA directly into the mammary gland lobule of a lactating goat. The highest production of hFIX in goat milk was 13.7 ng/ml 3 days after transfection, and the hFIX production in the goat mammary gland transfected with pMCIXm containing hFIX minigene was obviously higher than that transfected with pMCIX containing hFIX cDNA. Activity immuno-analysis and the barium citrate absorption method showed that > 90% hFIX protein in milk appeared to be a gamma-glycosylated and biological activity. This result confirmed the validity of the constructed vectors for further transgenic study, and this assay could also find its success in the evaluation of a foreign gene expression and secretion in the milk as a rapid detection system using liposome to transfect DNA directly into the goat mammary gland.
    No preview · Article · Feb 1997 · Chinese journal of biotechnology
  • D. Lu · X. Qiu · B. Zheng · H. Wang · J. Xue
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    ABSTRACT: A series of recombinant plasmids and retroviral vectors containing human clotting factor IX (hFIX), cDNA were constructed, and the efficiency of different promoters and enhancers in various constructors were compared. LTR promoter of MoMuL V showed a high transcription ability in mouse and rat fibroblasts, though CMV promoter has a better transcription ability in rabbit and human skin fibroblasts. The intron from both IL-2 gene and hFIX gene supposed to increase the expression level of hFIX in vitro was also studied. The coadjutant effect of double promoters and the combination use of selective gene in the constructors were also investigated.
    No preview · Article · Jan 1997 · Progress in Natural Science
  • X Qiu · D Lu · J Zhou · J Wang · J Yang · P Meng · J L Hsueh
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    ABSTRACT: To examine the safety and effects of gene therapy for hemophilia B by implantation of autologous fibroblasts genetically modified to secrete clotting factor IX (hFIX). Two hemophilia B patients LD and LW were selected from one family to accept gene transfer study. The hFIX protein of both patients were about 100 ng/ml plasma and hFIX activity was about 2%. The autologous skin fibroblasts of the two patients were genetically modified by retrovirus-mediated gene transfer with XL-IX and N2CMVIX vector (HBSF-IX). Human hFIX protein was measured by ELISA, hFIX activity was measured by one-stage clotting assay and barium citrate sorbent method. hFIX inhibitor was assayed by Bethesda methods. Human hFIX cDNA was detected by PCR. HBSF-IX cells were mixed with collagen for injection after safety assessments. The HBSF-IX cells from the two patients secreted hFIX at high levels in vitro. After implantation of autologous HBSF-IX cells, no treatment-related side effects were observed. Plasma hFIX protein in both patients increased over 2 folds after several injections of HBSF-IX cells and persisted for more than 420 days. Blood clotting activity increased significantly in both patients, hemorrhagic tendencies have been partially corrected after treatment. Further elevation of hFIX can be achieved by repeating the same treatment 420 days later in Patient LD. Implantation of autologous fibroblast genetically modified to secrete human hFIX offers a simple, safe and effective approach to gene therapy of hemophilia B.
    No preview · Article · Dec 1996 · Chinese medical journal
  • D Lu · X Qiu · B Zheng · J Xue
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    ABSTRACT: The construction of the high titer and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCIX, LIXSN and LCIXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, were constructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electroporation. Human clotting factor IX was detected in the cultured cells transduced with LNCIX and LIXSN but not in the cells transduced with LCIXSN. The viral titer of PA317/LNCIX was 800,000 CFU per mL. With ELISA detection, it was found that the cells transduced with this vector can express human clotting factor IX at the level of 3.3 micrograms per 10(6) cells in 24 h in human fibrosarcoma cells HT-1080 and 2.5 micrograms per 10(6) cells in 24 h in hemophilia B patients' skin fibroblast HSF cells, and more than 80% of them were biologically active. The viral titer and expression of human FIX were increased, and the construction of retroviral vector backbone was improved and the safety was guaranteed as compared to those vectors used previously. These vectors may produce a sufficient quantity of factor IX proteins to cause the phenotypic modification for hemophilia B patients.
    No preview · Article · Jul 1995 · Science in China. Series B, Chemistry, life sciences & earth sciences