Objective Conduct safety assessments of the established human skin fibroblast line. Methods A series of safety assessments were performed including cell morphology,chromosome karyotyping,soft-agar test,nude mice carcinogenic test,endotoxin test,mycoplasma determination,detection of viral agent,bacteria,fungi sterile tests and abnormal toxicity test.Results No abnormal changes were observed.Conclusion The fibroblast obtained from skin is a kind of safety and reliable target cell for gene therapy.

Background: Integrative traditional Chinese and western medicine may be a new approach to improve the eradication rate of Helicobacter pylori (Hp).Aims: To compare the efficacy and safety of Weifuchun tablet versus bismuth combined with standard triple regimen as the first-line therapy of Hp infection.Methods: A total of 141 patients with Hp infection and na(i)ve to treatment were randomly assigned into 3 groups receiving a 14-day eradication therapy.In standard triple therapy group, esomeprazole, amoxicillin and clarithromycin were given twice a day;while in Weifuchun group and bismuth group, Weifuchun tablet and bismuth potassium citrate were added, respectively, to the standard triple therapy.Hp eradication was assessed by 13C-urea breath test at least 6 weeks after the end of treatment.Hp isolates were tested for resistance to antibiotics.Results: One hundred and twenty-eight patients completed the study.Hp eradication rates in Weifuchun group, bismuth group and standard triple therapy group were 83.7%, 91.8% and 79.1%, respectively by ITT analysis and 88.4%, 97.8% and 84.6%, respectively by PP analysis.The eradication rate of Weifuchun group was lower than that of bismuth group and higher than that of standard triple therapy group, but the differences were not statistically significant (P>0.05).Only PP eradication rate of bismuth group was significantly higher than that of standard triple therapy group (P<0.05).The resistant rates of Hp to clarithromycin, amoxicillin and metronidazole were 33.3%, 2.9% and 70.5%, respectively.For eradication of clarithromycin resistant strains, bismuth group was superior to Weifuchun group and standard triple therapy group (100% vs.60.0% and 66.7%, P all <0.05).All three eradication regimens showed good compliance, and no significant difference in incidence of adverse events was found between the three regimens (P>0.05).Conclusions: Weifuchun tablet combined with standard triple regimen is safe and effective for use as first-line treatment for Hp infection, however, the eradication rate is relatively low in cases infected with clarithromycin resistant strains.Bismuth combined with standard triple regimen is a good alternative in areas with high clarithromycin resistance and regions where tetracycline is unavailable.

The gamma-secretase complex mediates the final cleavage of APP to generate the principal component of amyloid plaques in the brains of Alzheimer's disease patients.Four integral membrane proteins (PS,NCT,PEN-2 and APH-1) are essential and sufficient for gamma-secretase activity.To identify the promoter of human nicastrin gene (NCT),its 5' -flanking region has been characterized and a 270 bp fragment containing the TSS (transcription start site) for the promoter activity has been identified.EMSA assays confirmed that all four AP-1 binding sites and two NFAT sites in the NCT promoter region were able to bind relative transcription factors in vitro.Mutations,as well as treatment with PDTC,which adjust the regulatory effect of AP-1 and NFAT,altered NCT promoter activity in both HeLa cells and rat cortical neurons.The results demonstrated that AP-1 and NFAT are involved in the regulation of hNCT transcription and suggest that balanced activation of AP-1 and NFAT ensures a strict temporal and tissue-specific control of NCT transcription.

G mutation of SLC26A4, the parents and the second child were carriers of the same mutation, while the fetus had a wild-type form. Conclusion It is feasible to identify deafness related genes by screening for GJB2 and SLC26A4 mutation, thus providing correct prenatal diagnosis and avoiding deaf delivery of baby.

Objective To isolate and identify the potential binding partners of LRRK2,a gene linked to both dominant familial form and sporadic form of Parkinson's disease,thus to further our knowledge of its function.Methods We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait.The bait amplified by polymerase chain reaction(PCR) was then cloned into a yeast expression plasmid pGBKT7.After being sequenced and analyzed,pGBKT7-bait was transformed into the yeast strain AH109.Western blot was performed to confirm the expression of pGBKT7-bait in AH109 yeast strain.Then human fetal brain cDNA library was trarnsformed into that yeast strain.which could express pGBKT7-bait fusion protein.The yeast strain which contained pGBKT7-bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade)containing X-a-gal.We retested these positive colonies using 2 independent yeast strains AH109 contained pGBKT7-bait or pGBKT7,respectively.At last,these plasmids isolated from these true positive colonies were analyzed by bioinformatics.Results We obtained 9 true positive colonies,these colonies were sequenced, and we performed sequence Blast in GenBank.Three colonies of the 9 positive colonies were not in open reading-frames.Among other 6 colonies,there were known proteins including spermatid perinuclear RNA-binding protein(STRBP)and BCL2-associated athanogene 5 isoform b(BAG5),as well as unknown proteins including tyrosine phosphatase non-receptor type(PTPN23),1(3)mbt-like 3 isoform b(L3 MBTL3),RALY RNA binding protein-like isoform 1(RALYL),and Homo sapiens mRNA for KIAA1783 protein,partial cds(KIAA 1783).Conclusion True positive colonies of LRRK2 are successfully obtained by the yeast 2-hybrid.Our screened proteins may provide a new research clue for revealing biological functions of LRRK2,pathogenesis of Parkinson's disease,and other neurodegerations.

Hydroxyapatite nanoparticles were prepared in low Ca/P ratio by a kind of electrodeposition-hydrothermal process. The suspension of nanoparticles was cultured with SGC-7901 cells; metabolically active cells were evaluated by MTT analysis. Cells grew well and the nanoparticles in the concentration range of 10-100 microg/ml had no adverse effect on the cell viability. The results show that the nanoparticles have excellent biocompatibility with cells. Agrose gel electrophoresis analysis demonstrated that the nanoparticles had the potential to adsorb EGFP-N1 at the pH ranging between 2 to 7. Nanoparticle-DNA complex could transfer EGFP-N1 into the SGC-7901 cells, and the confocal microscopy analysis revealed that the cells with green fluorescence showed the efficiency of nanoparticle uptake to be about 80% of the efficiency of the Lipofectmine TM 2 000 uptake. In vivo, nanoparticles and DNA-nanoparticle complex were injected into mice respectively via tail-vein, and the mice grew well in two weeks. The liver, kidney, and brain of the mice were sampled and detected with electron microscopy, and all of these exhibited biodistribution of nanoparticles. This study demonstrates that Hydroxyapatite nanoparticles could be used as gene carriers.
Animals ; Biocompatible Materials ; Calcium Phosphates ; chemistry ; Drug Carriers ; chemistry ; Durapatite ; chemistry ; Genetic Therapy ; methods ; Mice ; Nanostructures ; Stomach Neoplasms ; pathology ; Transfection ; Tumor Cells, Cultured

Human ribosomal DNA (hrDNA) targeting vector pHr is a homologous recombinant plasmid for human genome which developed in the State Key Laboratory of Medical Genetics. pHr was used to construct a recombinant plasmid pHr-CMG expressing mda-7/GFP fusion gene and its efficacy in the hepatocellular carcinoma cell line Bel-7402 was investigated. The expression of mda-7/GFP fusion gene was detected by fluorescent microscope, RT-PCR and Western blotting, and its function was detected by cell-cycle analyses, MTT assay and Hoechst33258 staining. The results demonstrated that pHr-CMG vector could express MDA-7/GFP fusion protein effectually and the mda-7 gene could induce cell apoptosis and proliferation suppression in Bel-7402 cell line, which might be caused by the G2/M cell cycle arrest. These results also suggested that human ribosomal DNA targeting vector system and the pHr-CMG vector may be applied in further gene therapy researches for hepatocellular carcinoma.

In order to study the tumor suppression effect of p53 with CMV enhancer and hTERT promoter mediated by human-derived vector pHrn in liver cancer cell Bel-7402, report plasmid pchEGFP, tumor suppressor plasmids pchp53Arg and pchp53Pro were constructed by inserting expression cassette CMVe+hTERTp+EGFP, CMVe+hTERTp+p53Arg and CMVe+hTERTp+p53Pro into pHrn respectively. 24 h after cell transfection by lipofectamine 2000, GFP expression pattern was analyzed through fluorescence microscope and flow cytometry; RT-PCR and Western blot were taken to study the p53 expression pattern. The cell apoptosis by Hoechst 33258 and Annexin V-FITC/PI staining was also studied. Results show that the expression of GFP and p53 protein in Bel-7402were detected, but apparent cell apoptosis could not be found. The recombinant p53 mediated by human-derived vector could express in Bel-7402, but no significant tumor suppression effect was detected, which might result from the down regulation effect of the wild type p53 on hTERT promoter.

Ring dermoid of cornea (RDC) is an autosomal dominant disorder of cornea. The previous study identified a G185A mutation of PITX2 gene in a Chinese family with RDC. To investigate the pathological mechanism of PITX2 R62H mutation, a PITX2 prokaryotic expression plasmid were constructed and GST-PITX2 fusion protein were purified. EMSA was further conducted. The DNA-banding ability of PITX2 R62H was similar to that of the wild type PITX2 were found. The cell lines stably expressed PITX2 was also generated, and cell cycle were analyzed by flow cytometry, and the expression of ?-catenin and Cyclin D1 were detected by quantitative Real-time PCR. The results showed that proliferating ability of cells expressing PITX2 R62H was lower than that of cells expressing PITX2 WT, as well as ?-catenin and Cyclin D1 mRNA level. These findings revealed that PITX2 R62H mutation affected the Wnt/?-catenin→PITX2 pathway, promoted the genes expressing abnormally, and led to abnormal cell proliferation and the formation of RDC, which may play an important role in pathogenesis of RDC.

OBJECTIVETo study pantothenate kinase 2 (PANK2) gene mutations in Chinese patients with Hallervorden-Spatz syndrome (HSS).

METHODSPANK2 gene mutations were detected by PCR, DNA sequence analyses, restriction enzyme digestion and PCR-single strand conformation polymorphism in 5 patients, 3 unaffected family members and 51 unrelated healthy persons.

RESULTSNovel compound heterozygous PANK2 gene mutations, A803G and T1172A, in exons 3 and 5, respectively, were found in one patient. At the same time, 3 types of single nucleotide polymorphisms, -38 t>a in 5'-UTR, IVS1+42 c>a and G77C in exon 1, were confirmed; among them, -38 t>a, IVS1+42 c>a, were first reported.

CONCLUSIONPANK2 gene mutations can cause HSS in Chinese patients.

Adolescent ; Adult ; Base Sequence ; Child ; China ; DNA Mutational Analysis ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Pantothenate Kinase-Associated Neurodegeneration ; genetics ; Pedigree ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Young Adult