Construction of Prokaryotic Expressing Vector of Antisense Nucleic Acid of lasR and Its Effect on the Virulence of Pseudomonas Aeruginosus

To construct a pUCP18/lasR^antisense plasmid carrying the reversed gene and analyze its effect on the virulence of Pseudomonas aeruginosus, LasR gene was amplified from the genome of Pseudomonas aeruginosus by PCR and reversely recombined with plasmid pUCP18. The recombinant pUCP18/lasR^antisense was verified by enzyme digestion, PCR and sequencing. The biological effects of pUCP18/lasR^antisense were examined by using RT-PCR, NAD method and the assay of pyocyanin. Our results showed that the expected full length lasR fragment （721 bp） was extended from Pseudomonas aeruginosus gene with PCR. And it is consistent with LasR gene of Pseudomonas aeruginosa in GenBank （No. NC_002516）. The recombinant plasmid was successfully constructed and transferred into Pseudomonas aeruginosus. The antisense nucleic acid of LasR gene could reduce the virulence of Pseudomonas aeruginosus and might serve as a new target site for treatment purpose.

Apoptosis Induced by Bcl-2 Antisense Peptide Acid in HL60 Cells

Objective To study the differences and similarities of the antisense drugs with different structures on the biological functions of HL60 cells. Methods Cytotoxic effects were measured by cell viability assay. The expression levels of protein were assayed by immunofluorescence using fluoresce isothiocyanate label. The morphological changes in apoptotic cells were observed. Flow cytometric analysis of DNA fragmentation was also performed. Results Antisense peptide nucleic acid (PNA) targeting the coding region of the Bcl-2 mRNA could effectively inhibit the growth of HL60 cells, down-regulate the synthesis of Bcl-2 protein and induce apoptosis. After HL60 cells were treated with 10 μmol/L Bcl-2 antisense PNA or antisense oligonucleotide for 72 h respectively, apoptotic rates of HL60 cells were 17.80±1.53 and 13.17±1.12, respectively( P <0.05). Conclusion Antisense PNA targeting the coding region of Bcl-2 mRNA may have stronger antisense effects than the antisense oligonucleotides and could induce apoptosis of HL60 cells.

Effects of multidrug resistance, antisense RNA on the chemosensitivity of hepatocellular carcinoma cells

BACKGROUND: Multidrug resistance is a major obstacle in cancer chemotherapy. We examined whether the antisense RNA of multidrug resistance gene 1 (mdr1) could reverse multidrug resistance in the human hepatocellular carcinoma (HCC) cell line SMMC7721/ADM. METHODS: The recombinant adenoviruses pAdEasy- GFP-ASmdr1 product was produced by the adenoviral vector AdEasy system, which can express antisense RNA against the mdr1 gene. Following that, the recombinant adenovirus was transfected into the P-glycoprotein- producing multidrug resistance cell line, SMMC7721/ADM human HCC cells resistant to adriamycin (ADM) and daunorubicin (DNR). In order to investigate the reversal of multidrug resistance phenotype, we measured the expression of mdr1 mRNA by RT-PCR and the production of P-glycoprotein by flow cytometry. The sensitivities for ADM and DNR SMMC7721/ADM cells were examined by [3-(4, 5-dimethylthi-azol-2-yl)-2,5 diphenyl-terazolium bromide] (MTT) analysis. RESULTS: The low-level expression of mdr1 mRNA and P-glycoprotein production were observed in parental sensitive cells SMMC/7721 in addition to the overexpressionof mdr1 mRNA and P-glycoprotein in SMMC7721/ADM cells. The transfection of antisense-RNA into SMMC7721/ ADM cells resulted in decreases of mdr1 mRNA and P-glycoprotein, but increase of drug sensitivities. The sensitivities of transfected SMMC7721/ADM cells to ADM and DNR in IC50 reduced by 31.25% and 62.96% respectively. CONCLUSIONS: Mdr1 antisense RNA can increase the sensitivities of SMMC7721/ADM cells to anticancer drug by decreasing the expression of the mdr1 gene and inhibiting P-glycoprotein expression. This strategy may be applicable to cancer patients with P-glycoportein mediated multidrug resistance.

Construction of neuron specific vector of human antisense noggin gene expression

Thenoggin gene is present in the central nervous system in embryonic and postnatal mammals, and plays an important role in maintaining nervous system development and physiological function A 0.76-kb sequence of human noggin gene was cloned by polymerase chain reaction with the digestion site of Hind Ill and Xba I on the 5＇ end. The cloned fragment was reversely inserted into pCS2＋[Tal]-GFP plasmid, an neural cell-specific antisense eukaryotic expression vector. The plasmid expresses antisense for human noggin specifically in neurons, which may facilitate understanding of the physiological function of noggin.

EFFECT OF BcL-2 ANTISENSE DRUG WITH DIFFERENT STRUCTURE ON THE BIOLOGICAL FUNCTION OF K562 CELLS

Objective: To study the differences and similarities of the antisense drugs with different structures on the biological functions of K562 cells. Methods: Cytotoxic effects were measured by use of a cell viability assay. Flow cytometric analysis and agarose gel electrophoresis of DNA fragmentation were also performed. The expression level of protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Results: PNA targeting the coding region of the Bcl-2 messenger RNA could effectively inhibit K562 cell viability, down-regulate the synthesis of the Bcl-2 protein and increase cell apoptosis. By 72 h after the Bcl-2 antisense PNA treatment, K562 cells showed more reduction in the level of Bcl-2 protein compared with cells treated with the antisense ODN. After treatment with 10 μmol/L of Bcl-2 antisense PNA or antisense ODN for 72 h, apoptotic rates of K562 cells were 13.15±1.13 and 11.72±1.12, respectively. Furthermore, there was significant difference in the percentage of apoptotic cells between antisense PNA group and antisense ODN group. Conclusion: The results suggest that antisense PNA targeting the coding region of Bcl-2 mRNA has better antisense effects than the antisense oligonucleotides on inducing apoptosis of K562 cells. Key words Bcl-2 - Antisense peptide nucleic acid - Antisense oligonucleotide - K562 cells - Apoptosis CLC number Q255 Foundation item: This work was supported by the Key Foundation of Science & Technology Program of Guangzhou (No.2001-Z-037-01), and the Nature Science Key Foundation of Guangdong Province (No. 021195).Biography: LEI Xiao-yong(1970–), male, associate professor, doctor of medicine, Institute of Pharmacy and Pharmacology, Nanhua University, majors in tumor pharmacology.

Gene therapeutic approaches to inhibit hepatitis B virus replication

Acute and chronic hepatitis B virus(HBV) infections remain to present a major global health problem. The infection can be associated with acute symptomatic or asymptomatic hepatitis which can cause chronic inflammation of the liver and over years this can lead to cirrhosis and the development of hepatocellularcarcinomas. Currently available therapeutics for chronically infected individuals aim at reducing viral replication and to slow down or stop the progression of the disease. Therefore, novel treatment options are needed to efficiently combat and eradicate this disease. Here we provide a state of the art overview of gene therapeutic approaches to inhibit HBV replication. We discuss non-viral and viral approaches which were explored to deliver therapeutic nucleic acids aiming at reducing HBV replication. Types of delivered therapeutic nucleic acids which were studied since many years include antisense oligodeoxynucleotides and antisense RNA, ribozymes and DNAzymes, RNA interference, and external guide sequences. More recently designer nucleases gained increased attention and were exploited to destroy the HBV genome. In addition we mention other strategies to reduce HBV replication based on delivery of DNA encoding dominant negative mutants and DNA vaccination. In combination with available cell culture and animal models for HBV infection, in vitro and in vivo studies can be performed to test efficacy of gene therapeutic approaches. Recent progress but also challenges will be specified and future perspectives will be discussed. This is an exciting time to explore such approaches because recent successes of gene therapeutic strategies in the clinic to treat genetic diseases raise hope to find alternative treatment options for patients chronically infected with HBV.