Biomedical subjects

Ting Liu

Publications and source records attributed to Ting Liu.

At least 37 records · Page 2Linked to original sources

PubMed2006

[Implantation of FK506 drug delivery system into the anterior chamber for inhibiting corneal rejection in high risk rabbit corneal allograft].

OBJECTIVE: To study the effects of a biodegradable FK506 drug delivery system (DDS) on the inhibition of corneal rejection, to measure the concentration of FK506 in the aqueous humor and to study the relationship between intraocular concentration of FK506 and its immunosuppressive effects on corneal rejection. METHODS: Corneal neo-vascularization was induced by 5 - 0 silk sutures in 68 New Zealand rabbits to establish a high risk corneal transplantation model. A unilateral 7 mm diameter central penetrating corneal transplantation was performed with 7.5 mm diameter grafts from health New Zealand rabbit donors. Grafted rabbits were randomized into five groups as follows: Group A: untreated control animals; Group B: DDS anterior chamber recipients without drug; Group C: 1 mg cyclosporine DDS anterior chamber recipients; Group D: 0.1% FK506-olive oil drop recipients; Group E: 0.5 mg FK506 DDS anterior chamber recipients. Grafts were examined with a slit lamp every other day and clinical conditions were scored for up to 28 weeks. The aqueous humor and cornea of Group D and Group E were collected, and the concentration of FK506 was determined. The expression of cytokines IL-2R alpha, MCP-1, Fas and FasL mRNA was detected with in situ hybridization method. RESULTS: The median survival time was (17.9 +/- 4.7) d in Group A (untreated corneal allograft), (20.0 +/- 3.7) d in Group B, (56.3 +/- 8.8) d in Group C, (78.1 +/- 7.2) d in Group D, and over 180 d in Group E. Statistically significant difference (F = 926.37, P = 0.0000) in the survival time of allograft has been found between Group E and other groups. The concentration of FK506 in aqueous humor was (15.7 +/- 2.6) ng/ml in Group E at one week and remained stable for at least 24 weeks, much higher than that in Group D (<0.3 ng/ml). The concentration of FK506 in cornea was also higher in Group E than that in Group D. The expression of cytokines IL-2R alpha and MCP-1 mRNA was detected, but the expression of Fas and FasL mRNA was not detected in Group A. CONCLUSIONS: FK506 DDS implanted in the anterior chamber can significantly prolong corneal allograft survival in high-risk corneal graft rejection model. This intraocular DDS may be a valuable adjunct for the suppression of immune graft rejection in high-risk corneal transplants.

Animals↗

PubMed2006

[Meta-analysis of diagnostic capability of frequency-doubling technology (FDT) for primary glaucoma].

OBJECTIVE: To evaluate the overall diagnostic capabilities of frequency-doubling technology (FDT) in patients of primary glaucoma, with standard automated perimetry (SAP) and/or optic disc appearance as the gold standard. METHODS: A comprehensive electric search in MEDLINE, EMBASE, Cochrane Library, BIOSIS, Previews, HMIC, IPA, OVID, CNKI, CBMdisc, VIP information, CMCC, CCPD, SSreader and 21dmedia and a manual search in related textbooks, journals, congress articles and their references were performed to identify relevant English and Chinese language articles. Criteria for adaptability were established according to validity criteria for diagnostic research published by the Cochrane Methods Group on Screening and Diagnostic Tests. Quality of the included articles was assessed and relevant materials were extracted for studying. Statistical analysis was performed with Meta Test version 0.6 software. Heterogeneity of the included articles was tested, which was used to select appropriate effect model to calculate pooled weighted sensitivity and specificity. Summary Receiver Operating Characteristic (SROC) curve was established and the area under the curve (AUC) was calculated. Finally, sensitivity analysis was performed. RESULTS: Fifteen English articles (21 studies) of 206 retrieved articles were included in the present study, with a total of 3172 patients. The reported sensitivity of FDT ranged from 0.51 to 1.00, and specificity from 0.58 to 1.00. The pooled weighted sensitivity and specificity for FDT with 95% confidence intervals (95% CI) after correction for standard error were 0.86 (0.80 - 0.90), 0.87 (0.81 - 0.91), respectively. The AUC of SROC was 93.01%. Sensitivity analysis demonstrated no disproportionate influences of individual study. CONCLUSIONS: The included articles are of good quality and FDT can be a highly efficient diagnostic test for primary glaucoma based on Meta-analysis. However, a high quality perspective study is still required for further analysis.

Aged↗

PubMed2006

[Osteoblasts differentiated from human marrow bone mesenchymal stem cells support hematopoietic stem/progenitor cells from umbilical cord blood].

This study was aimed to construct a two-dimensional culture system by using osteoblasts induced from human marrow mesenchymal stem cells (MSC) and to investigate its support effect on survival of hematopoietic stem/progenitor cells for umbilical cord blood (UCB) ex vivo. MSCs were isolated from adult human bone marrow and were cultured, the second generation of MSCs were induced into osteoblasts which were irradiated with 20 Gy gamma rays in a Cobalt 60 source and confluenced into a feeder layer. CD34(+) cells were selected from fresh umbilical cord blood samples by using Microbead Kit of MiniMACS and seeded into the two-dimensional culture system to culture ex vivo without exogenous cytokines. By using colony-forming assay, high proliferative potential colony-forming cell assay, and long-term culture initiating cell assay, the ability of the two-dimensional system to culture HSCs/HPCs was observed. The results showed that the osteoblasts induced from bone marrow MSC in constructed two-dimensional culture system displayed more significant support effect on survival of hematopoietic stem/progenitor cells from umbilical cord blood (UCB) ex vivo, compared with other culture systems, especially on long term HSCs survival ex vivo. It is concluded that the two-dimensional culture system constituted by osteoblasts induced from human MSCs has certain ability of supporting maintenance and multipotency of HSCs/HPCs from umbilical cord blood in vitro, especially sustaining survival of HSC in long-term culture. It has also been proved that osteoblasts play a crucial role in regulation of HSC growth.

Bone Marrow Cells↗

PubMed2006

[Analysis of differential protein expression profile between retinoic acid resistant and sensitive acute promyelocytic leukemia cells].

BACKGROUND & OBJECTIVE: This study was to compare the protein expression profiles between retinoic acid (RA) resistant and sensitive acute promyelocytic leukemia (APL) cells by proteomic research method. METHODS: Total cellular proteins extracted from a RA sensitive cell line NB4 and a RA resistant cell line MR2 were separated by two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). High quality 2D-PAGE protein profiles were obtained and analyzed by PDQuest v7.1 analysis software to screen differentially expressed protein spots. Those sports were identified by mass spectrometry. RESULTS: 2-DE patterns of APL cell lines with high-resolution and reproducibility were obtained. The average spots for MR2 and NB4 cells were 890+/-45 and 912+/-56, respectively. 57 significantly differentially expressed protein spots were screened, among which 23 protein spots were identified to be up-regulated and 34 down-regulated in MR2 cells compared with NB4 cells. Ten proteins were identified by mass spectrometry, with a successful identification rate of 70%. The identified proteins could be classified into different categories: oncogenes,cell cycle regulator and signal transducer. CONCLUSION: The utilization of 2D-PAGE is effective in identifying the protein expression profiles between RA resistant and sensitive APL cells, therefore this study may provide a novel clue to elucidate the drug resistant mechanisms of all-trans retinoic acid.

Antineoplastic Agents↗

PubMed2006

[Experimental study of donor natural killer cells reducing graft-versus-host disease and enhancing engraftment].

The purpose of this study was to investigate whether pretransplant infusion of reactive natural killer cells (NK cells) from donor or recipient can reduce graft-versus-host disease (GVHD) and enhance engraftment in bone marrow transplantation (BMT). Recipient BALB/c mice were divided into 4 groups after received 6.5 Gy total-body irradiation (TBI): control group 1 was treated with nothing, control group 2 received BMT alone, experiment group 1 received BMT and autoreactive NK cells, experiment group 2 received BMT and alloreactive NK cells. Life span, clinical and pathologic changes of GVHD and chimerism rate of each group were evaluated. The results showed that all mice were survival in control group 1. The life span was shorter in experiment group 1 than that in control group 2 (P < 0.05) and longer in experiment group 2 than that in control group 2 (P < 0.01). GVHD was higher in score of experiment group 1 than in control group 2 (P < 0.05) but lower in experiment group 2 than that in control group 2 (P < 0.01). The donor chimerism rate in both two experiment groups were higher than that in control group 2 (P < 0.05), however, the donor chimerism rate was higher in experiment group 2 than that in experiment group 1 (P < 0.01). It is concluded that pretransplant infusion of alloreactive donor NK cells can prolong life span, reduce the degree of GVHD and enhance engraftment. But autoreactive recipient NK cells can shorten life span, aggravate the degree of GVHD and also enhance engraftment, which is weaker than that using alloreactive donor NK cells.

Animals↗

PubMed2006

[16S rDNA-RFLP analysis of structure and diversity of an aerobic microbial community degrading hexachlorobenzene].

Hexachlorobenzene is a chlorinated aromatic hydrocarbon that was widely used as a seed dressing for prevention of fungal growth on crops, and is also a component of fireworks, ammunition, and synthetic rubbers. Because of the bioaccumulation and persistence of hexachlorobenzene as well as its potential toxicity, hexachlorobenzene must be removed from environment. The potential for aerobic dechlorination of hexachlorobenzene by a hexachlorobenzene-adapted mixed culture was investigated. An aerobic microbial community which was able to grow at the presence of hexachlorobenzene was enriched from sediment from contaminated site after incubating about 2 months. During the growth of the mixed microorganisms on hexachlorobenzene, the accumulating consumption of oxygen, the microbial population curve and the release of Cl- were investigated. The data suggest the rapid degradation of hexachlorobenzene to support microbial growth and the aerobic decholrination of hexachlorobenzene was observed. The result showed that the mixed microorganisms were able to utilize hexachlorobenzene as sole carbon and energy source. It was shown that up to 55% of HCB could be degraded during 18 days incubation at 30 degrees C in mineral salts medium (pH 7.0) with 4.5mg/L HCB. The calculated rate of hexachlorobenzene biodegradation was 137.5 microg/ (L x d). The 16S rDNA genes were amplified from community DNA by using primers specific to bacteria and were subsequently cloned. The cloned 16S rDNA fragments were reamplified, and restriction analysis was performed following separate digestion with enzymes Hae III and Rsa I. Application of restriction fragment length polymorphism screening approach revealed 9 clusters, and 3 major clusters were sequenced. Nearly complete 16S rDNA sequence analysis show that the microbial community was dominated by Alcaligenes and Azospirillum groups. This is the first report describing aerobic dechlorination of hexachlorobenzene via dehalorespiration by a microbial community which was enriched from contaminated site. The microbial community can be used to degrade highly recalcitrant chlorinated pollutants.

Bacteria, Aerobic↗

PubMed2005

SIKE is an IKK epsilon/TBK1-associated suppressor of TLR3- and virus-triggered IRF-3 activation pathways.

Viral infection or TLR3 engagement causes activation of the transcription factors IRF-3 and NF-kappaB, which collaborate to induce transcription of type I IFN genes. IKKepsilon and TBK1 are two IKK-related kinases critically involved in virus- and TLR3-triggered activation of IRF-3. We identified a protein termed SIKE (for Suppressor of IKKepsilon) that interacts with IKKepsilon and TBK1. SIKE is associated with TBK1 under physiological condition and dissociated from TBK1 upon viral infection or TLR3 stimulation. Overexpression of SIKE disrupted the interactions of IKKepsilon or TBK1 with TRIF, RIG-I and IRF-3, components in virus- and TLR3-triggered IRF-3 activation pathways, but did not disrupt the interactions of TRIF with TRAF6 and RIP, components in TLR3-triggered NF-kappaB activation pathway. Consistently, overexpression of SIKE inhibited virus- and TLR3-triggered interferon-stimulated response elements (ISRE) but not NF-kappaB activation. Knockdown of SIKE potentiated virus- and TLR3-triggered ISRE but not NF-kappaB activation. Moreover, overexpression of SIKE inhibited IKKepsilon- and TBK1-mediated antiviral response. These findings suggest that SIKE is a physiological suppressor of IKKepsilon and TBK1 and plays an inhibitory role in virus- and TLR3-triggered IRF-3 but not NF-kappaB activation pathways.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Combined radiation and suicide gene therapy for Nasopharyngeal Carcinoma: in vitro and in vivo models.

Nasopharyngeal carcinoma (NPC) cells are highly radiosensitive. Historically, the main modality for NPC has been radiotherapy. Now, to explore a novel and more effective approach to NPC therapy, a combined strategy of suicide gene and radiation was developed. The yeast CDglyTK (yCDglyTK) gene controlled by a synthetic CMV-enhanced Egr-1 promoter (CE) was constructed whose expression in NPC CNE-2 cells was detected by Western Blot. The cytotoxicity of the combined radio-gene therapy was assayed by MTT. The conversion rate of 5-FC to 5-FU in vitro and the bio-distribution of 5-FU in vivo were determined by HPLC. An animal study in which yCDglyTK-expressing CNE-2 tumors were treated with 5-FC and radiation was also conducted. The results reveal that yCDglyTK is expressed in CNE-2 cells, the CEyCDglyTK/5-FC system has a potent anti-tumor action in NPC, and the radio-sensitization of Egr-1 promoter plays a key role in the killing of CNE-2 cells and in the conversion of 5-FC to 5-FU. Moreover, the treated tumors regressed significantly, and a significant difference of tumor volumes was observed between the CEyCDglyTK+5-FC+radiation group and the other four groups (P<0.05). The results also showed that suicide gene therapy and radiation have a synergic anti-tumor effect on NPC, and the combined strategy of radio-gene therapy is of great potential as a substitute for the traditional method, radiation alone, in NPC therapies.

Journal Article↗

PubMed2005

Hydration and dewetting near graphite-CH(3) and graphite-COOH plates.

The dynamics of water near the nanoscale hydrophobic (graphite-CH(3)) and hydrophilic (graphite-COOH) plates has been studied in detail with molecular dynamics simulations in this paper. It is shown that these designed surfaces (by growing a layer of methyl or carboxyl groups on top of graphite) can have a significant impact on the neighboring water dynamics, with the hydrophilic carboxyl surface having even more profound effects. The water hydrogen bond lifetime is much longer near both types of surfaces than that in the bulk, while on the other hand the water diffusion constant is much smaller than that in the bulk. The difference in the diffusion constant can be as large as a factor of 8 and the difference in the hydrogen bond lifetime can be as large as a factor of 2, depending on the distance from the surface. Furthermore, the water molecules in the first solvation shell of surface atoms show a strong bias in hydroxyl group orientation near the surface, confirming some of the previous findings. Finally, the possible water dewetting transition between two graphite-CH(3) plates and the effect of the strength of the solute-solvent attractions on the water drying transition are investigated. The relationship among the dewetting transition critical distance, van der Waals potential well depth, and water contact angle on the graphite-CH(3) surface is also analyzed on the basis of a simple macroscopic theory, which can be used to predict the dewetting transition critical distance.

Chemistry, Physical↗

PubMed2005

Inhibition of human lens epithelial B-3 cell proliferation by adenovirus-mediated transfer of antisense c-myc construct.

PURPOSE: To investigate the effects of adenovirus-mediated transfer of antisense c-myc construct on human lens epithelial B-3 (HLE B-3) cell proliferation, apoptosis and cell cycle. METHODS: HLE B-3 cell cultures were transduced with replication-defective adenovirus bearing either a nuclear-targeted beta-galactosidase (Ad-lacZ) or an antisense c-myc construct (Ad-AS-myc). The presence of beta-galactosidase activity in the transduced cultures was detected by immunohistochemical X-Gal staining, while c-myc mRNA and protein expression levels were evaluated by RT-PCR and Western blot analysis. HLE B-3 cell proliferation within 96 h after the transduction was analyzed by cell counting and MTT colorimetric assay. Apoptosis and cell cycle of the HLE-B3 cells were examined by flow-cytometric analysis. RESULTS: The mean transduction efficiency was 80% for HLE B-3 cells. Downregulation of c-myc mRNA and protein expression was noticed at 48, 96 and 144 h after the transduction with Ad-AS-myc. Cytostatic effects of Ad-AS-myc in HLE B-3 cells were obvious within 96 h after the transduction. An increased incidence of apoptosis and G1-phase arrest was identified in the Ad-AS-myc-transduced HLE B-3 cells. CONCLUSIONS: HLE B-3 cells were successfully transduced with adenovirus-mediated antisense c-myc construct. Ad-AS-myc transduction could significantly inhibit cell proliferation and induce cell apoptosis and G1-phase arrest in HLE B-3 cells. It may provide a novel approach for prevention of posterior capsular opacification.

Adenoviruses, Human↗

PubMed2005

FK506 in a biodegradable glycolide-co-clatide-co-caprolactone polymer for prolongation of corneal allograft survival.

PURPOSE: FK506 has been extensively used in preventing immune rejection for human organ transplantation. This study aimed to evaluate the effects of a biodegradable FK506 drug delivery system (DDS) implanted into anterior chamber for the prolongation of corneal allograft survival in high-risk keratoplasty. METHODS: Biodegradable glycolide-co-clatide-co-caprolactone polymer (PGLC) was used as drug carrier to be incorporated with 0.5 mg of FK506 powder. The drug release from the FK506-PGLC DDS was evaluated in vitro and in vivo. The FK506-PGLC DDS was implanted into the anterior chamber of 12 high-risk keratoplasty rabbits. The graft survival time and clinical features of the FK506-PGLC DDS group were compared with the untreated, PGLC DDS, cyclosporin A-PGLC DDS, and 0.5% FK506 drops groups. The histopathological examination was performed to evaluate the safety of the FK506-PGLC DDS. RESULTS: The mean graft survival time was longest (> 180 days) in the FK506-PGLC DDS group. In vivo, the FK506 concentration in aqueous humor peaked on day 28 (17.9 +/- 2.3 ng/ml) and kept a sustained release for at least 168 days. No adverse reactions were observed in the FK506-PGLC DDS group. CONCLUSIONS: Biodegradable FK506-PGLC DDS implanted into anterior chamber can effectively prevent immune rejection in high-risk keratoplasty model, presenting a promising approach for the prolongation of corneal allograft survival.

Absorbable Implants↗

PubMed2005

Calcium phosphate nanoparticles as a novel nonviral vector for efficient transfection of DNA in cancer gene therapy.

To explore an efficient gene vector in cancer gene therapy, a novel nonviral vector calcium phosphate nanoparticle (CPNP) was developed. Transmission electromicroscopy and Zeta potential demonstrated that CPNP was 23.5-34.5 nm diameters and had +16.8 mV positive surface charges. The analysis of the CPNPDNA complex showed CPNP could transfer foreign DNA into targeted cells with high transfection efficiency, as well as its DNA-binding property and protection of DNA from degradation. Furthermore, the CPNP-DNA complex had no obvious cytotoxicity for CNE-2 cells, while the liposome-DNA complex had certain cytotoxicity. When the CPNP combined with suicide genes yCDglyTK for nasopharyngeal carcinoma (NPC) therapy in vitro, just 24.76% of cells survived when the wild-type CNE-2 cells were treated with the CPNP-yCDglyTK complex plus the prodrug, 5-FC (200 mg/mL). Otherwise, the expression of yCDglyTK was detected in implanted CNE-2 tumors by reverse transcription-polymerase chain reaction (RT-PCR) analysis when the CNE-2 tumor was treated with an intratumoral injection of the CPNPyCDglyTK complex. Our results showed that the CPNP might be a potential vector for gene therapy.

Animals↗

PubMed2005

A wavelet approach for the analysis of folding trajectory of protein Trp-cage.

Understanding how protein folds into a functional native structure is arguably one of the most challenging problems remaining in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape in terms of various reaction coordinates such as the fraction of native contacts, the radius of gyration, the RMS deviation from the native and so on. In this paper, we present a wavelet approach towards understanding the global state changes during protein folding. The approach is based on the wavelet analysis on the trajectories of various reaction coordinates to identify the significant intermediate states or structural motifs in the folding process. We demonstrate through an example protein Trp-cage that this approach extracts crucial information about protein folding intermediate states as well as the time correlation among these states. Furthermore, the current approach reveals a meaningful structural pattern that had been overlooked in previous works, which provides a better understanding of the folding mechanism as well as the limitation of the current force fields.

Algorithms↗

PubMed2005

[A preliminary observation of bone marrow angiogenesis in patients with myelodysplastic syndromes].

OBJECTIVE: To observe the change of angiogenesis in the bone marrow of patients with myelodysplastic syndromes, and to explore its effect on this disease. METHODS: The sera of 20 patients with myelodysplastic syndromes and 20 normal controls were collected; the level of serum vascular endothelial growth factor (S-VEGF) was measured by ELISA; and the microvessel density (MVD) of marrow biopsy specimens was examined with the streptavidine-biovatidine immunoperoxidase method; the count of MVD was evaluated with image analysis system. RESULTS: The level of S-VEGF and count of MVD were higher than that of normal control, and both significantly elevated in MDS/MPD and RAEB-t subtypes. The S-VEGF was positively related with MVD (P<0.05), and S-VEGF and MVD were closely related with the percentage of blast in marrow and in peripheral blood. CONCLUSION: The result shows that there is abnormal angiogenesis in MDS bone marrow, and the elevated levels of S-VEGF and MVD are closely related with a progressive status in myelodysplastic syndromes.

Adult↗

PubMed2005

[Inhibition of proliferation of human lens epithelial cell line HLE-B3 by adenovirus-mediated transfer of antisense c-myc gene].

OBJECTIVE: To investigate the effects of adenovirus-mediated transfer of antisense c-myc gene on proliferation and survival of human lens epithelial cell. METHODS: Immortalized human lens epithelial cell line (HLE-B3) was infected by replication-deficient adenovirus of antisense c-myc gene (Ad-AS-myc). The expression of c-myc mRNA and protein was detected by RT-PCR and Western blotting respectively. Cell proliferation was analyzed by cell growth curve and MTT colorimetric assay, cell death and cell cycle of HLE-B3 were examined by flow cytometry. RESULTS: More than 80% of HLE-B3 cells were infected successfully after 48 hours incubation with Ad-AS-myc adenovirus. The expression of c-myc mRNA and protein was decreased. Ad-AS-myc suppressed the growth rate, enhanced the cell death (18.33% after 48 hours and 26.93% after 96 hours transduction). The proportions of G1 phase cells were increased to (60.50 +/- 7.59)% after 48 hours and (81.90 +/- 8.60)% after 96 hours transduction, while the proportions of S phase cells were decreased to (28.40 +/- 3.38)% after 48 hours and (16.75 +/- 8.97)% after 96 hours infection. CONCLUSIONS: Adenovirus-mediated antisense c-myc may inhibit proliferation and induce apoptosis of HLE-B3.

Adenoviruses, Human↗

PubMed2005

[Prokaryotic expression, purification and identification of recombinant Xenopus laevis and mouse vascular endothelial growth factors].

OBJECTIVE: To perform prokaryotic expression, purification and identification of Xenopus laevis and mouse vascular endothelial growth factors (VEGF). METHODS: The prokaryotic expression vectors were constructed by restriction endonuclease digestion and ligation, then the recombinant vector pET-xVEGF and the control vector pET-mVEGF were identified by restriction enzymatic digestion and DNA sequencing. The confirmed vectors were transformed into Escherichia coli. BL21 (DE3) and recombinant protein expression was induced by Isopropy-beta-D-thiogalactoside. The recombinant xVEGF and mVEGF were obtained and purified by Ni-NTA affinity chromatography under denature conditions and enterokinase specific digestion, respectively. Finally, the purified proteins' molecular weights and specificity were detected by SDS-PAGE and western blot analysis. RESULTS: Two new recombinant expression vectors, pET-xVEGF and pET-mVEGF, were constructed successfully. The xVEGF and mVEGF fusion proteins were expressed in E. coli. BL21 (DE3) stably, and the molecular weights of the purified xVEGF and mVEGF were identical to the expected values. The purity of final products reached a level higher than 95%. In addition, these two purified proteins could react with a specific antibody against mouse VEGF as expected. CONCLUSION: Recombinant Xenopus laevis VEGF and its control mouse VEGF protein may provide tools for further study of anti-tumor active immunity with this xenogeneic protein vaccine in mouse tumor models.

Animals↗

PubMed2005

[Construction of eukaryotic expression vector of siRNA specific to bcr/abl fusion gene].

OBJECTIVE: To construct eukaryotic expression vector of siRNA specific to bcr/abl and to initially investigate the effect of recombinant plasmid on bcr/abl and P210 protein expression in K562 cells. METHODS: siRNA (small interfering RNA) was designed according to the Tuschl's principle of RNAi-based medicine, and was converted into cDNA coding expression of shRNA (small hairpin RNAs)of siRNA for bcr/abl fusion gene. The cDNA was synthesized and inserted into plasmid pTER. The pTER117 and pTER363 of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymerase III, identified by the restriction map and the sequence analysis, and transfected into K562 cells by Lipofectamine. Expression of bcr/abl mRNA was assayed by RT-PCR; expression of P210 protein was detected by immunohistochemistry. RESULTS: The pTER117 and pTER363 of recombinant plasmid identified by the restriction map and the sequence analysis completely coincided with the designs. 24 hours after transfection in K562 cells, the recombinant plasmid could down regulate the expression of the bcr/abl mRNA and bcr/abl protein(P210) in K562 cells. CONCLUSION: The siRNA eukaryotic expression vector against bcr/abl mRNA has been successfully conctructed, and it effectively inhibits the expression of bcr/abl in K562 cells.

Base Sequence↗

PubMed2005

[Establishment of a drug resistant leukemia cell line caused by mdr1 gene].

OBJECTIVE: To establish a drug resistant leukemia cell line caused by mdr1 gene for the mdr1 reversing research. METHODS: The leukemia sensitive cell line K562 was transfected with mdr1 cDNA by liposome-induced gene transfection and selected by G418. The mdr1 phenotype was identified by RT-PCR, the P-gp expression was detected by flow cytometry, the function of P-gp was measured by Rh123 efflux experiment, and the sensitivity of cell lines to the drug was detected by MTT test. Results The transfected K562/mdr1 with mdr1 cDNA highly expressed mdr1 and showed a remarkable drug resistant feature. CONCLUSION: mdr1 plays an important role in drug resistance and the mdr1 cell line established by gene transfection can provide a better cell model for mdr1 reversing research.

ATP Binding Cassette Transporter, Subfamily B, Mem↗