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Objective To investigate the effects of antisense p21 oligodeoxynucleotide (p21 ASODN) on the expression of p21 protein and extracellular matrix in cultured human glomerular mesangial cells (HGMC) under high glucose medium. Methods HGMCs were transfected with 50 nmol/L or 100 nmol/L p21 ASODN or scrambled control oligodeoxynucleotide (SCODN) using lipofectamine 2000. After incubation under normal (5.5 mmol/L) or high (30 mmol/L) glucose media and different times, HGMCs lysates were collected and the expression of p21, fibronectin and laminin was examined by Western blot. The secretion of fibronectin and laminin by HGMCs in supernatants of culture media was also detected with EOSA. Results High glucose media significantly stimulated the expression of p21, increased the syntheses and secretion of fibronectin and laminin in cultured HGMCs in a time-dependent manner. Transfection of HGMCs with p21 ASODN not only decreased p21 protein level caused by high glucose media, but also attenuated the expression and secretion of fibronectin and laminin in supematants of HGMCs lysates and culture media. Meanwhile, SCODN had no significant effects on the expression of p21, fibronectin and laminin. Conclusions High glucose promotes the expression of p21 , fibronectin and laminin in cultured HGMCs. Transfection of HGMCs with p21 ASODN can decrease p21 protein level caused by high glucose media, and attenuate the expression of fibronectin and laminin in supematants of HGMCs lysates and culture media. Modulation of p21 expression in HGMCs may work as an effective way to mitigate the progression of diabetic nephropathy.
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Objective To study the effect and mechanism of antisense oligodeoxynucleotides (ASONs) inhibiting hepatitis B virus (HBV) expression. Methods We designed and synthesized antisense oligodeoxynucleotides directly against HBV PreS 2 gene and noncomplementary sequence control. 2.2.15 cells were chosen as cell model. Inhibitory effect of ASONs on HBV gene expression were assayed by ELISA. Cell apoptosis and proliferation were detected by Fascan Flow Cell Cytometer. Effect of ASONs on cell metabolism was detected by radioimmunoassay (RIA) and MTT assay. Results ASONs were able to effectively inhibit HBV expression. Their inhibitory rates of HBsAg and HBeAg were 66% and 91%, respectively. Noncomplementary sequence control group (both inhibitory rates were 11%) was not able to inhibit HBV expression. ASON might induce host cell apoptosis. Cell apoptosis rates on 3rd and 6th day were 6.10% and 6.43%, respectively. Proliferation index were 37% and 36%, respectively. Results of RIA and MTT showed that ASON had not cytoxicity on host cell. Conclusions Not only are ASON able to inhibit HBV gene expression with sequence specific but also clear HBV in the way of apoptosis.
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In order to explore the possibility and feasibility of administering antisense oligodeoxynucleotide to vascular adventia delivering by fibrin tissue adhesive, the authors first observed the physical characteristics of the adhesive, then the blue dye (methylenophin) was dissolved in the adhesive and the color change of the supernate was monitored. The adhesive with antisense oligomer inside was immerged in saline and the optical density (OD) value of the released oligomer was measured by ultraviolet spectrophotometer at different time points, then the in vitro controlled releasing curve was made. The preliminary in vitro test found the adhesive clot in 10 seconds when the two kinds of solution were mixed together, and the surface was membrane like under microscope, the clot inside was silk protein like configuration and was filled up with particle like liquid. The clot was elastic and had some reabsorbable property like that of sponge. The blue dye inside the clot gave a more and more significant dyeing of the supernate with time going. The in vitro releasing curve indicated that most oligomer was released within 72 hours. This implied that the adhesive clot is like a sponge reservoir of drugs, it could elongate the action time of antisense drug at vascular adventia.
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0 05). The results suggest antisense C myc oligodeoxynucleotide significantly suppressed the intimal hyperplasia in anastomosis by local fibrin glue. The investigation provides the basis for the early clinical trials of antisense C myc for the prevention of restenosis after anastomosis.
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Objective To investigate the effect of integrin-?1 antisense oligodeoxynucleotide(ASODN) on(human) pancreatic cancinoma transplanted subcutaneously in nude mice.Methods The models of human(pancreatic) cancinoma transplanted subcutaneously were established in nude mice,then divided randomly into 3 groups and different treatment was given respectively(control group,random oligodeoxynucleotide group and ASODN group).After treatment,the weight of nude mice and tumor volume were observed,and the tumor growth inhibitory rate and the tumor response rate were calculated.The expressions of integrin-?1 mRNA and protein in tumor tissue were determined by RT-PCR and Western-blot.Results The tumor growth inhibitory rate in the random oligodexynucleotide group and the ASODN group was 4.75% and 72.70%,respectively.The tumor decrease rate of the ASODN group was 10.91%.The expression level of integrin-?1 mRNA and protein was decreased in the ASODN group compared with other 2 control groups. Conclusions Our findings suggest that integrin-?1 antisense oligodeoxynucleotides result in marked inhibition of human pancreatic(cancinoma) growth in nude mice.It may be a novel treatment approach for human pancreatic carcinoma.
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AIM: To observe the effect of antisense bcl-2 oligodeoxynucleotides(AS-PS-ODN) on bcl-2 mRNA and protein expression, cell proliferation,viability and apoptosis in a small-cell lung cancer cell line NCI-H446. METHODS: Semi-quantitative RT-PCR was performed to detect the bcl-2 mRNA expression, the Bcl-2 protein was determined by immunocytochemistry and flow cytometry analysis, and the effect of bcl-2 AS-PS-ODN on cell proliferation, viability and apoptosis were investigated by colony assay , cell count, DNA content analysis and TUNEL. RESULTS: ① 1 ?mol/L bcl-2 AS-PS-ODN significantly down-regulated the expression of bcl-2 mRNA and protein. The inhibition rate of mRNA and protein were 69.5% and 62.7%, respectively. ② bcl-2 AS-PS-ODN decreased cell proliferation and viability , induced cell apoptosis.The apoptosis rate was 22.3%-32.7% in cells treated with 1?mol/L bcl-2 AS-PS-ODN. CONCLUSION: bcl-2 AS-PS-ODN down-regulated expression of bcl-2 mRNA and protein, inhibited cell proliferation and induced apoptosis in a small cell lung cancer cell line, NCI-H446.
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Aim To investigate whether F951,a novel Bcl-2 antisense oligodeoxynucleotide,down-regulates Bcl-2 expression in HL60 cells and inhibits HL-60 cells proliferation.Methods HL60 cells were cultured with F951 in variant doses.The proliferation of HL60 cells was assayed by MTT and Typan Blue exclusion test.Expression of Bcl-2 protein and its mRNA was measured by FACS and RT-PCR respectively.The apoptotic cells were detected by DNA ladder.Results After HL60 cells were treated with F951 in 5,10,20 ?mol?L~(-1) doses respectively for 1～5 days,they showed apparent inhibition of proliferation.With the improvement of the concentration of F951 and the prolongation of the time of treatment, F951 showed stronge effect in the aspect of inhibiting the HL60 cells proliferation.It was determined with MTT method that the inhibition rates of HL60 cells treated with 5,10,20 ?mol?L~(-1) F951 were 20.56%, 37.66%, 54.11% respectively. F951 significantly down-regulated the expression of Bcl-2 mRNA and protein in the HL60 cells.Typical DNA ladder was seen from gel electrophoresis. With the improvement of the concentration of F951,it showed more apparent effect in the aspect of inducing DNA ladder.Conclusion F951 can inhibit cells proliferation through down-regulating Bcl-2 gene expression and promoting cells apoptosis in HL60 cells.