Abstract:Aim To construct pEGFP-GPR109A recombinant plasmid by inserting GPR109A gene into pEGFP-N3 vector and establish a Chinese hamster ovary(CHO)cell line stably expressing nicotinic acid receptor GPR109A. Methods The pEGFP-GPR109A recombinant plasmid was constructed,which was subsequently transformed into DH5α E coli.After identification by PCR,digestion with restriction endonuclease and sequencing,the recombinant plasmid was transfected into CHO cells via lipofectamine 2000.The stable transfectants were screened by antibiotic G418.The fluorescent signal of cloned cell lines were detected by fluorescent microscope.The GPR109A gene mRNA expression was analyzed by RT-PCR and the fusion protein expression of green fluorescence protein and nicotinic acid receptor GPR109A(GFP-GPR109A) was detected by Western Blotting.The cell localization of fusion protein was detected by laser scanning confocal microscope. Results The results of PCR,restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pEGFP-GPR109A.Western Blotting showed that the fusion protein GFP-GPR109A was expressed stably in CHO cells.The fusion protein was mainly localized on cell membrane detected by laser scanning confocal microscope. Conclusion The eukaryotic expression vector pEGFP-GPR109A has been successfully constructed and a CHO cell line stably expressing fusion protein GFP-GPR109A was established,which facilitated the physiological and pathological function research of GPR109A and also provided important foundation for following up drug screening for atherosclerosis treatment.

Abstract:Aim To develop a novel method to effectively deliver enhanced green fluorescence protein (EGFP)C3 into Chinese hamster orary (CHO) cells in vitro by ultrasound-mediated microbubble destruction. Methods Expression of the EGFPC3 gene was quantified by laser confocal microscopy and FACS analysis. Cell viability was assayed by Trypan Blue staining. Results Ultrasound combined with microbubbles can enhance gene transfer in cultured cells, but the effect is vary according to the utrasound condition and concetration of microbubble. Optimal gene expression occurred with microbubble at the concentration of 10% and ultrasound parameter at 0.8 MHz, 1.0 W/cm 2,10% duty cycle, 60 s. Under this condition, the transfection rate of EGFPC3 gene with ultrasound-mediated microbubble destruction method was similar to that of transfection with lipofectamine (56.2%±2.6% versus 60.8%±4.1%, p>0.05) and the relative fluorescence intensity of EGFPC3 gene was as high as that of transfection with lipofectamine (2035±32 versus 2140±28, p>0.05). Furthermore both albumin microbubble and ultrasound had no effect on cell viability. The cell viablity were 94.1%±4.6% or 93.8%±3.1% when cultured with 10% albumin microbubble or under ultrasound at 0.8 MHz, 1.0 W/cm 2, 10% duty cycle, 60 s respectively, which were no significantly difference compared with controls. Conclusions These results suggest a possible new strategy for ultrasound-mediated microbubble destruction method in gene therapy.

Abstract:Aim To develop a novel method to effectively deliver enhanced green fluorescence protein (EGFP)C3 into Chinese hamster orary (CHO) cells in vitro by ultrasound-mediated microbubble destruction. Methods Expression of the EGFPC3 gene was quantified by laser confocal microscopy and FACS analysis. Cell viability was assayed by Trypan Blue staining. Results Ultrasound combined with microbubbles can enhance gene transfer in cultured cells, but the effect is vary according to the utrasound condition and concetration of microbubble. Optimal gene expression occurred with microbubble at the concentration of 10% and ultrasound parameter at 0.8 MHz, 1.0 W/cm 2,10% duty cycle, 60 s. Under this condition, the transfection rate of EGFPC3 gene with ultrasound-mediated microbubble destruction method was similar to that of transfection with lipofectamine (56.2%±2.6% versus 60.8%±4.1%, p>0.05) and the relative fluorescence intensity of EGFPC3 gene was as high as that of transfection with lipofectamine (2035±32 versus 2140±28, p>0.05). Furthermore both albumin microbubble and ultrasound had no effect on cell viability. The cell viablity were 94.1%±4.6% or 93.8%±3.1% when cultured with 10% albumin microbubble or under ultrasound at 0.8 MHz, 1.0 W/cm 2, 10% duty cycle, 60 s respectively, which were no significantly difference compared with controls. Conclusions These results suggest a possible new strategy for ultrasound-mediated microbubble destruction method in gene therapy.