Experimental Research RB94 Gene Transfection Into Retinoblastoma Cells Using Ultrasound-Targeted Microbubble Destruction

Objectives The purpose of this study was to explore the transfection of the recombinant expression plasmid pEGFP-C1/RB94 into human retinoblastoma cells using ultrasound-targeted microbubble destruction (UTMD).Methods1. We constructed a type of enhancement of EGFP and Rb94 co-expression vector, at the same time, we detected its expression in cultured eukaryocyte.2. The second part compared the difference between the new transfected method and the liposome transfected method.3. We explored the growth activity of HXO-Rb44 cells in different transfected methods.Results1. The sequence of the cloned DNA fragment is fully consistent with the sequence of Rb94 gene. The Rb94 gene was inserted into eukaryotic expression vector pEGFP-C1 exactly. Observed by fluorescent microscopy, the recombinant expression plasmid was transferred into HXO-Rb44 successfully. Efficacious expression of Rb protein was also examined by Western blotting.2. Transfection efficiency of microbubble as a gene vector was compared with that of the traditional gene vector liposome in the second part. Green fluorescence was more intense for the RB94 plasmid with microbubbles and ultrasound, the C1 plasmid with microbubbles and ultrasound and the RB94 plasmid with liposome than in RB94 plasmid with ultrasound, RB94 plasmid with microbubbles and control groups. The transfection efficiencies of the RB94 plasmid with microbubbles and ultrasound, the C1 plasmid with microbubbles and ultrasound and the RB94 plasmid with liposome groups were higher than those of the other groups. The difference between the three groups was not statistically significant. Western blot analysis showed that the protein expression of RB94 in the RB94 plasmid+ microbubbles+ ultrasound and RB94 plasmid+ liposome groups were more conspicuous than in other groups. The results from RT-PCR were well-matched with those of the western blot analysis.3. The growth activity of HXO-Rb44 cells in different transfected ways was compared. The MTT values of the RB94 plasmid+ microbubbles+ ultrasound and RB94 plasmid+ liposome groups were dramatically less than those of other groups. Cell cycle arrest in the G2-M phase and increased levels of apoptosis occurred in the RB94 plasmid+ microbubbles+ ultrasound and RB94 plasmid+ liposome groups.ConclusionThis study approved that UTMD can build up the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, indicating that the combination of UTMD and RB94 could be a useful method for application in the gene therapy of retinoblastoma.As a basic research, it still needs a lot of research before gene therapy for retinoblastoma really been applied in clinical work.