Differential Expression Of Loading-liposome In Hela Cells And Spiral Ganglion Neurons Of The Mouse

Objective: To provide basic data for the preparation of nanoparticle-based gene vectors, difference of in vitro transfection efficiency of loading liposomes in cervical cancer cells (Hela cells) and spiral ganglion neurons (SGNs) of the mouse were observed. Method: (1) a cell culture method of mouse SGNs was developed, and the cultured SGNs were identified by monoclonal anti-β3-Tubulin immuofluorescence staining. (2) transient transfection of Hela cells and SGNs were performed with recombinant plasmid pEGFPC2-NT3 comprising both neurotrophin3 (NT-3) and enhanced green fluorescent protein(EGFP), the recombinant plasmid was carried by the cationic liposome Lipofectamin 2000 (Invitrogen, USA) as a gene vector. The transfection efficiency of pEGFPC2-NT3 in Hela cells and SGNs were counted, respectively. (3) the differential expressions of NT-3 and EGFP in Hela cells and SGNs were observed respectively by means of immunohistochemistry (DAB method). Result: (1) SGNs began to adhere after 12 hour-culture. 72 hours later, the cell number reached the maximum level, i.e. 50/cm~2.β3-Tubulin immunofluorescence staining confirmed that this cultured cells were neurons. (2) the transfection efficiency was 60% in Hela cells, but 10%-15% in SGNs under the fluorescence microscope. (3) immunohistochemical staining showed that the transfection efficiency of GFP and NT-3 in Hela cells were much higher than that in SGNs, while GFP's transfection efficiency in the both kind of cultured cells was lower than that of NT-3. Conclusion: (1) for the same loading lipoplexes, the transfection efficiency in Hela cells was much higher than that in SGNs . (2) the transfection efficiency of therapeutic gene NT-3 was higher than that of reporter gene EGFP in the same cell line. The result indicate that a more effective gene vector was needed for transfection of exogenous genes into a neuron, and that the transfection efficiency showed by the reporter gene was not necessarily the true transfection efficiency or expression status of the therapeutic gene. These should be taken into account when analysing the result of gene tansfection experiment.