[Experimental investigation of pEGFP-bFGF gene transfer to human limbal stem cells]

Primary HLSCs were successfully cultured and assayed by AE5 in vitro. Constructed eukaryotic expressive vector of pEGFP-bFGF was transferred into the human limbal stem cells by the liposome-mediated technique, and 48 hours later, specific green fluorescence was observed by fluorescence microscope. The gene transfeetion efficiency was 20%-30%. Then the model of cells injury was created by use of NaOH. The cells were divided into four groups: Normal, bFGF, NaOH and bFGF+NaOH. The cellular viability in each group was measured by MTT colorimetry, and the cellular apoptosis rate and necrosis rate were observed by laser scanning confocal microscopy. The cellular viability in bFGF+NaOH group was higher than that in NaOH group (P < 0.05) ,while the cellular apoptosis rate plus necrosis rate displayed significant difference between the two groups (P < 0.05). The pEGFP-bbGF gene was noted to be successfully transferred into HLSCs and the cells were found growing well. These indicated that bFGF gene has a protective effect on the HLSCs injured by NaOH. We have also probed the feasibility of trying the treatment for ocular surface disease through gene engineering recombined tissue engineering.