pEGFP-bFGF基因转染人角膜缘干细胞的实验研究

Experimental Investigation of pEGFP-bFGF Gene Transfer to Human Limbal Stem Cells

本研究体外培养并用抗角质蛋白K3/K12鼠单抗(AE5)鉴定人角膜缘干细胞(HLSCs),采用脂质体转染法将pEGFP-bFGF基因转染培养的HLSCs,48 h后荧光显微镜下可见特异性的绿色荧光,基因转染率为20%～30%,同时制作NaOH细胞损伤模型,研究转染后的细胞对损伤的抵抗作用,转染后损伤的细胞存活状况高于未转染损伤组(P<0.05),而细胞凋亡及坏死率明显低于未转染损伤组(P<0.05)。pEGFP-bFGF基因能够转染培养的HLSCs,其表达产物对碱损伤的HLSCs具有保护作用,初步探讨了基因工程联合组织工程技术治疗眼表疾病的可行性。

Primary HLSCs were successfully cultured and assayed by AE5 in vitro. Constructed eukaryotic ex- pressive vector of pEGFP-bFGF was transferred into the human limbal stem cells by the liposome-mediated tech- nique, and 48 hours later, specific green fluorescence was observed by fluorescence microscope. The gene transfec- tion 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 micros- copy. The cellular viability in bFGF＋NaOH group was higher than that in NaOH group（P〈0.05） ,while the cellu- lar apoptosis rate plus necrosis rate displayed significant difference between the two groups（P〈0.05）. The pEGFP- bbGF gent 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.