碱性成纤维细胞生长因子反义寡核苷酸及其脂质体对体外培养的大鼠晶状体上皮细胞增殖的抑制作用

Inhibiting proliferation of cultured rat lens epithelial cells by bFGF antisense oligonucleotides and their liposomes

目的 探讨碱性成纤维细胞生长因子(bFGF)反义寡核苷酸及其脂质体对大鼠晶状体上皮细胞(LEC)增殖的抑制作用。方法 体外培养大鼠LEC,细胞传代后分别加入bFGF反义和正义寡核苷酸及其脂质体,以营养液和无药脂质体为对照培养24 h后,用噻唑蓝(MTT)法测定细胞的增殖情况,用逆转录聚合酶链反应(RT-PCR)法测定细胞bFGF mRNA的表达情况。结果 大鼠LEC原代培养24 h可见细胞生长,形态为多边形,培养2-3周细胞汇合成片;传代培养细胞可在1～2周汇合成片。MTT法测定显示,bFGF反义寡核苷酸组(Ⅰ组)、正义寡核苷酸组(Ⅱ组)及营养液组(Ⅴ组)的吸光度(A值)分别为0.138±0.074、0.325±0.097及0.370±0.079,Ⅰ组A值显著小于Ⅱ和Ⅴ组(P=0.024,0.005);bFGF反义寡核苷酸脂质体组(Ⅲ组)、正义寡核苷酸脂质体组(Ⅳ组)及无药脂质体组(Ⅵ组)的A值分别为0.128±0.032、0.258±0.120及0.348±0.017,Ⅲ组A值显著小于Ⅵ组(P=0.000)。RT-PCR法检测结果显示,以2μg总RNA为模板,循环30次,bFGF反义寡核苷酸、bFGF正义寡核苷酸及营养液的扩增产物的量分别为0.33、0.99及0.85μg;:bFGF反义寡核苷酸脂质体、bFGF正义寡核苷酸脂质体及无药脂质体的扩增产物的量分别为0.23、0.48及0.56μg。结论大鼠LEC可在体外培养;

Objective To evaluate the effects of basic fibroblast growth factor(bFGF) antisense oligonucleotides and their liposomes on the proliferation of cultured rat lens epithelial cells ( LECs) . Methods The rat LECs were cultured and bFGF antisense olignucleotides ( AONs) , sense olignucleotides (SONs) , AON liposomes, SON liposomes were added to second passage of cells supplied with DMEM while empty liposomes served as controls. MTT assay was used to examine the proliferation of LECs, and RT-PCR was performed to quantify bFGF mRNA expression in LECs 24 h after treatment Results The Shapes of rat LECs were polygonal after 24 h in culture. Cell confluence was reached in 2-3 weeks. After subculture, confluence was occurred in 1-2 weeks. The results of MTT assay were showed that the absorption (A) value of bFGF AONs was 0. 138 ± 0. 074, and that of bFGF SONs and DMEM control were 0. 325 ± 0. 097 and 0. 370 ± 0. 079, respectively. The A value of AONs was significantly less than SONs and DMEM alone ( P = 0. 024, P = 0. 005). The absorption (A) value of bFGF AON liposomes was 0. 128 ± 0. 032, and that of bFGF SON liposomes and liposomes alone were 0. 238 ± 0. 120 and 0. 348 ± 0. 017. The absorption (A) value of AON liposomes was not significantly different from that of liposomes negative control ( P = 0. 000). By RT-PCR, the amounts of PCR product for bFGF AONs, SONs and DMEM control were 0. 33 μg, 0. 99 μg, 0. 85 μg. The amounts of PCR product of bFGF AON liposomes, SON liposomes and liposomes only were 0. 23 μg, 0. 48 μg, 0. 56 μg. Conclusions The proliferation of cultured rat LECs is inhibited by the treatment of the antisense oligonucleotides, the inhibition is correlated with decreased expression of bFGF mRNA. (Chin J Ophthalmol ,2004,40:528-532)