超声联合色素上皮源性因子免疫纳米脂质体对脉络膜新生血管内皮细胞靶向杀伤作用的体外研究

In vitro study on ultrasound combined with pigment epithelium derived factor-immunoliposome targeting injury choroidal neovascular endothelial cells

目的体外实验研究超声联合色素上皮源性因子(pigment epithelium derived factor,PEDF)免疫纳米脂质体对脉络膜新生血管内皮细胞的靶向杀伤作用。方法制备能与脉络膜新生血管内皮细胞特异性结合的PEDF免疫纳米脂质体。向培养恒河猴脉络膜视网膜内皮细胞系RF/6A细胞加入免疫纳米脂质体,观察其与RF/6A细胞结合的能力。激光共聚焦显微镜观察免疫纳米脂质体的细胞内化过程。分别将游离PEDF、载PEDF脂质体、载PEDF免疫脂质体和超声联合载PEDF免疫脂质体作用细胞后,MTT法检测对RF/6A细胞生长的抑制作用,流式细胞术检测凋亡,并与仅用PBS作用细胞的对照组比较。结果制备的载PEDF免疫纳米脂质体可紧密结合RF/6A细胞,3h时,载PEDF免疫脂质体布满细胞浆。包含PEDF终浓度为80mg.L-1的游离PEDF、载PEDF脂质体、载PEDF免疫脂质体和超声联合载PEDF免疫脂质体对RF/6A细胞抑制率分别为(32.79±3.62)%、(48.71±3.84)%、(57.45±4.02)%和(90.83±5.61)%,凋亡率分别为(25.05±4.06)%、(30.21±3.13)、(36.85±4.12)%和(43.13±4.37)%。提示载PEDF免疫脂质体和载PEDF脂质体的抑制细胞生长和促进细胞凋亡作用均较游离PEDF强(均为P<0.05),且载PEDF免疫脂质体的抑制生长和促进凋亡作用更强(均为P<0.05)。超声可显著提高载PEDF免疫脂质体的细胞内化能力,并增强其抑制细胞生长和促进细胞凋亡的作用。结论体外实验证实超声联合PEDF免疫纳米脂质体可靶向杀伤脉络膜新生血管内皮细胞。

Objective To investigate the targeting injury of ultrasound combined with pigment epithelium derived factor （PEDF）-immunoliposome to choroidal neovascular endothelial cells in vitro. Methods PEDF immunoliposome which could specific binding with choroidal neovascular endothelial cells was made. Immunoliposome was added to cultured choroid-retinal endothelial cells （RF/6A） in rhesus monkey to observe its ability of binding with RF/6A cells. Cellular internalization of immunoliposome was observed under laser confocal microscopy. After being treated with liposome without PEDF, free PEDF, PEDF loaded liposome, PEDF loaded immunoliposome and ultrasound combined with PEDF loaded immunoliposome, their inhibitory effects on RF/6A were observed by MTF assay, apoptosis was observed with flow cytometry, and compared with control group, which was only treated with PBS. Results PEDF immunoliposome could closely bind with RF/6A cells. The inhibitory rates of free PEDF, PEDF loaded liposome, PEDF loaded immunoliposome and ultrasound combined with PEDF loaded immunoliposome （ final concentration of PEDF was 80 mg · L-1 ） on RF/6A cells were（32.79±3.62）% ,（48.71 ±3.84）% ,（57.45 ±4.02）% and （90.83% ±5.61%）, the apoptosis ratios were （25.05 ±4.06）% , （30.21 ± 3. 13）% , （36.85 ±4. 12）% and （43. 13 ± 4. 37 ）% , respectively, which indicated that inhibitory effect and apoptotic effect of PEDF immunoliposome and PEDF liposome on RF/6A cells were stronger than those of free PEDF （ both P 〈 0.05 ）, while PEDF immunoliposome had the strongest inhibitory effect and apoptotic effect（ all P 〈 0.05 ）. Ultrasound could significantly increase the cellular internalization of PEDF immunoliposome and enhance its inhibitory effect and apoptotic effect. Conclusion The PEDF immunoliposome combined with ultrasound can targeting injury choroidal neovascular endothelial cells in vitro.