纳米级siRNA与壳寡糖复合物体内外抗脉络膜新生血管作用研究

Inhibition of siRNA/Chitooligosaccharides （COS） complex nanoparticles in choroidal neovascularization

目的观察小干扰RNA与壳寡糖（siRNA与COS）纳米复合物体内外抗脉络膜新生血管的作用。方法实验研究。对2011年9月至2012年6月以提取人脐静脉内皮细胞（HUVEC）培养并传代。用激光粒度仪检测siRNA与COS纳米复合物的粒径与zeta电位。用琼脂糖凝胶电泳评价siRNA与COS质量比分别为1／1，1，5时所形成的siRNA复合物的情况。利用SRB方法评价COS分别为5μg／mL、25μg／mL、50μg／mL时，对人脐静脉内皮细胞增殖的抑制情况。以及50nmolsiRNA浓度条件下，siRNA／COS质量比为1／1、1／5时形成的复合物对人脐静脉内皮细胞增殖的抑制作用。采用单因素方差分析方法进行组间比较。利用荧光素眼底血管造影方法观察不同质量比的siRNA与COS（1／1、1／5）减轻血管荧光素渗漏的情况。结果激光粒度仪检测显示随着COS的量增加，siRNA与COS纳米复合物的粒径在93．9～459．8nm范围内变化，zeta电位在．19．5—17．8mV范围内变化。琼脂糖凝胶电泳显示当siR-NA与COS质量比为1／5时，siRNA能够完全与壳寡糖结合。质量比为1／1时，只有部分siRNA与壳寡糖相结合。SRB结果显示，两种质量比所形成的复合物对HUVEC增殖的抑制作用均强于对照组，siRNA与COS质量比分别为1：1，1：5时，增殖率分别为（68．7±12．2）％，（58．8±10．1）％vs对照组100％，差异有统计学意义（F=12．321，P=0．008）。COS5μg／mL、25μg／mL、50μg／mL三个剂量组均未显示出对HUVEC增殖的抑制作用（F=O．713，P=O．558）。2周时，siRNA与COS组，激光斑处荧光渗漏较对照组明显减轻。结论该研究表明siRNA与壳寡糖纳米复合物在抗脉络膜新生血管中可能起到积极的作用，且毒性较低。

Objective To investigate the chitooligosaccharides （COS） as a transfection agent me- diate the siRNA （target the VEGFR1） inhibit the proliferation of HUVEC. Methods HUVEC were extracted from neonatal umbilical cord. Size and zeta potential of siRNA/COS was measured by Mal- vern Zetasizer. Gel electrophoresis was used to evaluate the complex formation of siRNA/COS with different weight ratio （siRNA/COS=I/1 or 1/5）. SRB test was used to estimate the inhibition proliferation rate of HUVEC. 5μg/mL, 25μg/mL, 50μg/mL COS were added to HUVEC cells. And siRNA/ COS （weight ratio 1/1 and 1/5） also were cultured with HUVEC. The concentration of siRNA was 50nmol. The blank medium was as control. The therapeutic efficacy was assessed by FFA. Results The size and zeta potential were from 93.9 nm to 459.8um, -19.5mV to 17.8mV. The electrophoresisshowed that siRNA/COS weight ratio=l/5 was no free siRNA in the complex nanoparticles. While there was still some free siRNA in siRNA/COS weight ratio=1/1. Both of the siRNA/COS weight ratio =1/1 （proliferation rate 68.7%4-12.2% vs control group 100%） and siRNA/COS weight ratio =1/5 （proliferation rate 58.8%4-10.1% vs control group 100%） showed inhibition significantly （F =12.321, P =0.008）. COS showed no inhibition at 5μg/mL, 25μg/mL, 50/μg/mL （F =0.713, P =0.558）. One way ANOVA was used for the analysis. Two weeks later, the leakage of group siRNA/COS was less than control group. Conclusions The COS can mediate the siRNA inhibit the CNV with low toxicity.