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目的构建载内皮抑素(Es)重组表达质粒的壳聚糖纳米基因载体并评价其抑制内皮细胞增殖的活性。方法选用真核表达质粒p VAX1为重组载体,采用PCR及双酶切法制备能够表达Tat PTD-Es融合蛋白的真核表达载体p VAX1/Tat PTD-Es;离子交联法制备壳聚糖载基因纳米粒,琼脂糖凝胶电泳筛选处方,检测纳米粒的形态、Zeta电位及粒径;采用最佳处方比例制备载重组质粒纳米载体,转染人脐静脉内皮细胞HUVEC,采用ELISA法测定分泌型Tat PTD-Es蛋白的表达量,CCK-8法测定对HUVEC的抑制作用,以上两个实验均以LipofectaminTM2000为阳性对照。结果 p VAX1/Tat PTD-Es的真核表达载体构建成功,壳聚糖纳米粒Zeta电位(14.3±1.6)m V,粒径(145±12)nm,纳米粒转染HUVEC后细胞上清中Tat PTD-Es的浓度为(182.5±16.3)ng/m L,壳聚糖基因载体转染HUVEC后对细胞有明显的抑制作用,72 h的抑制率达(53.4±5.4)%(Z=-2.611,P=0.008)。结论构建的壳聚糖纳米基因载体能够成功转染HUVEC并能明显地抑制内皮细胞的增殖。
OBJECTIVE: To construct a chitosan nanoparticle vector containing endostatin (Es) recombinant plasmid and evaluate its inhibitory effect on endothelial cell proliferation. Methods The eukaryotic expression vector p VAX1 / Tat PTD-Es was constructed by PCR and double enzyme digestion method using p VAX1 as the eukaryotic expression vector. Gene nanoparticles and agarose gel electrophoresis were used to screen the prescriptions, and the morphology, Zeta potential and particle size of the nanoparticles were detected. The recombinant plasmid nano-carriers were prepared and transfected into human umbilical vein endothelial cells HUVEC by the best prescription ratio. Type Tat PTD-Es protein expression, CCK-8 method to determine the inhibition of HUVEC, the above two experiments were LipofectaminTM2000 positive control. Results The eukaryotic expression vector p VAX1 / Tat PTD-Es was successfully constructed. The Zeta potential of chitosan nanoparticles was (14.3 ± 1.6) m V and the diameter was (145 ± 12) nm. After transfection of HUVEC into the supernatant The concentration of Tat PTD-Es was (182.5 ± 16.3) ng / m L. The chitosan gene vector had a significant inhibitory effect on HUVEC transfected cells and the inhibition rate reached 53.4 ± 5.4% at 72 h (Z = 2.611, P = 0.008). Conclusion The constructed chitosan nano-gene vector can successfully transfect HUVEC and inhibit the proliferation of endothelial cells obviously.