负载Bcl-2 siRNA外泌体的制备及其对胰腺癌细胞生长抑制作用

Preparation of exosome-coated Bcl-2 siRNA and its inhibitory effect on the growth of pancreatic cancer cells

Bcl-2高表达与胰腺癌的发生、发展密切相关。本研究采用超速离心法从人胚肾(HEK293)细胞上清培养液中分离外泌体,利用电转技术制备了负载Bcl-2 siRNA的外泌体(exosiBcl-2),透射电镜观察表明,外泌体呈现典型内凹的杯状结构,Western blot分析表明外泌体特征蛋白CD9、CD81、CD63和TSG101呈现高表达,采用共聚焦显微镜、流式细胞术分析证实,exosiBcl-2可以高效转染人胰腺癌Miapaca-2细胞,转染效率为77.2%。MTS分析表明,exosiBcl-2可以显著抑制Miapaca-2细胞增殖,抑制率达到63%。本研究证实,exosiBcl-2可高效穿透细胞膜,通过抑制靶基因Bcl-2表达发挥抑制肿瘤细胞生长的作用。

High expression of Bcl-2 is associated with the development of pancreatic cancer,and downregulation of Bcl-2 is an effective approach for the treatment of pancreatic malignancy.In the present study exosomes were isolated from the cultured medium of human embryonic kidney cells(HEK293)by ultracentrifugation and exosome-coated Bcl-2 siRNA(exosiBcl-2)was synthesized using electroporation.The results showed that the particle size of exosiBcl-2 was 67.3±9.7 nm and the morphology of exosomes displayed a concave ring structure as determined by transmission electron microscopy(TEM).Western blot analysis indicated that exosomal proteins including CD9,CD81,CD63 and TSG101 were highly expressed.Confocal microscopy revealed that exosiBcl-2 was widely distributed in Miapaca-2 cells,and the transfection efficiency of exosiBcl-2 in Miapaca-2 was 77.2%as determined by flow cytometry.Treatment with exosiBcl-2 at a concentration of 100 nmol·L-1 resulted in an inhibitory effect on the growth of Miapaca-2 cells with an inhibition rate of 63%.ExosiBcl-2 treatment can downregulate Bcl-2 and upregulate Bax protein.This study provides evidence that exosiBcl-2 is able to inhibit the growth of pancreatic cancer cells and the nanoparticles have potential to be developed as a novel anticancer agent.