药学学报, 2020, 55(12): 2918-2923
引用本文:
左玲, 乔淦, 郭铭悦, 林秀坤, 刘明华. 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[J]. 药学学报, 2020, 55(12): 2918-2923.
ZUO Ling, QIAO Gan, GUO Ming-yue, LIN Xiu-kun, LIU Ming-hua. Preparation of exosome-coated Bcl-2 siRNA and its inhibitory effect on the growth of pancreatic cancer cells[J]. Acta Pharmaceutica Sinica, 2020, 55(12): 2918-2923.

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
左玲1, 乔淦1,2, 郭铭悦1, 林秀坤1, 刘明华1,2
1. 西南医科大学药学院, 四川 泸州 646000;
2. 厅市共建中枢神经系统药物四川省重点实验室, 四川 泸州 646000
摘要:
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。
关键词:    胰腺癌      Bcl-2      外泌体      细胞增殖     
Preparation of exosome-coated Bcl-2 siRNA and its inhibitory effect on the growth of pancreatic cancer cells
ZUO Ling1, QIAO Gan1,2, GUO Ming-yue1, LIN Xiu-kun1, LIU Ming-hua1,2
1. School of Pharmacy, Southwest Medical University, Luzhou 646000, China;
2. Central Nervous System Drug Key Laboratory of Sichuan Province, Luzhou 646000, China
Abstract:
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.
Key words:    pancreatic cancer    Bcl-2    exosome    cell proliferation   
收稿日期: 2020-06-17
DOI: 10.16438/j.0513-4870.2020-1011
基金项目: 四川省科技厅重点研发项目(2019YFS0116);四川省杰出青年科技人才项目(2019JDJQ33).
通讯作者: 刘明华,Tel:86-830-3193872,E-mail:liumhvip@163.com
Email: liumhvip@163.com
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参考文献:
[1] Golan T, HammeL P, Reni M, et al. Maintenance olaparib for germline BRCA-mutated metastatic pancreatic cancer[J]. N Engl J Med, 2019, 381:317-327.
[2] Schultheis B, Reuter D, Ebert MP, et al. Gemcitabine combined with the monoclonal antibody nimotuzumab is an active first-line regimen in KRAS wildtype patients with locally advanced or metastatic pancreatic cancer:a multicenter, randomized phase Ⅱb study[J]. Ann Oncol, 2017, 28:2429-2435.
[3] Foley K, Kim V, Jaffee E, et al. Current progress in immunotherapy for pancreatic cancer[J]. Cancer Lett, 2016, 381:244-251.
[4] Seufferlein T, Ettrich TJ. Treatment of pancreatic cancer-neoadjuvant treatment in resectable pancreatic cancer (PDAC)[J]. Transl Gastrointest Cancer, 2019, 4:21.
[5] Jain S, Pathak K, Vaidya A. Molecular therapy using siRNA:recent trends and advances of multi target inhibition of cancer growth[J]. Int J Biol Macromol, 2018, 116:880-892.
[6] Thery C, Amigorena S, Raposo G, et al. Isolation and characteri-zation of exosomes from cell culture supernatants and biological fluids[J]. Curr Protoc Cell Biol, 2006. DOI:10.1002/0471143030.cb0322s30.
[7] Van DB, Jasper G, Schlee M, et al. siRNA delivery with exosome nanoparticles[J]. Nat Biotechnol, 2011, 29:325-326.
[8] Alvarez-Erviti L, Seow Y, Yin H, et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes[J]. Nat Biotechnol, 2011, 29:341-345.
[9] Adams JM, Cory S. The Bcl-2 arbiters of apoptosis and their growing role as cancer targets[J]. Cell Death Differ, 2017, 25:27-36.
[10] Zuo J, Ishikawa T, Boutros S, et al. Bcl-2 overexpression induces a partial epithelial to mesenchymal transition and promotes squamous carcinoma cell invasion and metastasis[J]. Mol Cancer Res, 2010, 8:170-182.
[11] Westphal S, Kalthoff H. Apoptosis:targets in pancreatic cancer[J]. Mol Cancer, 2003, 2:6.
[12] Beh CW, Seow WY, Wang Y, et al. Efficient delivery of Bcl-2-targeted siRNA using cationic polymer nanoparticles:downregulating mRNA expression level and sensitizing cancer cells to anticancer drug[J]. Biomacromolecules, 2009, 10:41-48.
[13] Eloy JO, Petrilli R, Raspantini GL, et al. Targeted liposomes for siRNA delivery to cancer[J]. Curr Pharm Des, 2018, 24:2664-2672.
[14] Song F, Sakurai N, Okamoto A, et al. Design of a novel PEGylated liposomal vector for systemic delivery of sirna to solid tumors[J]. Biol Pharm Bull, 2019, 42:996-1003.
[15] Rytblat I, Wu N, Xu HL, et al. In vitro studies of polyethyleneimine coated miRNA microspheres as anticancer agents[J]. Nano Res, 2016, 9:1609-1617.
[16] Xiong MP, Forrest ML, Ton G, et al. Poly(aspartate-g-PEI800), a polyethylenimine analogue of low toxicity and high transfection efficiency for gene delivery[J]. Biomaterials, 2007, 28:4889-4900.
[17] Ohno S, Takanashi M, Sudo K, et al. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells[J]. Mol Ther, 2013, 21:185-191.
[18] Robert J. Biology of cancer metastasis[J]. Bull Cancer, 2013, 100:333-342.