Tesis

Efek Stimulasi Eksosom Dari Serum Sehat Pada Sel Nk Subyek Karsinoma Hepatoselular In Vitro = Stimulation effect of exosome from healthy sera to NK cells of hepatocellular carcinoma in vitro.

Kanker hati masih menjadi masalah kesehatan di dunia maupun di Indonesia yang memiliki prognosis yang buruk. Pengobatan karsinoma hepatoseluler yang ada saat ini masih belum optimal sehingga diperlukan pengembangan modalitas imunoterapi berbasis sel NK autologus terinduksi eksosom bagi pasien karsinoma hepatoselular. Dengan mengisolasi sel NK pasien karsinoma hepatoselular dari darah vena perifer setelah itu melakukan isolasi eksosom dari serum darah whole blood donor sehat. Setelah itu dilakukan karakterisasi eksosom dengan PSA dan flowcytometry. Kemudian dipaparkan eksosom dan IL-2 ke sel NK dan dinilai ekspresi reseptor aktivasi, reseptor inhibisi dengan flow cytometry, serta qRT-PCR melihat ekspresi perforin dan granzim B. Visualisasi interaksi sel NK terinduksi dengan fraksi sel mononuklear lain yaitu sel T, sel B, dan sel dendritik dengan imunofluoresens. Hasil karakterisasi eksosom didapatkan partikel berukuran < 100 nm, muatan listrik negatif, CD63+CD81+ (double positif). Terdapat peningkatan ekspresi reseptor aktivasi dan penurunan ekspresi reseptor inhibisi sel NK, dan peningkatan ekspresi relatif gen perforin dan granzim B pada sel NK terinduksi eksosom. Tidak didapatkan adanya interaksi sel berupa sinapsis imun antara sel NK terinduksi eksosom dengan limfosit CD4, CD8, dan CD19, serta CD11c pasien karsinoma hepatoselular. Ukuran partikel, muatan listrik, dan analisa proteomik mendukung karakteristik eksosom. Induksi eksosom ke sel NK pasien karsinoma hepatoselular menimbulkan perubahan ekspresi reseptor aktivasi, reseptor inhibisi, dan perforin serta granzim B. Hasil pengamatan imunofloresensi tidak menunjukkan adanya sinapsis imun antara sel NK yang terinduksi eksosom dengan fraksi mononuklear lain dari pasien kanker hepatoselular.
Kata kunci : Sel NK, Karsinoma hepatoselular, sinapsis imun, eksosom, vesikel ekstraselular


Liver cancer is still a health problem in the world and in Indonesia which has a poor prognosis. The current treatment of hepatocellular carcinoma is still not optimal, so it is necessary to develop exosome-induced autologous NK cell-based immunotherapy modalities for hepatocellular carcinoma patients. By isolating NK cells from hepatocellular carcinoma patients from peripheral venous blood, then isolating exosomes from whole blood serum of healthy donors. After that, exosome characterization was carried out by PSA and flowcytometry. Then exposed exosomes and IL-2 to NK cells and assessed the expression of activation receptors, inhibitory receptors by flow cytometry, and qRT-PCR to look at the expression of perforin and granzyme B. Visualization of induced NK cell interactions with other mononuclear cell fractions, namely T cells, B cells, and dendritic cells by immunofluorescence. The results of exosome characterization obtained particles < 100 nm in size, negative electric charge, CD63+CD81+ (double positive). There was increased expression of activating receptors and decreased expression of inhibitory receptors of NK cells, and increased relative expression of the perforin and granzyme B genes in exosome-induced NK cells. There was no cell interaction in the form of immune synapses between exosome-induced NK cells and CD4, CD8, and CD19, and CD11c lymphocytes in hepatocellular carcinoma patients. Particle size, electric charge, and proteomic analysis support the characteristics of the exosome. Induction of exosomes into the NK cells of hepatocellular carcinoma patients resulted in changes in the expression of activation receptors, inhibitory receptors, and perforin and granzyme B. The results of immunofluorescence observations did not show any immune synapses between exosome-induced NK cells and other mononuclear fractions from hepatocellular cancer patients.
Keywords: NK cells, hepatocellular carcinoma, immune synapses, exosomes, extracellular vesicles