배경 및 목적: 엑소좀은 정상 혹은 종양세포에서 유리되는 나노 크기의 낭포로 세포배양액이나 체액에서도 관찰된다. 현재까지 종양세포 유래 엑소좀에 대한 연구가 주로 진행되고 있으나, 정상세포에서 유래된 엑소좀의 기능에 대한 연구는 제한적이다. 자연살해세포 (Natural killer cell: ...
배경 및 목적: 엑소좀은 정상 혹은 종양세포에서 유리되는 나노 크기의 낭포로 세포배양액이나 체액에서도 관찰된다. 현재까지 종양세포 유래 엑소좀에 대한 연구가 주로 진행되고 있으나, 정상세포에서 유래된 엑소좀의 기능에 대한 연구는 제한적이다. 자연살해세포 (Natural killer cell: NK 세포)는 악성종양에 대하여 신속하게 대응하는 면역세포로 항암작용 특성에 대한 많은 연구가 진행되고 있으나, NK 세포 유래 엑소좀의 항암특성에 대한 연구는 전무한 상태이다. 이 연구에서는 NK 세포 유래 엑소좀의 악성 흑색종에 대한 체내 및 체외 항암성능을 평가하고자 하였다. 재료 및 방법: 악성 흑색종 세포인 B16F10 세포에 firefly luciferase (effluc)과 thy1.1 유전자를 이입하여 microbead를 이용하여 B16F10/effluc 세포주를 구축하였다. 리포터 유전자가 이입된 안정세포주에서 effluc 리포터 유전자의 발현을 RT-PCR, western blotting 및 luciferase activity로 평가하였다. NK 세포주인 NK-92MI 세포로부터 초원심분리 및 밀도차 초원심분리법으로 엑소좀을 (NK-92 Exo) 분리하여 투과전자현미경 및 western blotting로 평가하였다. 또한 NK-92 Exo에 사이토카인의 포함여부도 효소결합면역흡착제 검정법으로 확인하였다. NK-92 Exo의 시험관내 B16F10 세포 사멸효과를 발광영상 및 CCK-8 검사로 평가하였다. 또한 B16F10 세포를 이식한 누드마우스에서 NK-92 Exo의 종양사멸효과를 평가하였다. 결과: B16F10/effluc 세포주는 성공적으로 구축되었으며, 이는 RT-PCR과 western blotting으로 확인되었다. NK-92 Exo은 엑소좀의 마커인 CD63과 ALIX가 발현하였으며, 종양사멸능을 가진 perforin과 FasL 발현도 확인되었다. NK-92 Exo는 시험관내 및 종양이식 동물모델 모두에서 악성흑색종인 B16F10에 대하여 종양사멸효과를 나타내었다. 하지만 NK-92 Exo는 정상 세포에 대하여 독성을 나타나지 않았다. 결론: 본 연구의 결과는 NK 세포 유래 엑소좀이 악성 흑색종에 치료효과를 보이며 정상세포에는 독성이 거의 없음을 보여 주었는데, 이는 NK 세포 유래 엑소좀이 악성종양 치료 영역에서 새로운 면역치료 기법의 하나로 개발될 수 있음을 보여준다.
배경 및 목적: 엑소좀은 정상 혹은 종양세포에서 유리되는 나노 크기의 낭포로 세포배양액이나 체액에서도 관찰된다. 현재까지 종양세포 유래 엑소좀에 대한 연구가 주로 진행되고 있으나, 정상세포에서 유래된 엑소좀의 기능에 대한 연구는 제한적이다. 자연살해세포 (Natural killer cell: NK 세포)는 악성종양에 대하여 신속하게 대응하는 면역세포로 항암작용 특성에 대한 많은 연구가 진행되고 있으나, NK 세포 유래 엑소좀의 항암특성에 대한 연구는 전무한 상태이다. 이 연구에서는 NK 세포 유래 엑소좀의 악성 흑색종에 대한 체내 및 체외 항암성능을 평가하고자 하였다. 재료 및 방법: 악성 흑색종 세포인 B16F10 세포에 firefly luciferase (effluc)과 thy1.1 유전자를 이입하여 microbead를 이용하여 B16F10/effluc 세포주를 구축하였다. 리포터 유전자가 이입된 안정세포주에서 effluc 리포터 유전자의 발현을 RT-PCR, western blotting 및 luciferase activity로 평가하였다. NK 세포주인 NK-92MI 세포로부터 초원심분리 및 밀도차 초원심분리법으로 엑소좀을 (NK-92 Exo) 분리하여 투과전자현미경 및 western blotting로 평가하였다. 또한 NK-92 Exo에 사이토카인의 포함여부도 효소결합면역흡착제 검정법으로 확인하였다. NK-92 Exo의 시험관내 B16F10 세포 사멸효과를 발광영상 및 CCK-8 검사로 평가하였다. 또한 B16F10 세포를 이식한 누드마우스에서 NK-92 Exo의 종양사멸효과를 평가하였다. 결과: B16F10/effluc 세포주는 성공적으로 구축되었으며, 이는 RT-PCR과 western blotting으로 확인되었다. NK-92 Exo은 엑소좀의 마커인 CD63과 ALIX가 발현하였으며, 종양사멸능을 가진 perforin과 FasL 발현도 확인되었다. NK-92 Exo는 시험관내 및 종양이식 동물모델 모두에서 악성흑색종인 B16F10에 대하여 종양사멸효과를 나타내었다. 하지만 NK-92 Exo는 정상 세포에 대하여 독성을 나타나지 않았다. 결론: 본 연구의 결과는 NK 세포 유래 엑소좀이 악성 흑색종에 치료효과를 보이며 정상세포에는 독성이 거의 없음을 보여 주었는데, 이는 NK 세포 유래 엑소좀이 악성종양 치료 영역에서 새로운 면역치료 기법의 하나로 개발될 수 있음을 보여준다.
Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids. Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes release...
Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids. Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes released by normal cells. Natural killer (NK) cells display rapid immunity to metastatic or hematological malignancies, and efforts have been undertaken to clinically exploit the antitumor properties of NK cells. However, the characteristics and functions of exosomes derived from NK cells remain unknown. In this study, NK cell-derived exosome-mediated antitumor effects against aggressive melanoma was explored in vitro and in vivo. Methods:B16F10 cells were transfected with enhanced firefly luciferase (effluc) and thy1.1 genes, and thy1.1-positive cells were immunoselected using microbeads. The resulting B16F10/effluc cells were characterized using reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and luciferase activity assays. Exosomes derived from NK-92MI cells (NK-92 Exo) were isolated by ultracentrifugation and density gradient ultracentrifugation. NK-92 Exo were characterized by transmission electron microscopy and western blotting. An enzyme-linked immunosorbent assay was also performed to measure cytokines retained in NK-92 Exo cells. The in vitro cytotoxicity of NK-92 Exo against the cancer cells was determined using a bioluminescence imaging system (BLI) and CCK-8 assays. To investigate the possible side effects of NK-92 Exo on healthy cells, the BLI and CCK-8 assays using the human kidney Phoenix™-Ampho cell line were also performed. Flow cytometry and western blotting confirmed that NK-92 Exo induced apoptosis in the B16F10/effluc cells. In vivo, B16F10/effluc cell xenograft model was used to detect the immunotherapeutic effect of NK-92 Exo. NK-92 Exo was ingected into tumors directly, and tumor growth progression was monitored using the IVIS Lumina imaging system and ultrasound imaging. Tumor mass was monitored after in vivo experiments. Results:RT-PCR and western blotting confirmed effluc gene expression and protein levels in B16F10/effluc cells. B16F10/effluc activity was found to increase with increasing cell numbers, using BLI assay. For NK-92 Exo characterization, western blotting was performed on both ultracentrifuged and density gradient-isolated exosomes. The results confirmed that NK cell-derived exosomes express two typical exosome proteins, namely CD63 and ALIX. By western blot analysis that NK-92 Exo presented two functional NK proteins was demostrated, namely perforin and FasL. Moreover, the membrane expression of FasL was confirmed. The enzyme-linked immunosorbent assay results indicated that NK-92 Exo can secrete tumor necrosis factor (TNF)-α, which affected the cell proliferation signaling pathway. The antitumor effect of NK-92 Exo against B16F10/effluc cells in vitro was confirmed by BLI (p< 0.001) and CCK-8 assays (p< 0.001). Furthermore, in normal healthy cells, even after 24 h of co-culture, NK-92 Exo did not exhibit significant side effects. In the in vivo experiments, tumors in the vehicle control group were significantly increased,compared with those in the NK-92 Exo-treated group(p < 0.05). Conclusion:The results of the current study suggest that exosomes derived from NK cells exert cytotoxic effects on melanoma cells and thus warrant further development as a potential immunotherapeutic strategy for cancer.
Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids. Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes released by normal cells. Natural killer (NK) cells display rapid immunity to metastatic or hematological malignancies, and efforts have been undertaken to clinically exploit the antitumor properties of NK cells. However, the characteristics and functions of exosomes derived from NK cells remain unknown. In this study, NK cell-derived exosome-mediated antitumor effects against aggressive melanoma was explored in vitro and in vivo. Methods:B16F10 cells were transfected with enhanced firefly luciferase (effluc) and thy1.1 genes, and thy1.1-positive cells were immunoselected using microbeads. The resulting B16F10/effluc cells were characterized using reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and luciferase activity assays. Exosomes derived from NK-92MI cells (NK-92 Exo) were isolated by ultracentrifugation and density gradient ultracentrifugation. NK-92 Exo were characterized by transmission electron microscopy and western blotting. An enzyme-linked immunosorbent assay was also performed to measure cytokines retained in NK-92 Exo cells. The in vitro cytotoxicity of NK-92 Exo against the cancer cells was determined using a bioluminescence imaging system (BLI) and CCK-8 assays. To investigate the possible side effects of NK-92 Exo on healthy cells, the BLI and CCK-8 assays using the human kidney Phoenix™-Ampho cell line were also performed. Flow cytometry and western blotting confirmed that NK-92 Exo induced apoptosis in the B16F10/effluc cells. In vivo, B16F10/effluc cell xenograft model was used to detect the immunotherapeutic effect of NK-92 Exo. NK-92 Exo was ingected into tumors directly, and tumor growth progression was monitored using the IVIS Lumina imaging system and ultrasound imaging. Tumor mass was monitored after in vivo experiments. Results:RT-PCR and western blotting confirmed effluc gene expression and protein levels in B16F10/effluc cells. B16F10/effluc activity was found to increase with increasing cell numbers, using BLI assay. For NK-92 Exo characterization, western blotting was performed on both ultracentrifuged and density gradient-isolated exosomes. The results confirmed that NK cell-derived exosomes express two typical exosome proteins, namely CD63 and ALIX. By western blot analysis that NK-92 Exo presented two functional NK proteins was demostrated, namely perforin and FasL. Moreover, the membrane expression of FasL was confirmed. The enzyme-linked immunosorbent assay results indicated that NK-92 Exo can secrete tumor necrosis factor (TNF)-α, which affected the cell proliferation signaling pathway. The antitumor effect of NK-92 Exo against B16F10/effluc cells in vitro was confirmed by BLI (p< 0.001) and CCK-8 assays (p< 0.001). Furthermore, in normal healthy cells, even after 24 h of co-culture, NK-92 Exo did not exhibit significant side effects. In the in vivo experiments, tumors in the vehicle control group were significantly increased,compared with those in the NK-92 Exo-treated group(p < 0.05). Conclusion:The results of the current study suggest that exosomes derived from NK cells exert cytotoxic effects on melanoma cells and thus warrant further development as a potential immunotherapeutic strategy for cancer.
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