奈米鑽石的分子胞飲機制及細胞內運輸路徑

Abstract

奈米鑽石是一種碳組成的奈米材料，近幾年被開發作為生物醫學的應用，包括生物影像、生物標定及藥物運送等。然而奈米鑽石進入人類細胞中的機制與運輸路徑仍不清楚。在本論文中，我們利用平均100奈米尺寸大小的奈米鑽石，研究奈米鑽石在人類細胞中的分子胞飲機制與細胞內的運輸路徑。我們發現奈米鑽石處理細胞後，細胞內奈米鑽石的螢光強度及顆粒複雜度會隨著處理奈米鑽石的濃度與時間增加而增高，但奈米鑽石存於細胞質而不進入細胞核中。有趣地，奈米鑽石在細胞外或細胞內皆會與泛素結合。我們設計及改良蔗糖濃度梯度的離心分離方法，從處理奈米鑽石的細胞中分離出奈米鑽石-蛋白複合體，分析奈米鑽石-蛋白複合體，發現含有離氨酸-48及離氨酸-63之泛素鏈，也鑑定出參與泛素化作用的LRSAM1 之E3連接酶。我們也發現奈米鑽石-蛋白質複合體中含有參與細胞自噬作用的蛋白分子，包括p62與LC3。利用共軛焦顯微系統觀察到p62與LC3會結合到泛素化的奈米鑽石，使其形成自噬體。以基因轉殖送入缺乏泛素結合位的p62基因，發現表現泛素結合位缺失的p62蛋白無法與泛素化的奈米鑽石結合。在細胞受到細菌及病毒入侵時，細胞內的防禦機制會誘發OPTN、TBK1及NDP52 結合到泛素化的細菌及病毒，使其形成自噬體而進入溶酶體分解。我們鑑定出奈米鑽石-蛋白質複合體中含有OPTN、TBK1、 NDP52 等蛋白，在共軛焦顯微系統分析可觀察到OPTN及NDP52和泛素化的奈米鑽石位於相同的位置。奈米鑽石會經由LC3及LAMP-1 結合形成自噬溶酶體，最終存在於溶酶體中，但奈米鑽石不會存於內質網、粒線體或高基氏體等胞器。進入細胞的奈米鑽石經過數次繼代培養後，細胞內的螢光強度及顆粒複雜度會隨著細胞分裂而遞減，隨著細胞的繼代培養，10天後仍可發現細胞攜帶單一群聚的奈米鑽石於溶酶體中，但細胞中所攜帶的奈米鑽石，並不會影響細胞週期進行、細胞生長能力及細胞的傷害。此外，我們發現奈米鑽石可經由macropinocytosis及clathrin蛋白所調控的胞飲路徑進入細胞內，在缺少血清的細胞培養環境下，會增加細胞內clathrin、p62及LC3的蛋白表達量，並會促進奈米鑽石進入細胞內的含量，進入細胞之奈米鑽石在細胞有絲分裂時會平均分佈至兩個子代細胞中。綜合以上結果，本論文發現奈米鑽石是經由macropinocytosis及clathrin蛋白所調控的胞飲機制進入細胞內，進入細胞內的奈米鑽石會被泛素所結合，我們推測泛素化的奈米鑽石會調控p62、 LC3、OPTN、NDP52及TBK1形成奈米鑽石-自噬體，進行細胞內之運輸，奈米鑽石-自噬體會運送至溶酶體處理，最終存於溶酶體。本研究提出一個全新模式闡述奈米材料在細胞內運送及處理方式，是經由泛素化-自噬體-溶酶體的路徑。我們推測奈米鑽石引起細胞防禦的自噬作用，可能與病毒或細菌入侵細胞所引起的細胞保護機制有關，瞭解奈米材料進入細胞的分子胞飲機制與在細胞內的修飾作用及運輸路徑，將有助於評估奈米材料在細胞中的生理反應與提供可能的生物醫學應用。

Nanodiamond (ND), a carbon nanomaterial, has been developed for biomedical applications, including bio-imaging, bio-labeling, and drug delivery. However, endocytic and traffic pathways of NDs in human cells remain unclear. In this study, we used the average size of 100 nm NDs to investigate the molecular endocytic mechanisms and cellular traffic pathways in cells. After NDs treatment, we found the fluorescence intensities and particular complexities of NDs in cells that were increased via a concentration- and time-dependent manner. These ND particles were located in cytoplasm and not located in nucleus. Interestingly, ND can bind with ubiquitins (Ub) in vitro and in vivo. We have designed and modified a separation method by sucrose density gradient centrifugation to separate the ND-protein complexes from the ND-treated cells. We found ND-protein complexes that contained Ub K48 and Ub K63. In addition, the E3 ligase of LRSAM1 of ubiquitination was identified in the ND-protein complexes. We also found that autophagic proteins of p62 and LC3 were existed in the ND-protein complexes. Using confocal microscope system analysis, we found the p62 and LC3 conjugated with ubiquitinated NDs to form autophagosome. The expression of mutant p62 proteins by transfection with an Ub-binding domain deletion of p62 mutant vector did not bind to ubiquitinated NDs. During infection of bacteria or viruses, the cellular defense mechanisms can induce autophagy adaptors OPTN, NDP52, and TBK1 to targeting on the ubiquitinated bacteria or viruses to form autophagosomes and then fuse with lysosomes. We identified that OPTN, NDP52 and TBK1 proteins were presented in the ND-protein complexes. Moreover, OPTN and NDP52 proteins were co-located with ubiquitinated NDs analyzed by confocal microscope system. NDs can bind with LC3 and LAMP-1 to form autolysosome and NDs. Finally, NDs retained in lysosomes but did not locate in ER, mitochondria or Golgi apparatus. The fluorescence intensities and particular complexities of endocytic NDs were decreased through cell division by following sub-cultured for several generations. The cell retained a single ND’s cluster in lysosome after sub-cultured for several generations at 10 days. NDs could be carried inside of cells but did not alter cell cycle progression, cell growth ability and cell damage. In addition, we found that ND particles were taken into cells by macropinocytosis and clathrin-mediated endocytosis pathways. Serum starvation increased the protein levels of clathrin, p62 and LC3 and elevated the cellular uptake ability of NDs. ND particles were equal separating into two daughter cells of cell division approximately. According to above findings, this paper provides that ND particles are taken into cells by both macropinocytosis and clathrin-mediated endocytosis. The endocytic ND particles can be conjugated with Ub. We suggest that ubiquitinated NDs mediate p62, OPTN, NDP52 and TBK1 to form ND-autophagosomes for cellular traffic. ND-autophagosomes were delivered and subsequently located in lysosomes. This study provides an entirely novel model for transportation and processing of nanomaterials in cells through the ubiquitination-autophagosome-lysosome pathway. We suggest that the defense mechanism of autophagy induced by NDs may be related to cellular protective mechanisms for processing bacteria or viruses. Understanding the molecular endocytic mechanisms and intracellular modifications and traffic pathways of nanomaterials will help to evaluate the cellular physiological reactions of nanomaterials and provide potential biomedical applications.