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Abstract：
For decades, intranuclear inclusions in many normal and neoplastic cells have been considered to be mere invaginations of cytoplasm into the nucleus without any notable function or influence on disease. We investigated such inclusions in 75 specimens of hepatocellular carcinoma (HCC). In this context we demonstrate that these inclusions are true inclusions, completely closed and delimited by the nuclear membrane, containing degenerate cell organelles and lysosomal proteins. Moreover, their occurrence was positively associated with patient survival but not with tumour grade or stage. In a standardised area a mean of 124 inclusions per specimen was present in the tumorous liver tissue in contrast to 5 inclusions in the non-tumorous adjacent section and 89% of all scrutinised HCC showed at least one membrane-bound nuclear inclusion. Ultrastructural characterisation by transmission electron microscopy revealed degenerative materials such as residues of lysosomes, endoplasmic reticulum and Golgi apparatus within the inclusions. Due to the fact that the content of the inclusions appears to be more condensed than cytoplasm and contains fewer intact cell organelles, we assume that they are not mere invaginations of cytoplasm. Three dimensional (3D) reconstruction of isolated and immunofluorescence stained nuclei showed that the inclusions are completely located within the nucleus without any connection to the cytoplasm. The limiting membrane of the inclusions contained lamin B suggesting nuclear membrane origin. The content of the inclusions stained for the autophagy-associated proteins p62, ubiquitin, LC3B, cathepsin B and cathepsin D. Triple immunofluorescence staining followed by 3D reconstruction revealed co-localisation of p62, ubiquitin and LC3B in the same inclusion. Our observations uncover that these inclusions are real inclusions completely surrounded by the nucleus. We propose that the presence of autophagy-associated proteins and proteases within the inclusions contribute to beneficial survival.
摘要：
几十年来,许多正常和肿瘤细胞中的核内包涵体被认为仅仅是细胞质侵入细胞核，对疾病没有任何明显的功能或影响。我们调查了75例肝细胞癌（HCC）标本中的此类内含物。在这种情况下，我们证明了这些内含物是真正的内含物，完全封闭并由核膜界定，含有简并细胞器和溶酶体蛋白。此外，其发生与患者生存率呈正相关,但与肿瘤分级或分期无关.在标准化区域中，肿瘤肝组织中平均每个样本存在124个夹杂物，而非肿瘤相邻切片中存在5个夹杂物，并且所有仔细检查的HCC中有89％显示至少一个膜结合的核夹杂物。透射电子显微镜的超微结构表征显示变性物质，如溶酶体的残留物，内质网和高尔基体内的内含物。由于内含物的含量似乎比细胞质更浓缩，并且包含更少的完整细胞器，我们认为它们不仅仅是细胞质的内陷。分离和免疫荧光染色的细胞核的三维（3D）重建表明，内含物完全位于细胞核内，与细胞质没有任何连接。内含物的限制膜包含层粘连蛋白B，表明核膜起源。对自噬相关蛋白p62，泛素，LC3B,组织蛋白酶B和组织蛋白酶D。三重免疫荧光染色，然后进行3D重建，显示p62，泛素和LC3B在相同的包含中共定位。我们的观察发现，这些内含物是完全被原子核包围的真实内含物。我们认为，包涵体内自噬相关蛋白和蛋白酶的存在有助于有益的生存。
