Objective To observe hematoxylin's cytocidal and apoptosis-inducing effects on human urinary cancer cell-T24,and its cytocidal mechanism to the target cell.Methods Target cells were incubated in the medium 1640 for 24h,which contained hematoxylin in dosage of zero(blank),12.5,25,50,100,200μg/ml,respectively;under inverted microscopy to observe target cells'morphologic change,and then harvest them;by trypan blue tmpochrome method to determine hematoxylin's cytocidal activity to the target cells;by flow cytomelry to detect the effects of hematoxylin in its different levels on target cells'apoptosis.Results The control group(without hematoxylin)showed their target cells in a fusiform adherent growth,plump,close-arranged,and with a good transparence.With the addition and increment of hematoxylin,target cells turned round,not adherent,pyknotic,with a bad transparence,as well as chromatin condensation,the cells clumped.Cell death rate of control group was(2.63±0.29)%,with the increased dosage of hematoxylin the cell death rate of test groups was(10.00±4.82)%,(21.88±3.42)%,(76.41±4.82)%,(92.27±6.54)%,and(96.34±8.70)%respectively.Flow cytometry showed cell apoptosis rate in control group was 0.47%(occurred spontaneously),but hematoxylin in dose of 50μg/ml made the apoptosis rate increased markedly,to 43.1 8%,dead cell rate 48.47%,and survival cell rate 8.35%.With the increased hematoxylin dose,cell apoptosis rate decreased gradually,while dead cell rate increased.Conclusion Hematoxylin can inhibit the target cell by two routes:to induce apoptose or kill it.In a lower dose it is able to induce target cell to apeptose;hematoxylin in a dose over 100μg/ml can directly kill the target cell.Making this trial for checking the cell's morphologic changes benefits determining the optimal dosage level and optimal acting duration for the apoptosis induction.

ObjectiveTo observe the fine structure of the trunk kidney in zebrafish, and to identify its secreted exosomes. MethodsThe microstructure and ultrastructure of the trunk kidney in zebrafish were observed by light microscopy and electron microscopy, and the particle size of exosomes was detected by nanoparticle tracking analysis (NTA). ResultsThe trunk kidney was close and parallel to the spine in adult zebrafish. The nephron consisted of renal tubules and renal corpuscles. The renal tubules could be further divided into three types: proximal convoluted tubules, distal convoluted tubules, and cervical segments. The renal corpuscles were composed of glomerulus and renal capsules. The periodic acid-Schiff (PAS) staining results revealed that there were abundant glycogen granules in the proximal convoluted tubules, with brush-like outline in the apical surface of epithelial cells. Under transmission electron microscopy (TEM), there were exosomes distributed in the lumen of renal tubules, with numerous late endosomes and few number of multivesicular bodies (MVBs) in the cytoplasm of the epithelial cells concentrating on the apical side. Meanwhile, MVBs were also distributed in the apical regions of the renal tubules and the podocytes of the renal glomeruli. Immunohistochemical staining results showed that CD9, CD63 and TSG101 were strongly expressed in the lumen surface of the renal tubules, but weakly expressed in the corpuscles and lumen. NTA and TEM results showed that the exosomes isolated from zebrafish trunk kidney were saucer-like outline, and the particle size mode was 144.4 nm, which was consistent with the characteristics of morphological futures of exosome. ConclusionThe zebrafish somatic kidney has the typical structure of the mammalian kidney and is the urinary organ in the body. The renal tubules have the ability to secrete exosomes, and their formation is a process of releasing poly-vesicles to the free surface of epithelial cells into the extracellular space. This study laid a morphological foundation for further study of exosomes in urinary function in aquatic experimental animals as well as the development and application of related models.