RESUMO
Objective To evaluate the effect of exosomes from different sources on the growth, metastasis and immune microenvironment of hepatocellular carcinoma ( HCC ) in a mouse model of orthotopic transplanted hepatoma so as to provide new insight into the clinical immunotherapy of hepatocellular carcinoma. Methods The serum-derived exosomes were obtained by ultracentrifugation from Hepa1-6 cells and 3-week orthotopically tumor-bearing HCC mice. The morphology and size of exosomes were identified by transmission electron microscopy. One-week tumor-bearing HCC C57BL/6 mice were randomly divided into 3 groups ( 3 mice for each group ) , and respectively treated with tail vein injection of 120 μl PBS(control group), 120 μl Hepa1-6 cell-derived exosomes (1μg/μl, TEXcel group), and serum-derived exosomes (1μg/μl) (TEXserum group). The treatments were conduced once a week for 3 consecutive weeks. The mice were sacrificed on the 4th day after the treatments, and the liver tissue and lung tissues were dissected. The volumes of the liver cancer tissues were measured. The expression of PD-L1 protein and CD3+and FoxP3+T lymphocytes infiltration in the liver cancer tissues were respectively detected by Western Blot and immunohistochemical staining. The lung metastasis of the liver cancer was detected by hematoxylin-eosin staining. Results The diameters of the Hepa1-6 cell- and serum-derived exosomes both were about 100 nm, and were uniform vesicles with complete membrane structure. Compared with the control group, Hepa1-6 cell-derived exosomes had a certain inhibitory effect on the growth and lung metastasis of HCC, while there was no significant difference for the TEXserum group. Western Blot results showed that compared with the control group, PD-L1 protein expression was significantly up-regulated in both TEXcel group and TEXserum group. Immunohistochemical staining showed that compared with the control group, the infiltration number of Foxp3+-labeled regulatory T cells (Treg) had no significant changes in TEXcel group and the TEXserum group, and the differences were no statistically significant (all P>0.05). However, the infiltration number of CD3+-labeled T lymphocytes was significantly reduced in the two groups, and the differences were statistically significant (all P<0.05). Conclusion The Hepa1-6 cell-derived exosomes have a certain inhibitory effect on the growth and lung metastasis of HCC, and have no obvious regulation effects on the immune microenvironment of HCC. The serum-derived exosomes from orthotopically tumor-bearing HCC mice can promote the growth and lung metastasis of HCC and immunosuppress the microenvironment. The Hepa1-6 cell-derived exosomes are expected to be used for immunotherapy studies of liver cancer.
RESUMO
Parthenogenetic embryonic stem (pES) cells isolated from parthenogenetic activation of oocytes and embryos, also called parthenogenetically induced pluripotent stem cells, exhibit pluripotency evidenced by both in vitro and in vivo differentiation potential. Differential proteomic analysis was performed using differential in-gel electrophoresis and isotope-coded affinity tag-based quantitative proteomics to investigate the molecular mechanisms underlying the developmental pluripotency of pES cells and to compare the protein expression of pES cells generated from either the in vivo-matured ovulated (IVO) oocytes or from the in vitro-matured (IVM) oocytes with that of fertilized embryonic stem (fES) cells derived from fertilized embryos. A total of 76 proteins were upregulated and 16 proteins were downregulated in the IVM pES cells, whereas 91 proteins were upregulated and 9 were downregulated in the IVO pES cells based on a minimal 1.5-fold change as the cutoff value. No distinct pathways were found in the differentially expressed proteins except for those involved in metabolism and physiological processes. Notably, no differences were found in the protein expression of imprinted genes between the pES and fES cells, suggesting that genomic imprinting can be corrected in the pES cells at least at the early passages. The germline competent IVM pES cells may be applicable for germ cell renewal in aging ovaries if oocytes are retrieved at a younger age.
