Studies On Isolation, Characterization, Culture Of Rhesus Monkey Embryonic Stem Cell Line And Its Application

In this study, the embryonic stem cell from rhesus monkey blastocysts was isolated, cloned and characterized. The procedure for in vitro routine cell culture of rhesus monkey embryonic stem cell line, i.e. RS366.4, was developed and optimized. The effect of leukemia inhibitory factor (LIF) on trophoblast differentiation during human implantation was studied by using differentiated RS366.4 cell as a cell model. And photodynamic inhibitory effects of three perylenequinones on RS366.4 ES cells and other carcinoma cells were also investigated. The following main conclusions were reached:1. A total of 92 normal GV oocytes were obtained from 4 FSH-primed rhesus monkeys via in vitro culture, in vitro fertilization and in vitro maturation. 6 of hatched blastocysts with high quality were choosed from 22 oocytes when cultured in HECM-10 (with 20% BCS) and 1 of the embryonic stem cell line-like, i.e. RS5 cell line, was finally isolated from 6 of the inner cell mass. The RS5 ES cell line-likes appeared a high nucleus / cytoplasm ratio, a prominent nucleoli, and flatter colonies with individual, distinct cells. After 5 months continuous passage, this RS5 ES cell line-like remains a normal karyotype (42) and alkaline phosphatase (AKP) positive which means this RS5 cell colony remains undifferentiated. When plated at high density and cultured for a longer time, the RS5 ES cell line-like can differentiate into many types of cells.2. The procedure for in vitro routine cell culture, expansion, freezing of rhesus monky embryonic stem cell line, i.e. RS366.4 was established.3. LIF appears to be an important regulator of human embryonic implantation by directly modulating trophoblast differentiation. Studies on the relatioship between LIF and the differentiation of trophoblast were inconsistency. In order to avoid the shortcomings of cytotrophoblasts from early first trimester or term villous cytotrophoblast, the differentiated RS366.4 was used as new early cytotrophoblast model. The results from flow cytometry assay showed that the low concentrations of LIF had the ability to shift cytotrophoblast differentiation towards a syncytial pathway, while the high concentrations of LIF towards a invasive pathway. While, the results from immunohistochemical assay showed that immunoreactive cells for fibronectin, a mark of invasive extra villous cytophoblast differentiation, was positive when the differentiated RS366.4 cells were cultured in the presence of LIF.4. Perylenequinones are a type of excellent photosensitive pigments. The RS366.4 cells were first used to evaluate the photocytotoxicity of EA and the photodynamic bioactivity of EA was first reported in this study. After irradiated for different time, photoactivated EA at lower concentrations had no cytotoxic effects on R366.4 ES cells. Whereas, all of the three perylenequinones can induce apoptosis with a dose-dependent manner in these concentrations when Hce-8693 cells were inbubated with photoactivated EA, HA and HB respectively. The order of photodynamic inhibitory effects on Hce-8693 tumor cells would be approximately HA > EA > HB. EA can also induce apoptosis of B16 carcinoma cells with a dose-dependent manner. All of these results showed that all of EA, HA and HB had significant photosensitive anti-cancer activity.