| PART ONE: Cultivation of human embryonic neural stem cells from human embryos at different ages in vitro[Objective] To explore the methods of isolation, cultivation and expansion in vitro of human embryonic neural stem cells and to observe the characteristics of proliferation and differentiation of neural stem cells in vitro. To establish the techniques of isolation, cultivation and in vitro differentiation of human embryonic neural stem cells and to proliferate the human embryonic neural stem cells in vitro for further research.[Methods] The cortex tissues of human embryonic brain were isolated from human embryo at 4 months of gestation by means of water pocket abortion and human embryo of gestational age from 6 to 10 weeks by mifepristone abortion respectively. Unicellular suspension was obtained from the embryonic brain tissues by dissociating enzymatically and mechanically. Some of the suspension cells were frozen and the others were cultured. A serum free medium(DMEM/F12) containing EGF and bFGF(both 20ng/ml) was used to culture, expand and passage neural stem cells continuously in vitro. Cell morphology was studied with HE staining and under phase contrast microscope. The chromosomes of human embryonic stem cell were observed by conventional Giemsa stain. Immunocytochemistry and immunocytofluorescence were used to identify expression of Nestin(marker for the neural stem cells), NSE(marker for the neurons), GFAP(marker for the astrocytes) and CNP(marker for the oligodendrocytes). Flow cytometry and cell growth curve were performed to evaluate proliferation ability of the neural stem cells.[Results] The neurospheres that were isolated from the cortex of human embryonicbrain could expand and passage in the serum free medium with presence of EGF and bFGF. Nestin positive cells could be found easily in the neurospheres with immunocytochemistry and immunocytofluorescence assay. After attachment they could differentiate into GFAP, CNP and NSE positive cells respectively. The same neural stem cells could also be harvested from those cryopreserved cells after culturing in vitro. The results of Flow cytometry and cell growth curve show that the human embryonic neural stem cells could proliferate continuously in vitro. [Conclusion] The cells derived from the cortex of induced labor or abortive human embryonic brain show the capabilities of proliferation, self-renew and multipotential differentiation, these capabilities are the characteristics of neural stem cell, they were cultured in serum free medium(DMEM/F12) containing EGF and bFGF. The same neural stem cells can also be obtained from the cryopreserved cells of human embryonic brain. Especially, techniques developed for successfully cultured neural stem cells from drug abortive fetus were convenient and practical, and can be considered as a "neural stem cell depository" for further research.PART TWO: Proliferation and differentiation of human embryonic neural stem cell[ Objective ] To investigate the factors which may affect the proliferation and differentiation of human embryonic neural stem cells in vitro.[Methods] The cultured human embryonic stem cells were divided into four groups: Group EGF(only with 20ng/ml EGF, without bFGF), Group bFGF(only with 20ng/ml bFGF, without EGF), Group EGF+bFGF(with 20ng/ml EGF and bFGF), Group control(without EGF or bFGF). The optic dense(OD) of the cultured cells was detected by MTT assay every day from seeding to the sixth day. The cells from human embryonic brain were cultured without EGF or bFGF; their cell cycle dynamics were tested by Flow cytometry on the first day, third day and seventh day. Cell cycle dynamics of primary cultured human embryonic stem cells were tested by Flow cytometry on the third day and the seventh day. The human embryonic stem cells were plated on poly-L-Lysine coated glass coverslips in 24 cell culture plate, theimmunocytofluorescence was performanced on the first day, third day, seventh day, tenth day, fourteenth day. Quantitation of the astrocytes or neurons w...
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