| Neural stem cells (NSCs), a type of stem cells, are capable of of self-renewal which may be maintained for life-long, proliferation and differentiation like all other stem cells. Accumulating evidence suggests that NSCs are characteristic to differentiate into three main kinds of central nervous system cells: neurons, astrocytes and oligodendrocytes. The discovery and intensive research of neural stem cells may bring new hope for curing the diseases of cental nerve system. But the proportions of NSCs vary at different developing phases and in different location of central nerve systems and are always relatively low. Since NSCs being discovered, neurologists all over the world have attempted to establish in vitro culture system to produce NSCs rapidly and to generate high rate of neurons through differentiating NSCs. Leukemia Inhibitory Factor( LIF), a 20 kD protein containing 180 ammo acid residues, is a multifunctional glycoprotein that induces macrophage differentiation and suppresses the proliferation of the murine Mlmyeloid cell line. LIF plays an important role, along with interleukin-6(IL-6) and granulocyte-colony stimulating factor(G-CSF), in the regulation of early hematopoietic stem cells. Gangliosides are one of the important component of cell membrane and CNS is rich of gangliosides. Both Gangliosides and polypeptide growth factors are trophic agents involved in almost all stages of neural cell generation, differentiation, survival, and pathology. In most cases their physiological roles remain unclear. Monosialotetrahexosylganglioside(GM-l), one of themain component of ganglioside in mammal capable of passing blood brain barrier and embedding in the damaged neurons membrane, has distinct functions in resisting on neuronal injury, promoting rehabilitation and growth of neural cells. In the recent years, most of the research regarding NSCs has focused on murine animals, while only a few on human embryonic NSCs. The influences of LIF on proliferation and differentiation of NSCs were carried out abroad , while there are few literatures available in our country . No report regarding the effects of GM-1 on NSCs is available in our country to date. In the present study, 10w human embryonic NSCs were isolated from ventricular zone/subventricular zone(VZ/SVZ) with mechanical method and incubated in serum-free medium with different additional components, the growth of each group was compared with other groups' and the proportions of neuron and glial cells of each group after differention under same or different condition were compared with other groups', thus the aptness of mechanical method for NSCs incubation and the influences of LIF and GM-1 on growth and differentiation of NSCs were discussed, moreover, Nestin and PCNA of the cells cultured and NSE and GFAP of the cells after differentiation were exmined according to immunocytochemistry method, in this way , the NSCs were identified.Methods: The tissues were obtained from VZ/SVZ zones(VZ/SVZ) of lOw human embryon and dismissed with a polish-Pasteur pipette. According to the difference of additional components in basic medium(DMEM/F-l 2+827), the cells were divided into five groups: A added with bFGF(20ng/ml), B with bFGF(20ng/ml)+EGF(20ng/ml), C with bFGF (20ng/ml) +EGF (20 ng/ml)+LIF(10ng/ml), D with bFGF(20ng/ml)+EGF(20ng/ml)+ GM-l(33.5ng/ml), E with GM-1 (33.5ng/ml), the cells of each group were put into two bottles and were cultured(5%CCO2, 37 ).(1)A few of cell suspension were got out of group A, B and C respectively, the cell concentrations were modulated to 1 105 /ml, then the cells of each group were plated in one 96 well culture plate and cultured in CO2 Gas Incubator, 12wells for each group and 0.1ml for each well . At 4d and 8d, the NSCs mobility of groups A, B and C were detected according to MTT method, then the influence of LIF on NSCs proliferation were learned.(2) A few of cell suspension were got out of group B, D, E and F (the basic medium-DMEM/F 12+827) respectively, the cell concentrations were modulated to 1 105 /...
|
PDF Full Text Request