Influence Of NGF On The Proliferation, Differentiation And Apoptosis Of Neural Stem Cells

Objective: The discovery of Neural stem cells (NSCs) is the most important development of neurobiology in the end of 20^th century. NSC, a type of stem cells, is capable of self-renewal which may be maintained for life-long, proliferation and differentiation like all other stem cells. Accumulating evidence suggests that NSC is characteristic to differentiate into three main kinds of central nervous system cells: neurons, astrocytes and oligodendrocytes. It has vast clinical application foreground for it's ability of renew and differentiation. The mechanism and influencing factor of NSCs'proliferation and differentiation is unclear. Thorough research of which can make us regulate and control it in adult mammalian brain or in vitro in order to replce the degenerative and necrotic cells in brain, which provide a new direction for curing damage and degenerative diseases in CNS. Neral factor have important function in the proliferation and differentiation of embryonic human neural stem cells, a type that the Neurotrophic factor is among them. Neral growth factor(NGF), the first target-derived neurotrophic factor to be discovered, is one of the most important biological activity molecules in nervous system. It is necessary for nervous system to maintain the normal growth and function. NGF can promote the transfer and developm of embryo nerual crest sensory neuron, sympathetic ganglion and base prosencephalon cholinergic neuron. And participate in the nerve rebirth and thefunction repairs of the harm, to support the balance between nerve, immunity and the endocrine system.In the present study, methods including cell culture, immunocytochemistry and flow cytometry etc were used in the isolation and culture human neural stem cells. The proliferation, differentiation and apoptosis of influenced by NGF were examined. Finally the relation between NGF and life of NSCs were applied. Furthermore, this investigation was also to devise a new strategy for curing diseases in CNS.Methods: The tissues were obtained from VZ/SVZ zones(VZ/SVZ) of human embryon and cultured with serumHfree medium. The neurospheres were isolated with different dose of Dispase II and Trypsin. Observed the process and the sharp of cells and cultured singer neural stem cells to finding an ideal dose of in isolating neurospheres. Then get a number of viable singer cells on standby. Resuspend cells with serum-free medium at a density of 5* 105 cells/ml and seeded them in tissue culture plates. bFGF was added to plates as positive group; noting was added as blank group; different dose of NGF were added as experimental groups. The proliferation ability of the cells were measured by MTT methods at 3 days and 10 days; Counted the number of neurospheres at 10 days; Examed the rate of cell apoptosis at 5 days after having been cultured; Gathered all cells in plates at 7 days and used Streptvidin-Peroxidase (S-P) immunocytochemistry technique to detect the expression of NSE and GFAP, and counted the number of NSE(+) and GFAP(+) cells; supplemented media with 10% fetal bovine serum. Gathered all cells while cells adhered to the plates. Detected the expression of NSE and counted the number of NSE(+) cells. The statistical analysis was executed by SPSS 10.0 software. The a values less than 0.05 were considered significant.Results: 1. After 8—10 days , cells turned to be hundreds of suspended spherical clone, identified positive by immunocytochemistry Nestin.2. After cultured 7 days, the cells that be isolated from neurospheres by 1.2— 1.8 u/ml Dispas II formed hundreds of suspended spherical clone.3. The Stat. of the number of neurospheres and MTT test showed: the degree of proliferation in 50 ug/L NGF group was lower than that in other experimental groupsafter 3 days (PO.05). The proliferation of all groups was increased in a certain degree at 10 days than that at 3 days. There was no significant differences between 10 ug/L NGF group and the positive-control group and was significant differences between 10 ug/L NGF group and the blank group. Other experimental groups was on the contrary.4. The rate of cells differentiation was increased with the dose of NGF added. Differences were significant (PO.05). After cultured with media added serum, rate of NSE(+) cells were no significant differences among 5 % 10> 20 % 50 ug/L NGF groups; the rate in 2 ug/L NGF group is lower than other experimental groups (P<0.05).5. The analysis of cell apoptosis by Flow cytometry showed: there was no significant differences among 2% 5> 10 ug/L NGF groups, 20% 50 ug/L NGF groups have more cell apoptosis than others. The PI of 10 ug/L NGF group was higher than others.Conclusions: 1. The cells from human embryonic VZ/SVZ zone, grew in a good state in serum-free medium after having been dismissed according to the method of machine, The cells cultured in this experiment, which express Nestin.2. It's an ideal method to isolate meurospheres by using certain dose of Dispase II (1. 2 1.8 U/ml) for getting a number of viable singer cells.3. NGF was effective in the proliferation and differentiation of human NSC and a dose-result relation had been found. Low dose of NGF mainly facilitate the proliferation, of NSCs, High dose of NGF shows obvious influence on NSCs'differentiation.4. NGF may facilitate the apoptosis of NSCs to a certain extent with the increasing of NGF's dose.