The Toxicity Of A β 25-35 On Neural Stem Cells And Protective Actions Of Estrogen And Their Mechanisms

Neural Stem Cells is a kind of stem cells defined as neural cells with the potential to self-renew and to generate all the different cell types of the nervous system following differentiation: astrocytes, oligodendrocytes, neurons. The researching development on NSCs has opened a new door to treat neural-diseases. It is hoped that the NSCs that are cultured in vitro and then transplanted into brain or that existing in the adult brain are activated by some factors, will differentiate into neural cells with specific functions to repair injured brain for various reasons. Thus, the success of NSCs therapy is determined by the induced differentiation and the effect of micro-enviroment on the survival, proliferation and differentiation of it.The isolation and culture of NSCs in vitro successfully is the presupposition for its research. To avoid the influence of unknown factors exiting in serum on NSCs' differentiation, serum-free medium with EGF and bFGF as mitogens to stimulate proliferation and inhibit differentiation in vitro was introduced through a long period of search.Induced differentiation is the hot spot of NSCs research all the time. Among factors affecting the differentiation of NSCs, EGF and bFGF are the most thorough and extensive researched. In addition to stimulate proliferation, they also can induce differentiation of NSCs both in vitro and in vivo under different conditions. In addition, Retinoid Acid (RA) and Estrogen are necessary for neural development that means that they may play important roles in NSCs' differentiation. Amyloid β protein(A β ), a spontaneously aggregating peptidy of 39-43 amino-acid, is the primary component of senile plaues. This peptide is derived fromproteolytic processing of the amyloid precursor protein (APP). An increasing'amount of experiments show that A P can induce various toxic reaction in brain, thus play a causal role in the pathogenesis of Alzheimer's Disease(AD). In the meantime, the incapability of neurons to regenerate in AD patients means that it may inhibit the proliferation, differentiation and even survival of NSCs. Estrogen is an important antagonist of A P and can inhibit many toxicities induced by A P , so it has long been closely concerned.Our study isolated and cultured rat's embryonic NSCs successfully, then induced differentiation was investigated by addition of EGF, bFGF, 17β -estrodiol, ATRA respectively into medium with 10% FBS but not EGF and bFGF. In addition, Aβ 25-35 alone or allied with 17β -estrodiol was treated to investigate its toxicity and protective action of estrogen against and mechanisms of them.The objective of the study is to accumulate valuable data for researching induced differentiation of NSCs and to explore new ideas and methods for treatment of AD. Materials and MehodsNSCs were isolated from E-14 embryonic rat's brains, then cultured in serum-free medium with EGF and bFGF(20ng/ml respectively) as mitogens. When cultured in vitro for 8 days, NSCs were divided into three groups: the first one is the group to identify and evaluate NSCs' property and proliferating capability; the second one is the group to induce differentiation of NSCs by EGF, bFGF, 1 7β -estrodiol and ATRA respectively; the third one is the group to investigate the toxicity of A P 25-35 and protective action of 1 7β -estrodiol against Aβ . The above-mentioned groupswere cultured continuely according to formerly conditions, then conducting the following experiments:1. Identification of NSCs and evaluation of their proliferating capability BrdU (5mol/L) was added into the first group to label cells when group division was conducting. Two days later (that is, these cells had been cultured for ten days in vitro from isolation) these cells were collected and immunocytochemistry was performed by anti-nestin to identify NSCs and by anti-BrdU to evaluate NSCs' proliferating capability.2. Induced differentiation of NSCsFor NSCs of the second group cultured for 6 days after group division, all the growth factors were c...