Genetic Engineering Of Embryonic Stem Cells Used For The Basis Of Experimental Studies Of Ad Treatment

Embryonic stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies. The transplantion materials from ES cells can avoids the problem of transplant rejection by two approches. The first approch is to establish stem cell lines from the patient's own cells through therapeutic cloning. Another is to chang the genes of ES cells. So ES cells was thought as a very good source of transplantation materials and a very good vector cell to transfer therapeutic gene to the body. Alzheimer's disease (AD) is caused by a progressive and rather specific degeneration of certain neuronal populations in the central nervous system. One of the hallmrks of AD is the deposition of amyloid (A P) in senild plaques. It is generally postulated that the deposition of A P is ultimately responsible for the neuronal degeneration of AD. During the last few years several laboratories have presented compelling evidence indicating that brains with AD are subject to a pervasive level of oxidative stress. Melatonin(MT) exhibits a unique combination of antioxidant and antiamyloidogenic features which may be relevant to the design of preventive and therapeutic strategies in AD. Special treatment approach must be considered, for the half-life of MT is very short (0.5 - 5.6 seconds). In the present study, AANAT (the limited enzyme of MT) gene was transfected to ES cells. It was expected to produce therapeutic effect by two approches: antioxidant and antiamyloidogenic effect of MT, and the cell replacement therapy by neural precursor cells derived from ES cells. Ill ES cells can differentiate and reconstruct according to the environmental cue after being transplanted into the brain, but the transplantation of ES cells may result in teratoma. Comparing with ES cells and mature neural cells, neural precursor cells (NPC) are more suitable for neural transplantation. So in the first part of our work, the method to culture ES cells and to obtain high purity of NPC from ES cells were studied. Owing to the lack of animal model of AD, which has the deposition of A P in the brain, it becomes impossible to study the therapeutic effect of gene transfected ES cells on AD, So in the second part of our work, the cell model which overexpressed APP were established. In the last part of our work, The effects of the conditioned medium from AANAT gene transfeted ES cells and MT on the cell model were studied. The differentiation of AANAT transfected ES cells to NPC, and the transplantation of these cells to the brain of the animal model of AD, are the next step of our research work. Part one ES cell culture is a basic work in the ES cells research. Feeder layers are necessary for ES cell culture. Mouse embryonic fibroblasts (MEF) are commom used feeder layers. The limited usable passages of MES brought a great deal of work to ES culture and made it difficult to manipulate gene. In the present study, human embryonic fibroblasts were used to culture ES cells, it was found that ES cells on HEF are positive to alkaline phosphatase. The results of the present study showed that HEF could replace MEF to maitain the undifferentiated condition of ES cells. HEF has several benefits compared with MEF: (I) Simplifying the culture of ES cells. (2)The contamination of virus and mycoplasma could be av...