May 26, 2012
Isolation, Culture and Cholinergic-Induced Differentiation with BMP4 of Adult Rats’ Neural Stem Cells
Research has confirmed that neural stem cells have the ability to update itself and multiplex differentiation potential. Different cell factors can affect the proliferation and differentiation of neural stem cells in the process. Neural stem cells of neurons can express a variety of neurotransmitters, such as GABA, substance P, dopamine, etc, but did not add in special induction, most of the neural stem cells to differentiate into nature can GABA neurons, spontaneous grow into other phenotype neurons is very rare. Many studies show that by adjusting the cell factors can change the differentiation direction, neural stem cell differentiation for a specific type of cell. Some reports BMP 9 (BMP9) can by inducing acetylcholine shift enzyme and acetylcholine transport sac of gene expression, and increase the training of nerve cells acetylcholine synthesis; Brain BMP9 can increase indoor injection on live animals in the brain tissue of acetylcholine levels, think BMP9 is the central nervous system of the choline can differentiation factor. BMP in the family of neural stem cells with BMP4 whether the choline can differentiation and raise the nerve cells choline can express effect, has not been seen experiment report. This experiment through training and somatic cells in the body from animals know whether it has the role BMP4. First of all, from 2 months of SD rats hippocampus, the striatum, the third ventricle and the lateral ventricle master's degree thesis of neural stem cells in adult rats separation cultivation and BMP4 acetylcholine differentiation VZ, SVZ area to separate neural stem cells. The mechanical scattered and chemical digest method separating out of the cell, serum-free medium, with training; To Nestin, NF160 and GFAP antibodies as an antioxidant, immune cells chemical dyeing. The results showed that in optical differ under a microscope to see the cultured cells have the neural stem cells form characteristics and proliferation properties; Immune cells cultured cells chemical dyeing saw a Nestin positive reaction, NF 1 60, GFAP negative reaction, namely cell have neural stem cells in the immunological characteristics. Then, the neural stem cells in the medium to join or not join BMP4, observe the choline can differentiation condition. Will train the neural stem cells into two groups, BMP4 group to switch to join BMP4 induction medium, training, no BMP4 group still using the original medium training as a comparison; Eight days after training to cultivate cells were performed respectively ChAI... Antibody indirect immunofluorescence stain, ChAT antibodies and Nestin antibody SABC a DS double standard immune cells dyeing and mark of chemical FITC choline can flow cytometry detection.
The results show a more cells BMP4 group strong green fluorescent, show ChAT positive; And no BMP4 group less cells produce a weak ChAT fluorescence. Immune cell chemical dyeing of bids are three kinds of cells: one kind is ChAT antibody response positive cell, it is purple and blue cell plasma, and some of the cells are typical neurons morphological characteristics; The second Nestin antibody response positive cells, the red cell plasma, a round or sample precursor cells the shape of the cells in the part of the cells in process of division; The third kind for Nestin and ChAI, antibody response negative cells, the cells in the slurry not shading, cells have neurons morphological characteristics. Microscope count, BMP4 group of cell, ChAT reaction positive about 28%, Nestin reaction of positive about 38%; And no BMP4 group of cells, Ch door a, positive reaction of about 8%, and the positive reaction Nestin is about 52%. Flow cytometry BMP4 group ChAT was testing positive cells significantly higher than no rate BMP4 group (16%, 7%, P < 0. 05). The results show that: the generation of BMP4 separation, rats in vitro culture in the hippocampus and the striatum of the neural stem cells have such as choline can differentiation entrainment. Third, will BMP4 injection into adult rats in the hippocampus right the dentate gyrus to nerve cells to observe its master's degree thesis of neural stem cells in adult rats separation cultivation and BMP4 acetylcholine differentiation choline can express influence. Will only 20 adult male SD rats randomly divided into two groups: control group BMP4 group and salt water. After anesthesia, using three-dimensional localization technology will 1 wind BMP4 (BMP4 group) or physiological saline injection (control group) rat hippocampal right the dentate gyrus area. After 14 days take brain, paraffin slice, do ChAT antibody immunohistochemical study. With image analysis system calculation rat hippocampal left or right side the dentate gyrus ChAT positive cells of the total area, with 51, Al 'A 7.0 t test software. The results show positive neurons in the hippocampus ChAT CAI, CAZ, CA3 and the dentate gyrus all have distribution; BMP4 rats in the hippocampus right the dentate gyrus district door corpse positive cells Ch area significantly higher than the left (P < 0.01); Whereas the control rat hippocampal left or right side the dentate gyrus ChAT positive cells within the area of no significant difference (P > 0.05). Two groups of comparisons, BMP4 rats in the hippocampus in the right the dentate gyrus ChAI... Positive cells is significantly higher than the control group of the same parts (P < 0.01); And the left between the two groups in the hippocampus in the dentate gyrus ChAI, positive cells no significant difference in the area (P >.05). The results indicated that, the hippocampus the dentate gyrus area after the injection BMP4 accept, can induce the cells appear higher in the choline can express. In short, the results of this study indicate that: (l) from an adult rat striatum hippocampus, such as isolated area has morphological characteristics of neural stem cells and immunological characteristics of cells; (2) BMP4 join in the medium to induce the in vitro culture adult rats, the hippocampus striatum area such as neural stem cells in the choline can differentiation; (3) the hippocampus BMP4 into the dentate gyrus adult rats to induce in the neurons choline can express hike. Tip of the clan has induced BMP4 BMP neural stem cells in the choline can express or the effects of differentiation, worth in neural degenerative diseases of diseases such as animal experiment and clinical treatment for further study
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