Study About Calmodulin In The Oriented Differentiation To Neural Cells From Cortex Neural Stem Cells Of Neonatal Rat

ObjectiveNeural stem cells are the multipotential stem cells and can continually proliferate in serum - free medium, as well as keeping feature of stem cell, it can be differentiation into neuron, astroblast and oligodendrocyte induced by factors.Calmodulin ( CaM) was paid closely attention by people as a important Ca unite - protein. CaM has lots of functions, and participates in the process of Ca²⁺ giving play it's function. It has be proved, brain and endocrine are oranges in which there are most plentiful CaM. The typical animal cell contain 10⁷CaM molecule, it takes 1% in the total protein. CaM plays different roles in different tissues and cells, different physiological state and different stages of growth.Some dates presented the concentration of Ca²⁺ is related with the function of Actin, so CaM and Actin have correlation. Actin is a cellular skeleton protein. It takes part in the form of microfilaments, and maintains the function of cells with microtubules and intermediate. Actin plays a important role in growing and developing of cells as skeleton protein. It takes part in cell's motion, material transport and cell's division, differentiation, gene expression and so on.Lots of reports proved CaM is the most important finding till DNA rebuilding. But in the culture of NSCs and oriented differentiation, the reaction of CaM wasnt reported. Our test inquire the expression of CaM in the process of cortex NSCs oriently differentiation to neuron in order to provide experence basis for riching the theory of NSCs and working out the specific property of NSCs.Methods1. Primary and secondary cultured methods of cortex neural stem cells for newly born rat : shaving out a thin layer of cerebral cortex in sterility using newly born rat of Wistar in 24 hours, rinse, shears craniotripsotome, digestion in 0. 25% pancreatin , implantation cell into serum - free medium [ It contains DMEM/F12 (1:1) , bFGF (20ng/ml) , EGF (20ng/ml) and B27 (50 x ) ] in density of 1 ⁵ x 10 /ml , incubate in 37 degree, 5% CO2, and changed medium one times every other day , observed clone formed under phase contrast microscope, Collecting clone which culture 7 days, and we mono - cell suspension through centrifugation, implantation them in the same density above and made secondary culture, transfer of culture one times every 7 days, total made 4 transfer of culture.2. Induced cultured cortex neural stem cells differentiation; collecting nerve ball obtained by primary and secondary cultured, centrifugation, implantation mono -cell suspension in medium including BDNF(2ng/ml) and NGF( 10ng/ ml) and induced never stem cell differentiation, we changed the medium every other day, and observed the changes of cell differentiation form under phase contrast microscope.3. Immunocytochemistry methods (SABC) :(1). Evaluation of neural stem cells: Evaluation was made for stem cells of primary and secondary culture, and made Nestin histochemistry staining using the method of immunocytochemistry, then observed by Olympus light microscope.(2). Evaluation of neuron: Evaluation was made for neuron of 1st,3rd, 5 th ,7th, and made NSE histochemistry staining using the method of immunocytochemistry, then observed by Olympus light microscope.(3). Observed the expression of CaM during the procedure of cell differentiation : Using the method of immunocytochemistry to made CaM histochemistry staining of the cell differentiation of 1st,3rd,5th,7th, then observed by Olympus light microscope.4. Analysis of images and treatment of statistics: Gathered neuron of differentiation in the 1st.the 3rd,5th and 7th,after made immunocytechemical stain, we analyze the results of expression of CaM using Motic Images Aanvanced 3.0 system. Under microscope ( 400) , we gathered 50 cells in each group and measure IOD. These dates were analyzed by SPSS 11.0 for windows, and presented by x ± s, and analyzed the dates by analysis of variance between each two groups. If p <0.01, the result have sense.5. Observe the time - space changes of calmodulin and Actin in the procedure of nerve cell differentiation with immunoflurescence technique: The distribution of calmodulin and actin in cells during the process of nerve stem cells oriented differentiation to nerve cells was observed by fluorescence microscope when calmodulin mono - marked by Cy3 and actin mono - marked by FITC were made immunofluorescence staining. Calmodulin and Actin were marked with the fluorescence technique at the same time, we observed the three - dimensional structure of Calmodulin and Actin , and the relationship of time - space between them.Results1. Cultivating and evaluating the cells12 hours after the cultivation of cortex neural stem cells of newly born rat, there were no cell mass. But 24 hours, we could see a small quantity of cells were division into 2-4 cell mass, and the amount were kept increased with the time passed, the clone constituted in the number of ten to hundred cells would be formation in 7 days. These clone were growth in suspend, and cell form regulation, were round. These cell mass were made Nestin immunocytochemical stain, the result showed Nestin was strong positive.Breaking up cell mass and using culture solution contained serum,the cells stick to the wall swiftly in 4hours. In 2 day the differentiation of the neural stem cells grow processes. There were three kinds of cell; one is the kind of cell which are bigger and have more thicker processes than others, their number also is more. Another cells is smaller but they have longer and less processes, andtheir refractive power are stronger and lie on above - mentioned cells. The last kind is the cells which have shorter and thinner processes. These differentiation of neural stem cells were made NSE immunocytochemical stain, if the result showed NSE was positive, it is neuron. The body of neuron is small and ellipse or triangle. These neurons have 2-4 processes.2. The results of CaM immunocytochemical stain:In the process of cortex neural stem cells orientated differentiation to neuron , the expression of CaM located in nucleus and around nucleus in the 1 * day of differentiation; During the growth of neuron and processes, CaM located in cytoplasm and processes, and the expression of CaM in processes is present like joint. We can also find the expression in nucleus is less than the 1st day.3. The result of analysis of images;During increasing of culture days, the expression of CaM have clearly sense,p <0.01.4. The result of CaM and Actin marked with fluorescence was observed in confocal laser scanning microscope;Red fluorescent CaM and green fluorescent Actin in neuron were seen in distribution in the same saple. The distribution of both was the same, and was located in nucleus and around nucleus in initial stage of differentiation, and in cytoplasm and processes in the maturity of differentiation. Two kinds of fluorescence could been seen by excitation with double wave - length, orange light mixed with two kinds of light could be present in some spot. Two kinds of light were present in the same time in cytoplasm and processes, they were followed, sometimes overlapping but no confluence, this suggested they were correlation.Conclusion1 During the differentiation of neural stem cells into neuron, The expression of CaM is related straightly to the growth of neural branch and neural mature. CaM is beneficial to the growth of the differentiated neuron.2 The expression of CaM is cooperate with the expression of Actin. It made clear that CaM and Actin have relation. It also pointed out that the cooperative...