Effect Of Xinmailong Injection On Proliferation And Differentiation Of Neural Stem Cells In Hippocampus Of Newborn Rats

The adult neural cells can not regeneration and its function can not rebuild after the injury of them, the neural function of the patients with nervous system damage and neurodegenerative diseases is hard to recover. However, recent studies have found that, the central nerve system of mammals in the embryo and adulthood have neural stem cells (NSCs), which have the capacity of unlimited proliferation, self-renewal and multipotent differentiation. Neural stem cells originate from neuroepithelial cell of the embryonic period mammals, and can be differentiated into neurons, astrocytes and oligodendrocytes. The discovery of neural stem cells challenges the traditional theory and brings new prospects for the research of the neural cells regeneration and restoration. However, in natural condition, most of the neural stem cells differentiate into glial cells and the minor differentiate into neuron. Now there is unable to effectively control the NSCs oriented differentiate into specific nerve cells. Therefore, how to control the NSCs oriented differentiate into functional neurons is a hotspot and difficult problem in neuroscience. Xinmailong injection (XML) is a nucleotide compounds extracted from periplaneta americana. The experimental results validate the periplaneta americana extracts has anti-tumor, antibacterials, antiviral, anti-inflammatory, cell proliferative, tissue repair, scavenge free radicals antioxidation activity. This experiment is designed to observe the effect of Xinmailong injection on proliferation and differentiation of neural stem cells in petri dish, and whether it can be induced to neurons.ObjectiveTo establish the methods of in vitro culture of neural stem cells isolated from newborn rats. To examine the proliferation and differentiation of neural stem cells. To investigate the effects of Xinmailong injection on the proliferation and differentiation of neural stem cells from neonatal Sprague Dawley rats hippocampus.Methods1. The hippocampal tissue of newborn rat within24h were dissociated and cultured with serum-free medium using single-cell cloning technique and passaged in vitro. To observed the cell morphology under phase-contrast. Nestin, BrdU, microtubule-associated protein2(MAP-2), glial fibrillary acidicprotein (GFAP) expression of the3rd passage of the neural stem cells were detected using immunofluorescence cytochemistry staining.2. Hippocampal neural stem cells of the3rd passage were induced by xinmailong injection of final concentrations of0.5%,1%and2%with serum and divided into the various experimental groups and the control group. The solution was changed every3days, after5days later, to observe the cell morphology under the inverted microscopy.3. Observe the expression of the cell’s MAP-2and GFAP after differentiation by immunofluorescence cytochemistry staining. The amount of MAP-2positive cells was obtained respectively by Image Pro plus6.0, and to calculate the percentages.4. Monotetrazolium (MTT) assay was used to detect NSCs proliferation under the conditions of xinmailong injection.5. Hippocampal neural stem cells of the3rd passage were induced by xinmailong injection of final concentrations of2%with serum and divided into the various experimental groups and the control group. After5days later, to detect the NSE with the RTQ-PCR. 6. To detect apoptosis of the NSCs by the xinmailong injection with the flow cytometry.Result1. Neural stem cellsextracted from the neonatal Sprague Dawley rats hippocampus have the potential of self-renewal and multi-directional differentiation. The immunofluorescence cytochemistry staining of Nestin and BrdU was positive and the differentiated cell can express MAP-2and GFAP.2. The experimental group can differentiate more neuron and more immunofluorescence cytochemistry stained MAP-2positived cells were observed comparing with the control group. There were significant differences((?<0.05).3. The monotetrazolium (MTT) assay showed that the absorbance value of the experimental group was higher than the control group, and the results have statistically significant difference (p<0.05).4. The results of RTQ-PCR showed that the NSE of experimental group was higher than the control group, and the results have statistically significant difference.5. Flow cytometry analysis showed that the experimental group and the control group had not obvious difference.Conclusion1. Neural stem cells were successfully isolated from the newborn rats, and were proliferated, subcultured and induced to differentiate to neurons and glial cells in vitro.2. Xinmailong injection may promote the differentiation of NSCs into nerve cell.3. Xinmailong injection may facilitate the proliferation of NSCs.