Observation Of GAP-43 Expression On Differentiation Of Bone Mesechymal Stem Cell To Neuron-like Cells

At present there is no effective treatment to stop or reverse the course of diseases concerning retinal degeneration such as inherited retinitis pigmentosa, age-related macular degeneration, Usher syndrome and macular Sorby malnutrition and Star-gardt retinal degenerative changes. It is suggested in many studies that retinal transplantations, such as RPE transplantation, are practical therapeutic methods for diseases causing blind, such as retinal degeneration, age- related macular degeneration. However, there remain huge hurdles in implementing RPE transplantation due to social-economical issues concerning lack of donor, transplantation immunology and other ethical problems.With developments in the field of biological science, stem cell transplantation, serving as a regenerative therapy for retinal disease, becomes the hotspot topic for world wide researchers. Meanwhile, in a larger picture where it seems pressing to acquire new cell strains safe, stable and effective for transplantations, the bone marrow-derived mesenchymal stem cells become the focus of researches. It is generally agreed by researchers that owing to its capacity of differentiating into nerve cells, the post-operation therapeutic effect of MSC could be interpreted in the following two aspects: first, it takes the place of the deceasing cells; secondly, it generates cytokines hence protecting residual cells. We discussed the therapeutic mechanism of MSC via evaluating the changing trends in GAP-43 expression after the differentiation towards neural-like cells. Furthermore, we compared the differences in phenotype and cytokines due to different induction effect of chemical factors and cellulous factors.In this experiments, rat bone marrow-derived mesenchymal stem cells were cultured in vitro, evaluated by flow cytometry, to detect cell surface markers such asβ-mercaptoethanol and brain-derived neurotrophic factors in charging of inducing neuronal-like differentiation, observed to find changes in cell morphology via immunocytochemical techniques for the identification of cell differentiation as well as analysising neural nestin (nestin), neuron- specific enolase (NSE), neural silk protein (NF) expression and post- differentiation expression of GAP-43. BMSCs CD90 (+), CD71 (+), CD29 (+), CD44 (-) were detected, BMSCs can be induced to differentiate into neuron-like cells by the two methods, the differentiated cells can express GAP-43, NSE, NF. 1,2,4,6,8 hours after induction of expression of GAP-43 positive rate gradually increased in both groups, GAP-43 expression in the two groups were first increase after declining to 4 h after induction of expression peaking.Stem cell research is hot, and because of isolation and culture BMSCs is easy, compare with the embryonic stem cells and BMSCs has not ethnics restrictions, avoiding the immune rejection; higher stability, it is not easy to become tumor; greater plasticity, and hss a good future in clinical application. In vitro and in vivo transplantation experiments have confirmed that, under certain conditions, BMSCs can be differentiated to neuron-like cells. [1] A study found that the eyeball with retinal degenerative diseases which transplant BMSCs could promote the recovery of neurological function. Therefore, the BMSCs transplant may become a new therapeutic approaches to retinal degeneration diseases (such as inherited retinitis pigmentosa, age-related macular degeneration). But the transplant experiments show that BMSCs whith transplanted can survive in the retina and integrate in the original structure,but we can not judge the retina-like structure whith the BMSCs formatted have the characteristics of nerve cells, as well as the structure can make the correct nerve transmit, and the promotion of the improvement of neurological function may also be due to the differentiation process of the release of the role of neurotrophic substances, not the real"nerve regeneration."Although BMSCs can differentiation into neuron-like cells in vitro, but in this study neuron-like cells can only lasted a few hours (6 hours later under the microscope we can see differentiated cells become sphere-like, disengage from the culture plates), although some cells after induction can show neuron-like appearance, but whether it has mature neurons function is doubtful. Therefore, further work is to improve the differentiation system, the differentiation of cells can maintain a stable state of neuron-like cells, in order to have enough time to do further study on the function and characteristics.The traditional view that the process of differentiation of stem cells from the stem cells→progenitor cells→precursor cells→terminal differentiation of cells, which accompanied every step of asymmetric cell splitting. However, the experimental results show that, in 1 h after induction of cell that is beginning to show neuron-like appearance, after a gradual transition to a typical neuron-like morphology, thus speculate split in the asymmetric cell differentiation process does not hold a major position, Instead of a single experience a gradual BMSCs to neuron-like cells transformed. We may need a new awareness of stem cells, progenitor cells, the precursor cells and terminal differentiation of cells. Of course, this is still controversial, and further study is going on.Currently, the induction of MSC differentiation in vitro can be divided by chemical and cells, nerve-induced, this experiments were induced by both two methods, there was no significant difference from the morphological observation, and there was also no significant difference from the duration. In addition, some scholars tranplanted MSC into the eyeball without any induction, they believe that the condition in the eyes can provide the necessary cytokines, nerve factor whith can induce MSC into retinal cells, but the author believes that the eyeball with retinal degenerative diseases, because blood-ocular barrier damage, the environment is different from the normal eyes, it is questionable that the eyes with diseases can really provide conditions for differentiation.In addition, the authors found, the higher the rate of integration, the higher the rate of differentiation, with the secretion of nerve cell interaction of the factors needed to further explore.GAP-43 is a membrane protein, are belonged calmodulin- binding protein family, the molecular weight of 23.6 KDa, GAP-43 mRNA 1.5 Kb long, have three exons. GAP-43 synthesis from the body of neurons, transported to nerve endings by flow ,it is widely distributed in the nervous system,and have a high levels in the presynaptic membrane and the growth cone. GAP-43 is considered an internal decision factor in neuronal growth and regeneration, and is closely related to axon growth in the neurons in the process of development and regeneration, and it is molecular markers in nerve tissue in the synthesis as nerve injury , repair, regeneration [5-7]. The unique nature of GAP-43's is that it can be released calmodulin (calmodulin, CaM), CaM can stimulate actin and tubulin polymerization, and GAP-43 involved in the release of neurotran- smitters through integration or small capsule induced growth cone and the end of the presynaptic membrane promote endocytosis expansion [8]. GAP-43 on the previous study are mainly focus on the mature neurons, there are still many unresolved issues in regard to its physiological effects, the specific mechanism and regulation of factors, it has not research reports yet about the differentiation mechanism of stem cells into neural cell. The experimental used two different methods to induce BMSCs to differentiate to neutron-liked cells, the GAP-43 expression were detected in both the two groups, the positive rate of the two groups were gradually increased, and the strength of expression were first increased, then decreased, it get to the peak at about 4 h after induction, and the author believes that the duration of the neutron-liked cell was too short, so there were a large number of cells dead after 4h, it is not sure the real expression of GAP-43, it is important to make a long duration systems in further study, and the results clue on GAP-43 has important role in BMSCs differentiate into neuron-liked Cell and extension of the synaptic process.In a sense, BMSCs is the"rebirth"or"renewable", and GAP-43 is an important control factor in nerve regeneration, In this process of GAP-43 expression told us GAP-43 gene may be activated in BMSCs differentiation process, BMSCs play an important roll in differentiation.