The Effects Of Neuron-like Cells On The Activity Of C2C12Cells In Vitro

The development of skeletal muscle tissue engineering will provide some treating advice for many skeletal muscle diseases such as muscular dystrophy, spinal muscular atrophy, etc. Besides, plastic surgery for the treatment of trauma, tumor resection and denervation-induced loss of muscle tissue will also benefit from skeletal muscle tissue engineering. Many scholars are still committed to skeletal muscle in vitro and alternative research, attempt to create a function of the muscle tissue. After many years of exploration and efforts, a number of authoritative research team made considerable achievements, and constantly push forward the study of skeletal muscle tissue engineering progress:Powell used collagen and matrix to build a successful three-dimensional skeletal muscle and added mechanical stimulation to tissue, making sure the formation of muscle tissue, differentiation and the formation of contractile function in vitro; Dennis and Kosnik explored three-dimensional skeletal self organized model and added electric stimulation to the muscle tissue Myooids in the horizonl it would produce a sustained contraction of the excitatory and function. In Germany Bach’s team had co-cultured myoblasts fibrin and scaffold in three-dimension and obtained myotubes that had the multi-polar differentiated functional. However,the skeletal muscle tissue engineering still faces immense challengese,specially in matters of neuralization.Some researchers conducted co-culture experiments primary cultured neurons and myoblast neurons and confirmed the effects of the neurons on the myoblast on the biological characteristics. Wagner found high density tube acetylcholine receptors in the co-cultured group by co-culturing muscle cells and embryonic rat spinal cord. Das found the neuromuscular by co-culturing the rat nerve cells and muscle cells.In vitro, the cultivation environments of the myoblast with proliferation and differentiation cannot satisfy all the requirements primary cultured neurons at the same time, so that became a difficulty on the construction of skeletal muscle.PC12cells is Rattus norvegicus adrenal pheochromocytoma cell line originated from neural crest which it is easy to obtain and culture in vitro. In additon, they have strong proliferation and express TrkA and p75NTR receptors,so that the nerve growth factor can active the signaling pathway of the ras/raf/MEK/ERK after binded its receptors. PC12cells stoped the proliferation and changed cellular phenotype when formed neuron like appearance which looked like neurite structure, for instance, form a synapse like structure, have electric activity and so on. PC12cells can release the acetylcholine in some particular culture conditions, are widely are widely used in studying the neurological disorder in vitro. Zhou Tao studies have shown that the synaptic connections between PC12cells and primary neurons, JEON found that the differentiation PC12cells induced by the NGF can form synapse-like structure. Wang Jing co-cultured differentiation PC12cells and cardiac myocytes in vitro recycling of myocardial innervation of the research model of the donor cells. Glass found that PC12cells stimulate slow-myosin light chain2synthesis in chick breast muscle culture. Defez also found that PC12cells can influence the process of the differentiation of skeletal muscle. The research work referred in this section is pioneered in the domestic about co-culturing C2C12cells and PC12cells.Based on the above background, this article precisely launches the research, chose the differentiated PC12cells by NGF as a neuron model to study whether it interfere with the proliferation and differentiation of C2C12cells in vitro to provide new ideas for the construction of the nerves of muscle tissue. Objective:The effect of NGF on differentiation on PC12cells were identified by mmunofluorescence method.Methods:After thawed by general procedure, PC12cells were implanted in the culture bottle and cultivated in DMEM/F12complete medium supplemented with100U/mL penicillin,100ug/mL streptomycinand10%fetal bovine serum. They were routine cultured in the incubator at37℃and with5%CO2The PC12cells were inoculated on the small coverslip in six well plates and they were fine state.The concentrations of C2C12cells is2×104/L. Changed with DMEM/F12medium contained50ug/L of NGF when the cells were fused80-90%in order to induce differentiation of them.The morphological change of PC12cells were observed under invertphase contrast microscope. PC12cells were detected by immunofluorescence after differentiation culture after3days and7days.Results:The undifferentiated PC12cells were circular, short spindle or triangular.Some of them had neurites under a microscope. In this study we observed cell division in some morphologically differentiated neuronal PC12cells bearing long neurites at the effect of NGF. The cells are interwoven into mesh after3days and further differentiated after7days.Conclusions:In this study we observed cell division in some morphologically differentiated neuronal PC12cells bearing long neurites at the effect of NGF, and MAP-2positive expression, suggesting that PC12cells differentiate into neuron-like cells. MAP-2is a neuron-specific markers, mainly present in the neuron cell body and dendrites, can be used as marker proteins of neurons.Objective:Establish a co-culture system of PC12cells and C2C12cells. The cellular circle of C2C12cells was detected by FCM in order to be certain about the effect of PC12cells on proliteration of C2C12cells.Methods:The C2C12cells were allocated into three groups at random (1) control group:single-C2C12cells groups;(2) experimental group:Co-culturing the C2C12cells with the differentiated PC12cells;(3) negative control:Co-culturing the C2C12cells with the undifferentiated PC12cells. All these are the PC12cells were cultured in transwell chamber and the C2C12cells were inoculated with six well plates.The concentrations of C2C12cells is (1.0～1.2)×108/L.The transwell chamber(pore diameter size of0.4μm) cultured with PC12cells were in six well plates and the ratio of the former with the latter is nearly1:1. Changed the maturation medium depending on the different groups. The experimental group changed with DMEM/F12medium contained50ug/L of NGF in order to induce differentiation of PC12cells. The maturation medium of control group is same as the experimental group and the negative control not contained NGF. They were routine cultured in the incubator with CO2. In additon,the C2C12cells inoculated on the small coverslip in six well plates divided into different groups in the same as the above.The result of the FCM is shown by mean±standard deviation (x±s), between group compares the single factor analysis of variance (One-way ANOVA), if the difference was statistically significant, used the LSD inspection comparison between two, P<0.05think differences with a statistical significance.Results:C2C12cells proliferation were determined by flow cytometry:cell proliferation cultured for48h, n=3sample cells detected by flow cytometry (flow cytometry, FCM). Detector proliferation cycle, the statistical analysis shows that different groupsthe difference was statistically significant (F=103.00, P<0.05).The percentage of DNA G1period of C2C12cells in the experimental group were higher than those in the other two groups (P<0.05) and a similar proliteration index index(Prl)(P<0.05). The differentiated PC12cells inhibited the proliferation of C2C12cellsConclusions:The differentiated PC12cells inhibited the proliferation of C2C12cells by flow cytometry and to provide new ideas for the construction of the nerves of muscle tissue.Objective:Establish a co-culture system of PC12cells and C2C12cells. The expression of Myogenin and Desmin in the C2C12cells co-culture with PC12cells were detected by RT-PCR,qPCR and mmunofluorescence method in order to be certain about the effect of PC12cells on differentiation of C2C12cells.Methods:The C2C12cells were allocated into three groups at random (1) control group:single-C2C12cells groups;(2) experimental group:Co-culturing the C2C12cells with the differentiated PC12cells;(3) negative control:Co-culturing the C2C12cells with the undifferentiated PC12cells. All these are the PC12cells were cultured in transwell chamber and the C2C12cells were inoculated with six well plates.The concentrations of C2C12cells is (1.0～1.2)×108/L.The transwell chamber(pore diameter size of0.4μm) cultured with PC12cells were in six well plates and the ratio of the former with the latter is nearly1:1. Changed the maturation medium depending on the different groups. The experimental group changed with DMEM/F12medium contained50ug/L of NGF in order to induce differentiation of PC12cells. The maturation medium of control group is same as the experimental group and the negative control not contained NGF. They were routine cultured in the incubator with CO2. The expression of Myogenin and Desmin in the C2C12cells co-culture with PC12cells were detected by RT-PCR and qPCR: Collected the C2C12cells in3days and7days. In additon,the C2C12cells inoculated on the small coverslip in six well plates divided into different groups in the same as the above.The result of the PCR is shown by mean±standard deviation (X±s) between group compares the single factor analysis of variance (One-way ANOVA), if the difference was statistically significant, used the LSD inspection comparison between two, P<0.05think differences with a statistical significance.Results:The expression of Myogenin and Desmin in the C2C12cells co-culture with PC12cells were detected by RT-PCR and qPCR. The expression of Myogenin (F=185.153, P<0.05and F=6020.343, P<0.05) and Desmin(F=2149.081, P<0.05and F=57.612, P<0.05) in the C2C12cells in the experimental group were higher than those in the other two groups in3days and7days in same timepoint,the the expression of Myogenin, Desmin protein show that the differentiated PC12cells activated the differentiation of C2C12cellsConclusions:The expression of Myogenin and Desmin in the C2C12cells in the experimental group were higher than those in the other two groups in3days and7days in same timepoint, show that the differentiated PC12cells activated the differentiation of C2C12cells.