The Research On The Expressing Difference Of Proteins And Electrophysiological Characteristic In Course Of Inducing BMSCs Into RGC-like Cells

Many studies proved that not only ES cells cultured from embryonic tissue but also bone marrow mesenchymal stem cells that were ability of induced into neural cells. There were many advantages in BMSCs compared with other stem cells. It was convenient to obtain materials, and no effects in the ethics. And we could get a large number of cells in a short time. In addition, BMSCs might reach all the position of the brain through the blood-brain barrier and cover the whole retina by the blood-retina barrier. Because of no immunological rejection of allotransplatation and heterotransplatation, BMSCs could be applied in many fields, such as the seed cells in tissues engineering, cell therapy and gene therapy.It had been reported that BMSCs might be induced into cells expressing photoreceptor-specific markers in vivo. BMSCs were labeled with green fluorescent protein and then injected into the subretinal space of rats. After two weeks, they covered the entire retinal area, and lots of them formed structures similar to the photoreceptor layer and expressed a photoreceptor -specific marker.The research of development neurology verifies that the development of the retina has time phase. The retina ganglions appear earliest, and the photoreceptor cells presents late relatively in the development of the embryo. It is possible to induce BMSCs into the RGC-like cells for it was successful in inducing BMSCs into the photoreceptor cells.At present, in the experiments reported about the BMSCs, the induced neuron-like cells had all expressed the relevant neural proteins and been similar to nerve cells. But the function and the electrophysiological feature of the mature neuron had not been proved. And then we have the following questions, such as whether the BMSCs could be induced into RGC-like cells, the expressing difference between the induced RGC-like cells and the original RGCs, in addition the electrophysiological function of the induced cells. Therefore, the following experiments are taken.1.Retinal cells of rat neonates induce the bone marrow mesenchymal stem cells into RGC-like cellsThe full marrow from Wistar rat neonates were isolated by Percoll centrifugation and passaged repeatedly. And the antigenic phenotypes of the cultured cells were analysed by immunocytochemical. The result showed they were positive for CD71, CD44, and negative for CD34,CD45, which improved that the cultured cells were BMSCs.In vitro, the BMSCs were induced by retinal cells of rat neonates in transwell. The morphological changes of the BMSCs were observed under phase contrast microscope, the specific markers of induced cells were identified immunocytochemically with NF, nestin,β-III Tubulin and Thy1.1.Nestin is a kind of middle silk protein as the peculiar marker of the neural stem cells. And NF is a marker of neurons, for it is an important skeleton protein forming neurons and synapses.β-III Tubulin is one of the primitive neural epithelium markers. It was proved that the induced cells might be neurons, for the positive expressing of nestin, NF andβ-III Tubulin.Thy-1 is the most abundant glucoprotein on the mammal neuron surface. It is location in the RGCs of mammals mainly and related to the growth, development, aging and death of the RGCs. Thy-1.1 antigen is only expressed on the rat RGCs, for which it is widely used to verify RGCs. The induced cells expressed not only neurons, markers but also RGCs, markers, so we named them RGC-liked cells.2.The research on the expressing difference of proteins when inducing BMSCs into RGC-like cellsWe identified the discrimination of protein profiles about BMSCs, induced RGCs-like cells and RGCs in the article. Two types of protein arrays, CM10 and IMAC30, were employed and they were analyzed with Biomark Wizard System.It was resulted that 16 marker proteins were upregulated in the induced RGC-like cells than BMSCs, of which 10 marker proteins were also upregulated in the RGCs. They were 6869Da, 8675Da, 9917Da, 12317Da, 13747Da, 15321Da, 16751Da, 17190Da, 17724Da, 11272Da and 15282Da. Among them, CM10 protein chip captured 9 markers and IMAC30 captured 2 markers.They are the distinctive neurnal markers probably, in addition the expression amount of the induced cells is less than the inherent RGCs among 10 sign molecules. So we assume that cell skeleton proteins and conduction signals changed in the course of inducing BMSCs into RGC-like cells. Although the 10 sign molecules in BMSCs are the same as the RGC-like cells, the expressing quantity is less after all. It might be considered that the RGC-like cells are a little difference from the inherent RGCs.3.The research on the electrophysiological characteristic when inducing BMSCs into RGCs-like cellsThis study characterized functional ion channels in rat cultured BMSCs with whole-cell patch clamp. Three type of currents were found, including a tetrodotoxin-sensitive sodium current (Ina-TTX), a delayed rectifier K+ current (IKDR) and L-type Ca2+ current (Ica-L). The first two are inward currents, and the last one is outward currents. Others research had reported seven types of currents in summary. They are Ina-TTX, Ica-L, Ikca, Ito, heag1 and IKDR. Possible reasons for the heterogeneity may be related to the fact that they are cells at different phases of the cycle. It was reported that ion channel expression changed with cell cycle progression. Some differences of electrophysiological propertied in BMSCs between this observation and previous reports are likely related, at least in part, to the reasons described above to slight differences in cell culture conditions. Three types of currents are all increased in the course of inducing and approach to the inherent RGCs. It is probably a way from unripe BMSCs to ripe RGCs.In summary, we induced BMSCs into RGC-like cells by retinal cells of rat neonates in transwell. The induced cells approached to inherent RGCs on the distinctive expressing proteins and the electrophysiological characteristic, but no more than RGCs. The RGC-like cells show the expressing proteins and electrophysiological characteristic as similar as the inherent RGCs.