Isolation And Biological Characterization Of Retinal Stem Cells And Growth Effects Of Lead Usnic Acid On RSCs From Fetal Bovine

The refractory diseases of the retina that retinitis pigmentosa, age-related maculardegeneration, glaucoma optic atrophy, retinal blunt trauma and other eye diseases graduallythreat to human health. Nowadays In vitro cultured retinal cells separation and induced todifferentiate into retinal damage elements, and then transplantation in vivo, replace its damagetissue, so as to rebuild the retina research of structure. Usnic acid as a new therapeuticmedicine of target cells in China, the outlook for stem cells therapy remains to be fastidious.Using usnic acid to induce growth of retinal stem cell is probably a promising way of eyediseases therapy. Finally we have get effective treatment on these eye disease and to provide anew thought in restore the patient’s visual function.This study aimed to research that retinal stem cells (RSCs) were isolated from fetalbovine using mechanical separation, and the biological characteristics of retinal stem cells(RSCs) from bovine fetus. The surface markers of RSCs, Nestin, Chx-10and Pax-6, weredetected by immunofluorescence and RT-PCR. The multi-lineage differentiation potential ofRSCs was investigated and induced to differentiate into astrocytes, neurons andoligodendrocytes. And also RSCs were regarded as the research object, Cells were culturedby using suspension culture method, systematically expounded the different concentrations ofusnic acid induced apoptosis in RSCs and its mechanism. Observed cell morphology changesby using inverted phase contrast microscope; detected the changes of cells structure bytransmission electron microscopy and nucleic acid by agarose gel electrophoresis; RT-PCRdetection of the bax, bcl-2, p53gene mRNA level changes. The results are as follows:1. The RSCs were isolated from30-50days fetal bovine by mechanical separation andsubcultured up to passage10in vitro. And get a lot of high purity retinal stem cells.2. Illustrate the growth rhythm of retinal stem cells in vitro, and the growth curve was "S"type.3. Karyotype analysis suggested that the retinal stem cells from fetal bovine were diploid(2n=60), containing1pairs of autosomes and29pairs of sex chromosomes. The sexchromosome type is XY(♂)/XX (♀). The results supported the stable heredity of the cell line.4. The cells were identified using immunofluorescence and RT-PCR. Specific markers ofthe retinal stem cells, including Nestin, Pax-6and Chx-10are expressed in retinal stem cells.The RT-PCR results showed that the retinal stem cells were Sox2, Nestin, Six6and Pax6positive. 5. The multi-lineage differentiation potential of RSCs was investigated and induced todifferentiate into astrocytes, neurons and oligodendrocytes. Results shown that they expressmarkers of retinal stem cells, and they have the ability to self-renew and can give rise to threetypes of nerve cell in vitro.6. The morphology of RSCs treatment by different concentrations of usnic acid show thatcells culture in suspension, assumes the circular, good refraction, cells body is smaller andnucleus larger. The cell surface smooth and no obvious protrusions.7. The TEM of RSCs treatment by different concentrations of usnic acid show that cellscytoplasm full and uniform, with large and clear nucleolus, nuclear membrane integrity, theorganelles had no significant change.8. RT-PCR results showed that after the effect of usnic acid, bcl-2, bax, Fas and caspasegenes had no significant change.