The Study Of Differentiation Induction Of Tansforming Growth Factor-β2 To Mouse Retina Stem Cell Cultured In Vitro

ObjectiveTo observe whether TGF-β2 (Transforming Growth Factor-β₂)can promote the retina stem cell(RSC) differentiation. And take the embryo cow blood serum as contrast, to observe the magnitude of the differentiation induction effect of it to retina stem cell.Material and method1.The isolating culture and identification of RSCTo kill Kunming mouse having 17 days of gestational age, obtain embryoes and quickly dissect fetus murine eyes. the retina stem cells are separated from the mouse embryo eye under the operation microscope and carried on to raise, hand down from generation to generation. After completing the sixth generation, the retina stem cells will be carried on Nestin and Chx-10 immunity fluorescent test.2.The differentiation induction and identification of RSCTo centrifuge and wash the sixth generation of cells, then revaccinate renewedly to the culture bottle. Next to carry on the induction differentiation, dividing into the embryo cow blood serum(The thickness is 10ï¼…) induction differentiation group, the TGF-β₂ (10 ngPml) induction differentiation group, and blank comparison group. And carrying on anti- GFAP (Glial Fibrillary Acidic Protein), anti- Opsin, anti-β-tubulinâ…¢, the anti- PKC (Protein Kinase C) immunity fluorescence dyes, to verificate the end cell.3. To observe the result under the fluorescence microscopeTo randomly count 10 unoverlapping visual fields(x20),and calculate the proportion of anti-GFAP,anti-Opsin,anti-β-tubulinâ…¢and anti-PKC positive cells in the total cells, i.e.the positive cell ratio. Applying statistical analytical method, comparing the positive cell ratio's statistical difference of differentiated natural cells inducted by different conditions. Observe the magnitude of the differentiation induction effect of different induction conditions to retina stem cell.Result1.The shape observation and immunity fluorescent identification of the farina seem cell groups cultured in vitroWhen the separated retina stem cell was carried on the raise, it was possible to form the suspended growthing cell group and after transfered of culture many time the cell multiplication ability was not to be seen to weaken. To the sixth, the cutured cells' growth state is very well, the refraction is strong, and the cell boundary of cell groups marg is distinctness, respectively fluorescein staining to the Nestin and Chx-10 of cell group. The Nestin and Chx-10 dyeing masculine indicated the cultured cell is primitive cell in the early embryonic development retina.2.The fluorescence detection of the three group differentiated cellsThe cultured cells after being inducted by 10ï¼…embryos cow blood serum and TGF-β₂ (10ngPml) can differentiate to the mature cells. The immunity fluorescence examination demonstrated differentiated cells was possible to express the specificity antigen GFAP, Opsin, PKC andβ-tubulinâ…¢separately for the neuroglia cell, photoreceptor cell, bipolar cell and the nodal cell. Only very small amounts of differentiated cells has be seen in the blank comparison group not added the embryo cow blood and TGF-β₂。3. The result under the fluorescence microscope and statistics analysisThe positive cell rate of anti- GFAP, anti- Opsin, anti-β-tubulinâ…¢, the anti- PKC inducted by TGF-β₂ (10 ngPml) is put up statistic analysis with by the embryo cow blood serum(10ï¼…).The P is less 0.05, having statistic difference.Conclusion1,TGF-β₂ and the embryo cow blood serum all can induce the retina stem cell to differentiate to the retina cell.2,TGF-β₂'s induction differentiation action is stronger than the blood serum induction alone.