| The major radial glial cells in retina are Müller cells, they play many roles in nutrition, support, isolation and protection of neurons in normal condition. Recent studies show that glial cells in brain take part in neurogenesis, in some special area, neuroglial cells show characters of stem cells. Müller cells in adult avian have strong regeneration potentials, suggesting they may act as adult stem cells in retina. Several different lines of evidence indicate that Müller cells share a lineage and a precursor with retinal neurons. The latest studies show that Müller cells by in vitro aggregate-culture could be converted into neuronal lineage, indicating Müller cells in mammalian have a potential to dedifferentiate into neurons. However, few studies have been reported on Müller cells dedifferentiation in mammalians since regeneration in mammalians is not as strong as in low vertebrates. In our study, we investigate stem cell potentials and special differentiation of Müller cells in photoreceptor degeneration rats; and investigate regulation of stem cell property of Müller cells by Sonic Hedgehog pathway both in vivo and in vitro, which may possibly contribution to the treatment of degenerative retinal disease such as retinitis pigmentosa.The methods we used are as following:(1) Establishment of photoreceptor degeneration animal model. Photoreceptor degeneration was induced by intraperitoneal injection of 60mg/kg N-methyl-N-nitrosourea into Sprague-Dawley rats. (1)HE staining, transmission electron microscopy and TUNEL were used to detect photoreceptor apoptosis process in outer nuclear layer. (2)The effects of MNU to other retinal neurons were determined by fluorescent immunostaining of marker of retinal neurons and synapses. (3)Changes of melanopsin on ganglion cells were detected by real time RT-PCR and immunostaining.(2)Photoreceptor degeneration induced stem cell potential of Muller glia. (1) Double staining of glutamine synthetase (GS) with glial fibrillary acidic protein(GFAP), nestin and proliferating cell nuclear antigen (PCNA) were used to determine gliosis of Müller cells. The mechanism of proliferation was assessed by immunoflurescence staining and western-blot of Cyclin D1 and cyclin D3. (1)Dedifferentiation of Müller glia was detected by double staining of PCNA and rhodopsin, PKCa, syntaxin, Thy1.1 respectively, synaptophysin antibody was applied to detect the formation of synapses around new born neurons. (3)Müller cells were isolated from Z/EG C57 mice, the purity of cultured Müller cells was assessed by RT-PCR and FACS, and then Müller cells were transplanted into vitreous cavity. Double staining of LacZ antibody and marker of retinal neurons was used to observe survival, migration and differentiation of Müller cells in vitreous cavity. Transplantation of retina progenitors isolated from E18 was as the control.(3) Regulation of Sonic Hedgehog to Müller cells in vitro. (1)Expression change of Shh receptor Patched (Ptc) was assessed by in situ hybridization and RT-PCR in sections from animal model. (2)Muller glia was isolated from P0-P7 SD rats, Ptc and Shh downstream transcripts Gli were detected in Müller cells by RT-PCR. (3)Expression of Ptc and Gli in Müller cells was assessed by real time RT-PCR after treated by different dose of SHH-N. (4)The proliferation of Müller cells induced by SHH-N was evaluated by Brdu/GS, PCNA/GS double staining and cell cycle examination. (5)Expression change of Cyclin D1 and Cyclin D3 on SHH-N treated Müller cells was evaluated by GS/cyclin D1, GS/cyclin D3 double staining and western-blot.(6)Expression of stem cell marker (Pax6, nestin, Sox2, ABCG2, and Musashi)on Müller cells after SHH-N treatment was investigated by fluorescent immunostaining and RT-PCR. (7)Dedifferentiation of Müller cells into photoreceptor was analyzed mRNA of Nrl, Crx and rhodopsin, transcripts of photoreceptor development.(4) Effects of SHH-N on Müller cells in vivo. Within 6 hours after animal model was established, SHH-N solution was injected into vitreous cavity of right eye, left eye was injected saline as control. (1)FACS and TUNEL staining were used to analyze the apoptosis index between the control and experimental group. Caspase-3 and caspase-8, which were induced after MNU treatment, were assessed by western-blot. (2)Double staining of GS/PCNA was applied to analyze the number of proliferating Müller cells between two groups. Differences of Cyclin D1 and Cyclin d3 expression were analyzed by western-blot. (3)Dedifferentiation of Müller cells to photoreceptors was assessed by double staining of PCNA and rhodopsin between two groups.Results:(1)60mg/kg MNU intraperitoneal injection to SD rats can induce photoreceptors in ONL apoptosis in a short time, apoptosis cells reached peak 1 day after injection, photoreceptor disappeared on day 7. MNU did not affect morphology of other retinal neurons including bipolar cells, amacrine cells, horizontal cells and ganglion cells. However, photoreceptor loss down-regulated expression of melanopsin in ganglion cells, which may affect circadian rhythms.(2) Photoreceptor damage induced Müller cell gliosis: upregulation of GFAP and nestin, Müller cells reenter cell cycle to proliferate. The proliferation of Müller cells reached peak on day 2, and through upregulation of cyclin D1 and Cyclin D3. On day 15, proliferating Müller cells decreased gradually, some proliferating Müller cells began to express marker of rods rhodopsin, on day 28 and 40, rhodopsin positive cells increased, and synaptophysin was also detected around new born rods. However, dedifferentiation of Müller cells to other retinal neurons was not detected. Müller cells isolated from Z/EG mice coexpressed LacZ and GS. The purity of cultured Müller cells assessed by FACS was 97%, which indicated cultured Müller cell was not contaminated by other neurons. Transplantation of LacZ labeled Müller cells migrated from GCL, IPL and outer margin of inner nuclear layer; some donor cells expressed rhodopsin. Retinal progenitors from E18 rats were induced into retinal neurons and Müller cells in vitro. However, transplanted progenitors could migrate, but rarely expressed marker of retinal neurons.(3) In situ hybridization and RT-PCR revealed that Ptc upregulated in Müller cells after photoreceptor loss. Cultured Müller cells expressed Ptc and GU1.5, 10, 20nM SHH-N were added to the culture respectively, real time PCR showed that Ptc and Gli mRNA increased in a manner of dose dependent. Cell cycle assessed by FACS and GS/PCNA, GS/Brdu double staining revealed SHH-N promote the proliferation of Müller cell, while effect of 20nM is more evident. Proliferation of Müller cell was contributed by upregulation of Cyclin D1 and Cyclin D3. Stem cell marker Pax6, nestin, Sox2 and ABCG2, Musashi mRNA were expressed in SHH-N treated Müller cell. Nrl, Crx and rhodopsin transcripts of rods development were also expressed in Müller cell after SHH-N induction.(4) Injection of SHH-N into vitreous cavity decreased photoreceptor apoptosis obviously compared to the control group eye which was injected saline. Caspase-3 and caspase-8 were downregulated by SHH-N injection. Proliferation of Müller cells in vivo was promoted after SHH-N injection, especially at late stage, 15, 28, 40 days after MNU, differences between two groups became more obvious. SHH-N could induce upregulation of Cyclin D1 and D3 more than the control. SHH-N promoted Müller cells dedifferentiate into more photoreceptors.
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