Protective Of CNTF Gene-modified Bone Marrow-derived Mesenchymal Stem Cells With Sodium Iodate Induced Retianl Degeneration In SD Rats

Retinal degeneration is a series of diseases,characterized by the progressive degeneration and apoptosis of retinal ganglion cells,bipolar cells,photoreceptor cells,pigment cells,etc.Including retinitis pigmentosa,age related macular degeneration,cone rod dystrophy and Stargardt disease,etc.Aiming at this kind of diseases,the mainly treatment is symptomatic treatment and ease the retinal cells' s degeneration.From now on,there has been no effective prevention of the diseases' s progression and recovery the retinal function.Stem cells,a self-renewal and multi-directional differentiation potential of undifferentiated or poorly differentiated cells in higher multicellular organisms,as an ideal seed cells in tissue regeneration,have unique advantages in application of retinal degeneration diseases.It through the supply and replace the degeneration cells to treat retinal degeneration.It's also may stimulate the remaining cells' s activity through paracrine mechanism,including secrete various of neurotrophic factors,such as ciliary neurotrophic factor(CNTF),bone derived neurotrophic factor(BDNF),basic fibroblast growth factor(b FGF),etc.Prompt their expressions in the microenvironment of the damaged retina,promote the retinal tissue repair and play the role of neuroprotection.CNTF is one of the most studied retinal nerve protection factors.A large study evidence showed that,CNTF mainly on retinal ganglion cells with neural protection,and it's also shown that it have nerve protective effect with the retinal pigment epithelium cells,photoreceptor cells and so on.It can improve the survivability of the pathological retinal cells.This study intends to explore the retinal degeneration model through tail vein injection of sodium iodate in rats,and ensure the protection of lentivirus based CNTF gene modified bone marrow mesenchymal stem cells with the degeneration retinal through vitreous cavity injecting.Chapter 1.Sodium iodate induced the retinal degeneration and CNTF expression research in SD ratsObjective:Through tail vein injection of sodium iodate caused retinal degeneration in SD rats,preliminary understand the morphological and electrophysiological changes,and ensure the expression of CNTF in degeneration retina.Methods:Tail vein injection of sodium iodate(50mg/kg)caused SD rats' s retinal degeneration,randomly divided into experimental groups(1D;3D;1W;2W;4W)and control group,6 rats each group.At the point time,F-ERG was used to detect the retinal function,Eyeball tissues' s H-E stain to observe retinal morphology,Paraffin sections' s TUNEL stain to observe apoptosis in each layer,CNTF immunohistochemical stain observe it's expression in each layer.The m RNA expression of CNTF were analyzed by Real-time PCR.Results:1.Each group of rats before the trial,There were no significant differences of latency and amplitude of b waves with Rod-R or Max-R(P>0.05).Compared with the normal group,From the 3D after injection,there were significantly latency prolonged and amplitude reduced of b waves with Rod-R and Max-R.2.HE dye showed:Compared with normal group,From the 1D after injection,there were apoptosis of RPE cells,and the 3D,the RPE cells were almost dead,IS/OS and outer nuclear layer structure arranged sparsely,retinal thickened and edema.To the 1W,retinal edema exacerbated,the outer and inner layer structures disordered,raised like waves and loose,REP layer and outer nuclear layers visible part small cells proliferation.To the 2W,outer nuclear layers density decreased.The 4W retinal thickness thinned,obviously in the posterior pole,outer and inner nuclear layer thickness became thinning,structure became loose and disorder.3.TUNEL staining showed:Normal SD rat retinal apoptosis cells rarely in each layer,pigment epithelium layer cells TUNEL staining deepened and RPE cells began apoptosis at the 1D after sodium iodate injection.To the 3D,RPE layer appears obvious apoptosis.and at the 1W,RPE cells almost completely died,almost involved the outer nuclear layer.To the 2W,he outer nuclear layer appears obvious apoptosis.At the 4W,The apoptosis accumulate into pieces,and apoptosis appears in inner nuclear layer.4.The RT-PCR results showed,Compared with normal group,CNTF expression has no obvious abnormality in the 1D after injection(P>0.05),It is on the rise in the 3D(P>0.05).With time goes on,It has rise to the top after 1W,Which is about 4.53 times with the normal control group,and then decreased progressively.In the 4W,Compared with normal group,It has no obvious abnormality(P>0.05),and at a relatively low status.5.Immunohistochemistry showed that CNTF is mainly expressed in ganglion cell layer and outer nuclear layer and RPE layer in normal adult SD rats retina.With the extension of time after the sodium iodate injection,CNTF expression gradually increases in outer nuclear layer,and part of hyperplasia of glial cells almost expressed.In the 1W,the CNTF expression positive rate highest in outer nuclear layer cells,and then decreased progressively,and clump together in significantly proliferate position.Conclusions:As a inorganic oxidant,In SD rats,sodium iodate can selectively damage retinal pigment epithelial cells,and then photoreceptor cells,The main damage in outer layer.The histopathological patterns are the RPE cells loose,and then disappear completely.and then,outer nuclear layer cells apoptosis,disorder,raise like waves,and loose.With the time goes,the change is aggravating,the outer nuclear layer get thin and cells reduced obviously.The longer effect of sodium iodate the injury is heavier.At the beginning of retinal degeneration CNTF expression in a relatively hingh state,especially in outer nuclear layer,rised to the top after 1W,and then decreased.The application of sodium iodate via tail vein injection in SD rats,we can simulate process of retinal degeneration,Sodium iodate induced rat retinal cell degeneration associated with CNTF secret level.Chapter 2.Protective of CNTF gene-modified bone marrow-derived mesenchymal stem cells with rat retianl degenerationObjective:Through intravitreal injection of CNTF gene-modified bone marrow-derived mesenchymal stem cells with SD rat retianl degeneration,preliminary understand the cells protective effect.Methods: On the 7 day after sodium iodate injection(50 mg/kg),SD rats were randomly divided into control group,sham-operation group(DMEM group),CNTF group and GFP group.control group with no intravitreal injection,and sham-operation group injected DMEM solution,cell injection group injected with CNTF-GFP gene-modified BMSCs and GFP gene-modified BMSCs respectively.In the 3D?1W?2W?4W?8W after injection,F-ERG was used to detect retina functio,HE staining to observe the retina structure;In the 2W and 8W,eyeballs paraffin section TUNEL staining to observe retinal layers' s apoptosis,eyeballs paraffin section and retinal flat observe GFP location with fluorescence microscope.In the 2W,take each group retinal tissues,RT-PCR detect Caspase-3 expression.Results: 1.The F-ERG result:In the 3D?1W?2W?4W?8W,the CNTF group have statistically significant latency and amplitude of Max-R b waves.In the 2W,the latency is at the lowest level and the amplitude is at the highest,like wave shape(F=4.446,P<0.05;F=2.984,P<0.05);CNTF and GFP group have great with latency and amplitude of Rod-R/Max-R b waves difference with control group and DMEM group in the 2W.CNTF group compared with GFP group,there were Max-R b waves latency and amplitude difference(t=-3.686,P < 0.05),but no of Rod-R(t=1.598,P>0.05).In the 8W,CNTF group(P<0.05),not GFP group(P>0.05),have Max-R b waves latency and amplitude difference compared with control group and DMEM group? 2.HE dye showed:cells injection group have cells distributed in the inner limiting membrane at all time points,and part of them gathered on the lens posterior,a small number of cells migrate to the inner plexiform layer.The outer nuclear layer cell counts showed,the CNTF and GFP group have significant improved compared with DMEM and control group(P < 0.05)in the 3D/1W/2W.Compared with GFP group,CNTF group also have statistical differences in the 1W and 2W(P<0.05).In the 4W and 8W,Compared with DMEM and control group,there were almost differences of CNTF group(P<0.05),but no of GFP group(P>0.05).3.TUNEL staining showed:At the 2W after injection,the CNTF and GFP group outer nuclear layer cells TUNEL staining positive rate have great reduced(P<0.01),compare with the DMEM and the normal group,and It's almost happened at the compare of the CNTF group with the GFP group(P<0.01).At the 8W,positive rate almost continue with the group of CNTF compared with the DMEM and the normal group(P<0.01),but GFP group has no significant difference compared with the DMEM and the normal group(P>0.05).4.The PCR results showed:Compared with the normal group,at the 2W after intravitreal injection,the CNTF/GFP/DMEM and the control group all have enhanced the expression quantity of Caspase-3(P<0.05).The CNTF and the GFP group have difference with the DMEM and the control group(P<0.05),and the CNTF group have difference with the GFP group(P<0.05).5.The transplanted cells distribution:paraffin sections showed,at the 2W,the CNTF and the GFP group all have GFP positive cells in front of the inner limiting membrane and some of them gathered on the posterior of the lens,a few of them can be seen in the inner plexiform layer.At the 8W,the CNTF group also can see little GFP positive cells in front of the inner limiting membrane and the inner plexiform layer,but not see with the GFP group.Retinal flat showed:At the 2W,the CNTF and the GFP group have many GFP positive cells scattered,the CNTF group have higher density with the GFP group.At the 8W,the CNTF group also can see some GFP positive cells,but the GFP almost disappeared.Conclusions:Through intravitreal injection of CNTF gene-modified BMSCs with SD rat retianl degeneration,it can alive in the intravitreal and gather in front of the inner limiting membrane,and part of them can migration into the inner plexiform layer,they can rise the F-ERG level,delay retinal photoreceptor cells degeneration and apoptosis,reduce the level of apoptosis related proteins caspase3,play the role of protection with the retianl degeneration.