| Retinal degeneration is a group of diseases that result in irreversible vision loss, and leads to the death of photoreceptors or retinal ganglion cells. Current therapies for these diseases mainly focus on the etiology or palliative ways to slow down the process of retinal cell death. With the progress in stem cells research, bone marrow-derived mesenchymal stem cells(MSCs) have received much attention due to their properties such as plasticity, neuroprotection and immunomodulation. As resident phagocytic cells of the retina, microglia play an important role in the pathogenesis of retinal degeneration. It has been rarely reported that whether MSCs could interact with retinal microglia, activate related signaling pathway, and maintain homeostasis in the retina. CX3CL1 could control the degree of microglia activation through binding to CX3CR1, which is the specific receptor of CX3CL1. In this study, we employed an in vitro coculture system to evaluate the effect of CX3CL1 gene-modified MSCs(CX3CL1-MSCs) on regulating the function of activated microglia in retina. Moreover, we transplanted CX3CL1-MSCs into the subretinal space of light-injured rats, and observed the protective effect of CX3CL1-MSCs. Additionally, we intended to investigate the effect of CX3CL1/CX3CR1 signaling pathway.Chapter 1 Construction of CX3CL1 gene-modified MSCs by lentivirusObjective To investigate the construction of CX3CL1 gene-modified MSCs and the biological properties.Methods MSCs from SD ratswere collected by the method of whole bone marrow adherent, and underwent flow cytometric analysis for surface markers and induction for osteogenic/adipogenic differentiation. The expression of neurotrophic factors on MSCswas analyzed by indirect immunofluorescence assay. Both CX3CL1 plasmid and vectorplasmid were digested byrestriction endonuclease. The productswere obtained, connectedand thentransformed into Escherichia coli DH5α.Positive clones wereidentified by PCR and sequence comparison. 293 T cells were co-transfected withthree plasmids system of lentivirus by liposome. The supernatant was collected, and concentrated by centrifugal filtre units. The titer of lentivirus was determined by Real-time PCR. The lentivirus was employed to infect MSCs. The infection efficiency wasobserved under fluorescent microscope. CX3CL1 secretion was identified with ELISA, the expression of CX3CL1 m RNA was measured with Real-time PCR.Results MSCs were successfully obtained by the method of whole bone marrow adherent. Flow cytometric analysis indicated that 3th passage of MSCs were positive against CD90 and CD44, with a positive rate of(95.21±2.13)% and(96.75±3.30)%, while negative against CD34 with a positive rate of(1.84±0.92)%. After induction, MSCs could differentiate into osteoblast and adipocyte cells. MSCs could express CNTF,BDNF,GDNFand b FGFin vitro. Lentivirus containing CX3CL1 was successfully generated. The titer of lentivirus was 2×108TU/ml at least. CX3CL1 lentivirusefficiently infected MSCs. The best multiplicity of infection(MOI) was 10. Both the secretion of CX3CL1 protein and the expression of CX3CL1 m RNA in transfected MSCs were higher than those in control MSCs(F=338.300;640.300,P<0.05).Conclusions CX3CL1 gene could be integrated into MSCs genome, and transcribed accuratelyafter CX3CL1 lentivirus infection.CX3CL1 could be stablyexpressedand secreted by gene-modified MSCs.Chapter 2 Effect of CX3CL1 gene-modified MSCs on the biological function of activated retinal microgliaObjective: To evaluate the effect of CX3CL1/CX3CR1 signaling on the interaction between MSCs and retinal microgliain vitro.Methods:Retinal microglia from SD rats were cultured with mixed culture of retinal glial cells and purified by shaking,and the expression of CD11 b, Iba1 and GS was detected by indirect immunofluorescence assay. Retinal microglia were stimulated byLPS, and then cocultured with MSCs, CX3CL1 gene-modified MSCs(CX3CL1-MSCs) or CX3CL1-blocked MSCs in Transwell apparatuses for 24 hours. The functional changes of retinal microglia, including proliferation, phagocytosis and migration, were examined. NO release was detected by using Griess reagent. The m RNA and protein expression of cytokines were analyzed by Real-time PCR, Western blot and ELISA.Results: Retinal microglia from SD rats were harvested successfully by mixed culture and swing bed shaking. The cells were positive against CD11 b and Iba1, with a positive rate(96.18±3.40)% and(94.05±4.18)%, while negative against GS. LPS stimulation activated retinal microglia in vitro. The proliferation of activated microglia was suppressed by MSCs coculture(F=32.280, P<0.05), while the capacities of phagocytosis and migration were enhanced(F=72.200; 20.020, P<0.05). MSCs treatment downregulated the m RNA expression of TNF-α,IL-1βand i NOS(FTNF-α=169.400;FIL-1β=29.050;Fi NOS=79.060, P<0.05), but upregulated the m RNA expression of CNTF, BDNF and CX3CR1(FCNTF=20.710;FBDNF=63.530;FCX3CR1=78.580,P<0.05). Meanwhile, the protein expression of CNTF and BDNF was increased(FCNTF=24.460;FBDNF=20.510, P<0.05), the release of NO,TNF-α and IL-1β wassuppressed(FNO=23.670;FTNF-α=84.960;FIL-1β=30.760, P<0.05). These effects were strengthened by CX3CL1-MSCs, and reversed by CX3CL1 blockage on MSCs.Conclusions: MSCs might restore homeostatic functions of retinal microglia mainly through CX3CL1/CX3CR1 signaling.Chapter 3 Subretinal transplantation of CX3CL1 gene-modified MSCs protects against light-induced retinal damageObjective To investigate the protective effects of CX3CL1 gene-modified MSCs(CX3CL1-MSCs) on the light-injured retinal structure and function.Methods SD rats were exposed to(9500±500)Lux intensive white light for 24 hours with pupil dilation. Histological changes in the light-injured retinas were observed using hematoxin-eosin(HE) staining. Terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL) was conducted to identify apoptosis in retinal cells.MSCsand CX3CL1-MSCs were transplanted into the subretinal space of light-injured rats. The migratory capacity of MSCs and CX3CL1-MSCs was observed. By flash electroretinogram(F-ERG) and TUNEL methods, their rescue effect of the host retina was compared with untreated light-injured rats and DMEM-injected rats. Activated microglia in the retina were stained by ED-1 antibody. Western blot was performed to quantify cytokines expressed by the retina post-transplantation, including CNTF,BDNF,IL-1β and TNF-α. Immunofluorescence assay was conducted for locating CNTF or BDNF in the retina.Results Compared with the normal retinas, the reduction of the outer nuclear layer(ONL) thickness was observed in light-injured retinas, which was companied with an increase of apoptotic cells(t=8.009;8.753, P<0.05). The number of cells that migrated into the entoretina in CX3CL1-MSCs group was higher than that in MSCs group(t=3.674, P<0.05). CX3CL1-MSCs exerted superior effects in inhibiting a decrease of ONL thickness, apoptosis of the retinal cells and microglial activation. F-ERG analysis showed significantly better function in CX3CL1-MSC-injected group than other groups at 1 week, 3 weeksand 6 weeks post-transplantation. At 3 weeks post-transplantation, the protein expression of neurotrophic factors in host retina that received CX3CL1-MSCs was stronger than in the retina that received MSCs or DMEM(FCNTF=31.490;FBDNF=26.040, P<0.05). Conversely, the protein expression of proinflammatory factors was downregulated(FIL-1β=62.730;FTNF-α=21.940, P<0.05). After CX3CL1-MSCs transplantation, CNTF and BDNF were distributed in all retinal layers and integrated MSCs.Conclusions Subretinal transplantation of CX3CL1-MSCs may enhance structural and functional protection against light-induced retinal damage. |
PDF Full Text Request