ABSTRACT
Objective:To explore the effects of conditioned medium of human bone marrow mesenchymal stem cells (BMSCs) on the proliferation, adhesion and differentiation of immortalized human Müller cell line (MIO-M1).Methods:The differentiation was induced in the third-passage BMSCs with osteogenic, chondrogenic and adipogenic medium and identified by alizarin red, alcian blue and oil red O staining, respectively.The expression levels of mesenchymal stem cell markers CD73, CD90 and CD105 and hematopoietic cell markers CD34, CD45 and human leukocyte antigen-DR (HLA-DR) were assayed by flow cytometry.The expressions levels of Müller cell markers SOX9, glutamine synthetase (GS), vimentin and cellular retinaldehyde-binding protein (CRALBP), retinal stem cell markers SOX2, nestin and CHX10, and cell proliferation marker cyclin D3 (CCND3) in MIO-M1 cells were detected by immunofluorescence staining.The MIO-M1 cells were divided into standard medium group, 293T conditioned medium group, and BMSC conditioned medium group and were incubated in the medium according to grouping.The cellular area, circularity, elongation factor and perimeter were analyzed quantitatively.The cell cycle was detected by flow cytometry, and the cell proliferation was determined by neurospora experiment and 5-ethynyl-2'-deoxyuridine (EdU) staining.The expression of vascular cell adhesion molecule 1 (VCAM-1) at protein and mRNA levels in the culture supernatant was detected by enzyme linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR), respectively.The expression of retinal neuron markers protein kinase C (PKCα), Rhodopsin, microtubule-associated protein 2 (MAP2) and β-tubulin (Tuj1) was detected by immunofluorescence staining and qRT-PCR.Results:CD73, CD90, CD105 showed an enhanced expression, and CD34, CD45 and HLA-DR showed weakened expression in the BMSCs.The BMSCs differentiated into osteoblasts, chondrocytes and adipocytes.Expression of SOX9, GS, vimentin and CRALBP, SOX2, CHX10, nestin and CCND3 was found in the MIO-M1 cells.Compared with standard medium group and 293T conditioned medium group, MIO-M1 cells cultured in BMSC conditioned medium group changed into an elongated spindle-shaped or multipolar morphology with reduced cell area, increased elongation index and decreased circularity, showing statistically significant differences among them ( F=6.973, 12.370, 6.311; all at P<0.01). There were increased neurospheres formed by MIO-M1 cells in BMSC conditioned medium group compared with standard medium group and 293T conditioned medium group at different time points ( Fgroup=134.300, P<0.001; Ftime=82.910, P<0.001). Compared with the standard medium group and 293T conditioned medium group, the EdU-positive rate and proliferation index of MIO-M1 cells in BMSC conditioned medium group were significantly increased, with statistically significant differences ( F=6.973, 74.110; all at P<0.05); the VCAM-1 protein expression in cell supernatant and the relative expression level of VCAM-1 mRNA in BMSC conditioed medium group were significantly increased ( F=13.720, 7.896; all at P<0.05); the mRNA expression levels of PKCα, Rhodopsin, Tuj1 and MAP2 were higher in MIO-M1 cells of BMSC conditioned medium group under the condition of differentiation ( F=14.490, 5.424, 14.330, 7.405; all at P<0.05). Conclusions:BMSCs conditioned medium can change the morphology of MIO-M1 cells and promote their proliferation, adhesion and differentiation into retinal neurons.
ABSTRACT
Background Glial scaring induced by the activation and proliferation of astrocytes after optical nerve damage is one of causes of neural axons difficult to regeneration.Researches showed that α-crystallin can promote the regeneration and pass through scaring zone of retinal ganglion cells (RGCs) axons,and we speculate α-crystallin protect optical nerve tissue against scaring process.Objective This study was to investigate the influence of α-crystallin for the activation and secretion of inflammatory factors of astrocytes.Methods Optical nerver tissue was isolated from 3-5 day-old SPF Long Evans rats to culture and purify astrocytes.The cells were identified by detecting the expression of glial fibrillary acidic protein (GFAP) with immunofluorescence technique.The cells were cultured with regular culture medium in the normal control group,and 5 μg/ml lipopolysaccharides (LPS) was added in the LPS group,while 5 μg/ml LPS and 1 ×10-4 g/L α-crystallin were added in the α-crystallin group,and the cells were consecutively cultured for 24 hours.The proliferation (absorbance,A) of the cells was assayed by cell counting kit-8 (CCK-8).The expression of GFAP in the cells was detected by immunofluorescence technique and quantitated by Western blot.The contents in the cell supernatants of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISA.Results The morphology and size were well-proportioned in 3-4 generation of cells with the GFAP positive rate over 95％.The A values were 1.335±0.070,1.643±0.069 and 1.390±0.004 in the normal control group,LPS group and α-crystallin group,and the A values in the LPS group were significantly higher than those in the normal control group and α-crystallin group (t =3.315,3.681,both at P＜0.05).Immunofluorescence examination showed that the fluorescence intensity was evidently enhanced in the LPS group compared with the normal control group and α-crystallin group and presented the largest cell bodies in the LPS group.The relative expressions of GFAP in the cells were 0.851 ±0.076 in the LPS group,which were higher than those in the normal control group and α-crystallin group (0.786±0.091,0.569±0.049).Compared between the LPS group and α-crystallin group,there is a significant difference between the two groups (t =3.115,P＜ 0.0l).In addition,compared with the LPS group,the contents of TNF-α and IL-1β in the suspensions were significantly reduced in the normal control group and α-crystallin group (all at P＜0.05).Conclusions α-Crystallin protein can inhibit the activation and secretion of optic nerve astrocytes stimulated by LPS.