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Objective To investigate the effect of Semaphorin3A (Sema3A) on axon growth of primary retinal ganglion cells (RGCs) in mice.Methods C57/BL6 mice within 24 hours after birth were sacrificed and the eyeballs were removed,RGCs was isolated from retina and cultured in vitro.The primary cultured RGCs was identified by Brn3a immunofluorescence staining.Seven days after culture,the RGCs was divided into control group,0.05 μg/ml Sema3A group,0.10 μg/ml Sema3A group and 0.50 μg/ml Sema3A group,and the processing time was 2 hours.Immunofluorescence staining was used to detect the expression of neuron-specific marker β3-tubulin and dendritic marker MAP2,β3-tubulin+/MAP2-was identified as axons.The length of axons was measured by Image J software.The axon lengths of 0.10 μg/ml Sema3A group and control group at 0.5,1 and 2 hours after treatment were calculated.The primary cultured cells were divided into control group,Sema3A treatment group,Y27632 treatment group and combined treatment group according to different drugs.The average axon lengths were compared among the groups.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO).Results Brn3a was positively expressed in primary cultured RGCs as a specific cell marker.Seven days after culture,RGCs tended to mature,with complete elongated neuronal processes and branches.The axon lengths of 0.05,0.10 and 0.50 μg/ml Sema3A groups were (69.35±1.49),(60.45±1.42) and (93.65±1.86) μm,which were significantly shorter than (109.80±2.29) μm of the control group (all at P＜0.01).The axon length of 0.10 μg/ml Sema3A group was (165.00 ±4.39)μm and (97.63 ±2.79)μm at 1 hour and 2 hours after treatment,respectively,which was significantly lower than (210.40 ±4.44) μm and (199.00 ± 4.36) μm of control group at corresponding time points (both at P＜0.01).There was a significant difference in the axon length of control group,Sema3A treatment group,mixed treatment group and Y27632 treatment group (F =142.50,P＜0.01).The RGC axon length of Sema3A treatment group was significantly lower than that of control group and combined treatment group (both at P＜0.01).Conclusions Sema3A can inhibit axon growth of primary retinal RGCs in mice,and ROCK signaling pathway inhibitors can alleviate such restrain effect.
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Sjögren's syndrome is a kind of autoimmune disease, whose main clinical symptoms are dry mouth, dry eye and chronic parotid glandular inflammation. The conservative treatments include artificial tears or saliva,oral administration of corticosteroids,and immunosuppressantsl with limited effectiveness. Along with the development of molecular biology, vast attentions are being paid to researches on gene therapy for Sjögren's syndrome, hopefully to bring gospel to patients with Sjögren's syndrome. This article reviews the recent research progresses on transcatheter delivery of recombinant adenovirus vector with aquaporin gene in experimental treatment of Sjögren's syndrome.
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Humans , Adenoviridae , Aquaporins , Genetics , Autoimmune Diseases , Therapeutics , Catheters , Genetic Therapy , Genetic Vectors , Sjogren's Syndrome , TherapeuticsABSTRACT
OBJECTIVE To study the roles of copper transporter 1 (CTR1 )and Cu2 + transporting ATPase αpolypeptide (ATP7A)in lead exposure-induced copper accu mulation and oxidative stress in rat C6 glio ma cells.METHODS Methyl thiazolyl tetrazoliu m (MTT)assay was performed to determine the proper Pb doses (without affecting cell viability)by treated the cells with lead acetate 0 -100 μmol·L -1 for 24 h and 48 h.Superoxide dis mutase (SOD)activity or malondialdehyde(MDA)level were detected respectively by xanthine oxidase technique and thiobarbituric acid (TBA) method.Ato mic absorption spectrophoto metry was e mployed to determine the intracellular levels of Pb and Cu ions.Real-ti me quan-titative PCR and Western blot were used to detect the mRNA and protein levels of CTR1 and ATP7A, respectively.RESULTS The cell viability significantly decreased when the doses of Pb treat ment was higher than 10 μmol·L -1 ,so 10 μmol·L -1 was chosen as a working concentration of Pb exposure in this study.Co mpared with those in the normal controls,a moderately decreased T-SOD activity and an increased MDA level was determined in the cells treated with Pb 10 μmol·L -1 or Cu 5 μmol·L -1 alone, while a significant drop of T-SOD activity and a re markable increase of MDA level was found in the cells co-exposed to Pb and Cu (P<0.01 ).Pb exposure for 24 and 48 h increased the cellular Cu uptake by 1 .2 and 2.5 fold,respectively (P <0.01 ).Evidences fro m RT-PCR showed that Pb exposure for 24 and 48 h upregulated the CTR1 mRNA level by 23.2% and 58.7%,and downregulated the ATP7A mRNA level by 58.1 % and 50.0%,respectively.Results fro m Western blot confirmed that Pb exposure also resulted in an increased CTR1 expression and a decreased ATP7A expression at protein level (P<0.01 ).CONCLUSION Pb exposure lead to Cu accu mulation,by affecting the expression levels of CTR1 and ATP7A,and increased oxidative stress in C6 cells.
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Objective To explore the roles of p53 in ionizing radiation induced MCF-7 cell cycle uncoupling. Methods The p53 knock-down models was established in MCF-7 with retrovirus packaged particles from 293T cells through calcium acid phosphate co-precipitation, then Western blot was used to detect the protein expression. Flow cytometry(FCM) was used to analyze the cell cycle uncoupling and polyploid after irradiation. Results Compared with p53+/+ group, the percentages of G0/G1 cells in p53 -/- group decreased, while those of S and G2+M increased (P < 0.01). In polyploidy analysis 2N cells decreased, whereas both 4N and 8N cells =increased (P<0.01). Compared with sham-irradiation, 4 Gy X-ray led to the decrease of G0/G1, S cells, and the increase of G2+M cells. The increase of 2N cells and decrease of 4N and 8N cells were observed in both p53+/+ and p53-/- cells. Compared with p53+/+ +IR group, the decrease of G0/G1 and S cells and the increase of C2 + M cells were significant (P < 0.01) in p53-/-+ IR groups. 2N cells decreased, 4N cells increased, but no changes in 8 N cells occurred. Conclusion Radiation might induce G2 arrest and cycle uncoupling, p53 plays a role in the regulation of G2 arrest, but no role in cycle uncoupling.