| Rett syndrome(RTT)is an X-linked neurodevelopmental disorder with severe neurological dysfunction in females.RTT is caused by mutations in MeCP2,a nuclear protein that,primarily binds to methylated CpG and plays a critical role in mediating transcriptional repression by recruiting histone deacetylase complexes.Lacking bindings to methylated DNA hinders the regulation of downstream target genes,and ultimately leads to brain dysfunction.At present,the role of MeCP2 in the process of brain development and how to cause RTT is still unclear.There is no approved treatment targeting to RTT.Relieving the symptoms of RTT will become an important treatment means.Therefore,we explored the effects of IMPX977 on the expression of MeCP2 protein in rats.At the same time,MeCP2 protein was studied by co-immunoprecipitation and mass spectrometry to explore the biological function in vivo.The experiment is divided into four parts:Part 1 The expression of MeCP2 protein in the tissues of wide type male ratsObjective In order to elucidate the cause of RTT,the function and localization of MeCP2 protein,we studied the distribution of MeCP2 protein in wide type rat organs.We also determined the expression level of MeCP2 by Western blotting analysis and quantitative real-time PCR(qRT-PCR).Method Six week-old wild type male rats were fed for three days,and anesthetized by chloral hydrate.We rapidly separated hippocampus,cerebullum,cortex,heart,liver,spleen,lung,kidney and muscle on ice.Then we detected the expression of MeCP2 protein by Western blotting technology;at the same time,total RNA was extracted from various tissues.We detected the relative MeCP2 mRNA expression using RT-PCR analysis.Result The results of Western blotting analysis showed that MeCP2 protein was mainly expressed in cerebellum,cortex and hippocampus,followed by spleen,lung and heart.The result of RT-PCR analysis showed the high expression of MeCP2 mRNA in hippocampus,cortex and cerebellum.Conclusion The MeCP2 protein plays an important role in the neural development of rat,and there is no significant correlation between MeCP2 protein and mRNA levels,which the irrelevancy is probably due to post-transcriptional regulation in tissue-specific manner.Part 2 The effect of IMPX977 on MeCP2 expression in different tissues of male ratsObjective To investigate the effect of different doses of IMPX977 on MeCP2 expression in various tissues in male rats.Method Male rats were divided into four groups:control group,olive oil group(negative control group),low dose group(10 mg/kg IMPX977)and high dose group(30 mg/kg IMPX977).All rats were orally administrated for two weeks and after the last administration,rats were anesthetized with chloral hydrate,then the cortex,cerebellum,heart,lung and spleen were isolated.Through Western blotting analysis,we researched the effect on MeCP2 expression of different doses of IMPX977 in several tissues.At the same time,the total RNA of cerebellum and cortex was extracted,and then analyzed by RT-PCR.Result The results of Western blotting showed that compared with the control group,the expression of MeCP2 protein was increased significantly in cerebellum and heart of rat in olive oil group(P<0.05,P<0.05),whereas it decreased significantly in rat heart and spleen tissues in high dose group.At the same time,the expression of MeCP2 protein was significantly up-regulated in the cortex of the low dose group.RT-PCR test results showed that compared with the normal control group or negative control group,the level of MeCP2 protein was increased significantly in rat cerebellum in low dose group.But compared with the control group,the expression of MeCP2 protein was decreased obviously in low dose group and high dose group in rat cortex.Conclusion Low dose of IMPX977(10 mg/kg)may induce the expression of MeCP2 protein in male rat cortex.Part 3 The effect of IMPX977 on MeCP2 expression in different tissues of female ratsObjective To investigate the effect of different doses of IMPX977 on MeCP2 expression in various tissues in female rats.Method Female rats were divided into four groups:control group,olive oil group,low dose group(10 mg/kg IMPX977)and high dose group(30 mg/kg IMPX977).All rats were orally administrated for two weeks and after the last administration,rats were anesthetized with chloral hydrate and sarcrificed.Then the cortex,cerebellum,heart,lung and spleen were isolated.Through Western blotting analysis,we researched the effect on MeCP2 expression of different doses of IMPX977 in several tissues.At the same time,the total RNA of cerebellum and cortex was extracted and analyzed by RT-PCR.Result The results of Western blotting showed that compared with the normal control group,the expression of MeCP2 protein showed a trend to increase in cerebellum in the other three groups,but without statistical significance(P>0.05,P>0.05,P>0.05).RT-PCR results showed that compared with the normal control group there was no obvious difference for MeCP2 expression in rat cerebellum of the other three groups.While the MeCP2 protein expression was significantly decreased in rat cerebellum and cortex in the negative control group and high dose group.Conclusion There was no significant difference in cerebellum and cortex of IMPX977 treated female rats.Part 4 Screening of MeCP2 interacting proteins by co-immunoprecipitation and mass spectrometry and evaluating the MeCP2 interacting protein dataObjective To study the MeCP2 interacting proteins in cortex tissue,and to explore the role of MeCP2 in cortex tissue.Method Male SD rat and C57BL/6 mice and female cynomolgus monkeys were fed for a week,and then the rats were anesthetized with chloral hydrate(0.3-0.4 mL/100g).The cynomolgus monkeys were anesthetized with ketamine hydrochloride(3 mg/kg),and then the cortex was rapidly separated on ice.Through IP-MS(co-immunoprecipitation-mass spectrometry)technology,Western blotting analysis,SDS-PAGE electrophoresis,Coomassie blue staining,we identified the components by LTQ and explored MeCP2 interacting proteins of different cortex tissues.And the proteins were evaluated by bioinformatics methods.Results Coomassie staining results showed clear MeCP2 band.Western blotting results showed that MeCP2 protein was enriched in mouse input samples.And through mass spectrometry data analysis,we found that the MeCP2 protein interacts with components of signal transduction,transcriptional regulation,cell growth and development,cell composition et al.,and it closely related to the EGFR signal transduction pathway,FGF signaling pathway,Wnt signaling pathway,Cadherin signaling pathway.They may be involved in Parkinson disease and Huntington disease et al..Conclusion These interacting proteins provided clues to reveal the biological function of MeCP2 and how it is involved in Rett syndrome.And these results provide a scientific basis for our further research. |
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