RESUMO
Huntington's disease (HD) is a deadly neurodegenerative disease with abnormal expansion of CAG repeats in the huntingtin gene. Mutant Huntingtin protein (mHTT) forms abnormal aggregates and intranuclear inclusions in specific neurons, resulting in cell death. Here, we tested the ability of a natural heat-shock protein 90 inhibitor, Gedunin, to degrade transfected mHTT in Neuro-2a cells and endogenous mHTT aggregates and intranuclear inclusions in both fibroblasts from HD patients and neurons derived from induced pluripotent stem cells from patients. Our data showed that Gedunin treatment degraded transfected mHTT in Neuro-2a cells, endogenous mHTT aggregates and intranuclear inclusions in fibroblasts from HD patients, and in neurons derived from induced pluripotent stem cells from patients in a dose- and time-dependent manner, and its activity depended on the proteasomal pathway rather than the autophagy route. These findings also showed that although Gedunin degraded abnormal mHTT aggregates and intranuclear inclusions in cells from HD patient, it did not affect normal cells, thus providing a new perspective for using Gedunin to treat HD.
RESUMO
ObJeetive To report a simple and efficient method to label two different types of retinal ganglion cells (RGCs) in mouse retina.Methods Eyeballs were harvested from normal adult C57 BL/6 J mouse,the retinas were isolated,four radial cuts were done,the retinas were pasted on the nitrocellulose membrane with the ganglion cell layer upturned.The immunofluorescence double staining and laser confocal nmicroscope was used to reveal conventional retinal ganglion cells and intrinsically photosensitive retinal ganglion cells (ipRGCs) using Brn3a and Melanopsin.Results The double staining results of whole mount retina showed that conventional RGCs and melanopsin immunopositive ipRGCs had a complementary distribution in mouse retina,these two subtypes of RGCs were predominantly present in the ganglion cell layer.The numbers of ipRGCs was just about 1％-2％ of conventional RGCs,and the axons of ipRGCs toward the direction of the optic disc,several dendrites toward the inner plaximem layer.Conclusion The immunofluorescence double staining of whole mount retina is a simple,stable and efficient method to label two different types of mouse RGCs.
RESUMO
Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells that can be generated from adult somatic cells.They have similar characteristics and function to embryonic stem cells (ESCs).Over the past decade,iPSCs were widely concerned in regenerative medicine and stem cell field.Especially the patient specific iPSCs have several advantages over ESCs,such as convenient source,do not exist immune rejection and ethical issues,even keep certain individual genotype.At present,tremendous progress have been made about the application of iPSCs in a variety of retinal diseases.Here,this article reviews pluripotent stem cell sources of RPE,photoreceptors and retinal ganglion cells and current transplantation strategies,the safety problems and prospects.
RESUMO
Objective To investigate the effect of di-2-ethylhexyl phthalate (DEHP) on the proliferation of SK-N-SH human neuroblastoma cells and its underlying mechanism. Methods Cells were cultured in estrogen-free improved Dulbecco's Modified Eagle's Medium and then divided into 5 groups: no treatment (control group); treated with 17β-estradiol (E_2 group); treated with DEHP (DEHP group); treated with both E_2 and phosphatidylinositol-3-kinases (PI3K) inhibitor LY294002 (E_2 + LY294002 group); treated with both DEHP and LY294002 (DEHP + LY294002 group). The absorbance value (AV) was measured on day 0, 2, and 5. DNA proliferation index (PI) and apoptotic index (AI) were determined by flow cytometry on day 5. Caspase-3 protein, protein-serine-threonine kinase (Akt) and phosphor-Akt (Ser473) protein expression were analyzed by Western blot on day 5. Results The AV of All groups increased on day 2, and 5. The AV of E_2 and DEHP groups were higher than that of the control group (P<0. 001), but the AV of E_2 + LY294002 and DEHP + LY294002 groups were lower than those of E_2 and DEHP groups (P<0.01) on day 2 and 5. On day 5, PI of E_2 and DEHP groups were also higher than that of control (P<0.01). However, PI of E_2 + LY294002 and DEHP + LY294002 groups were lower than those of E_2 and DEHP group (P<0.01) on day 5. There was no significant difference in AI and caspase-3 protein expression among the groups. At the same time, phosphor-Akt (Ser473) protein expression of E_2 and DEHP groups increased obviously, compared with the control group. Compared with E_2 and DEHP groups, E_2 + LY294002 and DEHP + LY294002 groups decreased significantly. However, Akt protein expression was equal among those groups. Conclusions DEHP can promote the growth of SK-N-SH cells to a level similar to that of E_2, with activation of the PI3K/Akt signaling pathway.
RESUMO
Objective To study the estradiolNGF regulatory cascade in retinal endothelial cells. Methods Retinachoroids vascular endothelial cell line RF/6A from rhesus was cultured in M199 medium contain 20% FBS.mRNA of ER,NGF and its receptors,TrkA and P75 NTR were detected with RTPCR.The cells were incubated with NGF,VEGF,antibodies against NGF or VEGF,and K252a(100nmol/L),the specific inhibitor of TrkA,separately or in different combination.MTT based cell counting assay was used to study the viability of the cells.The apoptosis was evaluated by FACS,mass migration by wound healing assay,and tubogenesis by AngioMatrix assay. Results We amplified the specific fragments of cDNA of ER,NGF and NGF receptor TrkA using RTPCR.10?nmol/L1??mol/L estradiol augments the proliferation and increases the viability of RF/6A in a dosage dependent manner.In the wound healing based migration assay,we found the similar alteration.This effects of estradiol was partially blocked by NGF neutralized antibody and K252a.Apoptosis rates were at the similar level among the groups.For the tubogenesis of RF/6A,we found no augmentation by NGF,and no blocked augmentation by estradiol.Conclusion NGF,first identified as the survival factor of nerve system,now also seemed to be an activator of retinal endothelial cell,is under the regulation of estrogen.The proliferation and migration,but not the tubogenesis of retinal endothelial cells are regulated by this regulatory cascade.
RESUMO
AIM: To investigate the role of Cx43 in the myocardialization of the proximal outflow tract(OFT) septum in the mouse heart.METHODS: C57/BL6 mice of ED11.5 to 1 day after birth were used in this study,which included Cx43 knockout homozygotes((Cx43-/-)),heterozygotes((Cx43+/-)) and wildtypes((Cx43+/+)).Pathohistological analysis was used to examine the structure of the hearts.The expression of alpha-sarcomeric actin(?-SCA),active caspase-3 and activator protein-2(AP-2) were detected by immunohistochemistry.RESULTS: Most(Cx43-/-) mice died within 24 h after birth with a swelling and blockage of the conotruncal region,which led to the obstruction of OFT and enlargement of right ventricle.HE staining showed plenty of abnormal tissues in this region forming many pouches.No apparent malformations were observed in(Cx43+/-) and(Cx43+/+) mice.The expression of ?-SCA in the proximal OFT septum was delayed obviously in(Cx43-/-).The apoptotic cells existed in the proximal OFT septum of(Cx43+/+) mostly during ED12.5 to ED15.5.However,there were less apoptotic cells observed in(Cx43+/-),and few in(Cx43-/-).The expression of AP-2,marker of neural crest cells,was increased in (Cx43-/-) and abnormally located in the proximal OFT septum.CONCLUSIONS: Cx43 KO mice are characterized by hyperplasia in conotruncal region,which may be associated with the delayed myocardialization of OFT septum.The decreased apoptosis and the abnormal distribution of cardiac neural crest cells are likely to contribute to the abnormal myocardialization in mice with Cx43 defects.
RESUMO
Objective To estabolish an optimal animal model of oxygen-induced retinopathy(OIR) suitable for examining pathogenesis and therapeutic intervention for retinopathy of prematurity(ROP). Methods Fifty-four 7-day-old C57BL/6J mice were devided into two groups. Twenty-seven mice in hyperoxic group were exposed to 75% oxygen for 5 days and then to room air for another 5 days. Twenty-seven mice in normoxic control were exposed to room air for 10 days. The proliferative neovascular response was estimated by observing the vascular pattern in adenosine diphosphate-ase(ADPase) stained retina flat-mounts and quantitated by counting the number of new vascular cell nuclei extending into the internal limiting membrane in cross-sections. Results Angiography in ADP ase stained retina flat-mounts delineated the entire vascular pattern. Hyperoxia-induced neovascularization occurred at the junction between the vascularized and avascular retina in the mid-periphery in all mice exposed to hyperoxia. After 5 days of exposure to hyperoxia at postnatal day 12(P12),the larger central radial vessels became tortuous and constricted and central perfusion became decreased obviously. After return to room air for 2 days at P14,neovascularization was seen. This response was maximal at P17. There was a mean of 44 neovascular nuclei per cross-section extending into the vitreous in hyperoxia compared to less than 2 nuclei in the normoxia control ( P
RESUMO
Objective The purpose of this experiment is to study the in vivo differentiation and myelin formation of rat striatal neural precursor cells after transplantation into homogeneous retina,observe the order of myelination and its influence on the structure of retina,establish an animal model of CNS myelin formation in vivo. Methods Passage cultured striatal neural precursor cells from embryonic Sprague-Dawley rats were transplanted into the vitreous cavity of neonatal rats.In different stages after transplantion,myelin formation in retina was observed under light and electron microscope and analysed with different stained methods.Results Bundles of myelin appeared in parts of retina 4 weeks later.The distribution and morphology of myelined area expanded with prolonged survival time after cell transplantation.Oligodendrocyte wrapped the naked axons and formed normal myelin limited in the nerve fiber layer.Myelination influenced the distribution of local retinal ganglion cells.Conclusion Striatal neural precursor cells could differentiate into oligodendrocytes and formed myelin after transplanted into retina and the naked axons in retina promoted the myelin formation.This model provides a new method to study the myelin formation and myelin-axon interaction in vivo.
