Roof plate secretion of bone morphogenetic proteins(BMPs)directs the cellular fate of sensory neurons during spinal cord development,including the formation of the ascending sensory columns,though their biology is not...Roof plate secretion of bone morphogenetic proteins(BMPs)directs the cellular fate of sensory neurons during spinal cord development,including the formation of the ascending sensory columns,though their biology is not well understood.Type-ⅡBMP receptor(BMPRⅡ),the cognate receptor,is expressed by neural precursor cells during embryogenesis;however,an in vitro method of enriching BMPRⅡ^(+)human neural precursor cells(hNPCs)from the fetal spinal cord is absent.Immunofluorescence was undertaken on intact second-trimester human fetal spinal cord using antibodies to BMPRⅡand leukemia inhibitory factor(LIF).Regions of highest BMPRⅡ^(+)immunofluorescence localized to sensory columns.Parenchymal and meningeal-associated BMPRⅡ^(+)vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers,CD34/CD39.LIF immunostaining identified a population of somas concentrated in dorsal and ventral horn interneurons,mirroring the expression of LIF receptor/CD118.A combination of LIF supplementation and high-density culture maintained culture growth beyond 10 passages,while synergistically increasing the proportion of neurospheres with a stratified,cytoarchitecture.These neurospheres were characterized by BMPRⅡ^(+)/MAP2ab^(+/–)/βⅢ-tubulin^(+)/nestin^(–)/vimentin^(–)/GFAP^(–)/NeuN^(–)surface hNPCs surrounding a heterogeneous core ofβⅢ-tubulin^(+)/nestin^(+)/vimentin^(+)/GFAP^(+)/MAP2ab^(–)/NeuN^(–)multipotent precursors.Dissociated cultures from tripotential neurospheres contained neuronal(βⅢ-tubulin^(+)),astrocytic(GFAP+),and oligodendrocytic(O4+)lineage cells.Fluorescence-activated cell sorting-sorted BMPRⅡ^(+)hNPCs were MAP2ab^(+/–)/βⅢ-tubulin^(+)/GFAP^(–)/O4^(–)in culture.This is the first isolation of BMPRⅡ^(+)hNPCs identified and characterized in human fetal spinal cords.Our data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres,characterized by surface BMPRⅡ^(+)hNPCs.Our study has provided a new methodology for an in vitro model capable of amplifying human fetal spinal cord cell numbers for>10 passages.Investigations of the role BMPRⅡplays in spinal cord development have primarily relied upon mouse and rat models,with interpolations to human development being derived through inference.Because of significant species differences between murine biology and human,including anatomical dissimilarities in central nervous system(CNS)structure,the findings made in murine models cannot be presumed to apply to human spinal cord development.For these reasons,our human in vitro model offers a novel tool to better understand neurodevelopmental pathways,including BMP signaling,as well as spinal cord injury research and testing drug therapies.展开更多
背景:课题组以往研究证实,骨形态发生蛋白4对生长发育期下颌骨的生长有促进作用,而骨形态发生蛋白2是否能与骨形态发生蛋白4相互促进下颌骨生长目前未见有相关报道。目的:检测生长发育高峰期骨性Ⅱ类错畸形患者血液中骨形态发生蛋白2...背景:课题组以往研究证实,骨形态发生蛋白4对生长发育期下颌骨的生长有促进作用,而骨形态发生蛋白2是否能与骨形态发生蛋白4相互促进下颌骨生长目前未见有相关报道。目的:检测生长发育高峰期骨性Ⅱ类错畸形患者血液中骨形态发生蛋白2和骨形态发生蛋白4的表达情况,以探究骨形态发生蛋白2和骨形态发生蛋白4的表达量与下颌骨生长的关系。方法:生长发育高峰期骨性Ⅰ类错畸形患者为Ⅰ组,以下颌后缩为主的骨性Ⅱ类错畸形患者为Ⅱ组,每组18人。采用实时荧光定量PCR(RT-PCR)分别检测两组血液骨形态发生蛋白2和骨形态发生蛋白4的表达。结果与结论:骨性Ⅱ类错畸形组中骨形态发生蛋白2 mR NA的表达量明显低于对照组骨性Ⅰ类错畸形组(P<0.05),骨性Ⅱ类错畸形组中骨形态发生蛋白4 m RNA的表达量明显低于对照组骨性Ⅰ类错畸形组(P<0.05),骨性Ⅱ组中骨形态发生蛋白2与骨形态发生蛋白4的表达有显著相关性。结果证实,骨形态发生蛋白2和骨形态发生蛋白4在生长发育高峰期的表达量均降低与下颌骨发育不足有密切关系,且骨形态发生蛋白2和骨形态发生蛋白4相互协同,共同参与调节下颌骨的生长。展开更多
目的研究TGF-1β、BM P-2和typeⅡco llagen在退行性腰椎滑脱(degenerative lum bar spondy lo listhes is,DLS)和腰椎间盘突出症(lum bar d isc hern iation,LDH)黄韧带中的表达及其意义。方法37例手术切除的腰椎椎板间部黄韧带标本分...目的研究TGF-1β、BM P-2和typeⅡco llagen在退行性腰椎滑脱(degenerative lum bar spondy lo listhes is,DLS)和腰椎间盘突出症(lum bar d isc hern iation,LDH)黄韧带中的表达及其意义。方法37例手术切除的腰椎椎板间部黄韧带标本分为3组,第1组为退行性腰椎滑脱组(DLS)10例;第2组为腰椎间盘突出症组(LDH)17例,第3组为正常对照组10例,其中7例取自腰椎骨折手术病人,3例取自意外死亡者。应用EnV is ion二步免疫组化的方法检测其TGF-1β、BM P-2和typeⅡco llagen的表达情况,普通光镜观察,计算出各标本的表达阳性率和表达强度,数据以x-±s标准差及表达强度表示,结果分别用Spss统计软件和R id it进行分析。结果TGF-1β、BM P-2和typeⅡco llagen的阳性表达产物见于成纤维细胞、成软骨细胞和软骨细胞中,而Ⅱ型胶原染色还可同时见于基质。TGF-1β、BM P-2和typeⅡco llagen在DLS组中的表达明显高于LDH组和正常组(P<0.01或P<0.05),Ⅱ型胶原基质染色明显深于LDH组和对照组。LDH组的TGF-1β和typeⅡco llagen的表达阳性率和表达强度与正常组之间差异无显著性(P>0.05),而BM P-2的表达阳性率和表达强度在LDH组与正常组之间具有统计学意义(P<0.01)。结论黄韧带所受到的异常机械牵张力可以增加TGF-1β在黄韧带细胞中的合成,而TGF-1β则促进退行性腰椎滑脱黄韧带中的Ⅱ型胶原合成,导致黄韧带的退变和肥厚。BM P-2在退变黄韧带中的表达异常增高,可能与黄韧带的软骨化倾向有关。展开更多
基金supported by grants from the National Health and Medical Research Council(NHMRC)of Australia(Nos.571100 and 1048082)the Baxter Charitable Foundation(to TCL)+1 种基金Medical Research grants from the Rebecca L.Cooper Medical Research Foundation(to MWW,TCL,and MDL)supported by a Charles D.Kelman,M.D.Postdoctoral Award(2010)from the International Retinal Research Foundation(USA)。
文摘Roof plate secretion of bone morphogenetic proteins(BMPs)directs the cellular fate of sensory neurons during spinal cord development,including the formation of the ascending sensory columns,though their biology is not well understood.Type-ⅡBMP receptor(BMPRⅡ),the cognate receptor,is expressed by neural precursor cells during embryogenesis;however,an in vitro method of enriching BMPRⅡ^(+)human neural precursor cells(hNPCs)from the fetal spinal cord is absent.Immunofluorescence was undertaken on intact second-trimester human fetal spinal cord using antibodies to BMPRⅡand leukemia inhibitory factor(LIF).Regions of highest BMPRⅡ^(+)immunofluorescence localized to sensory columns.Parenchymal and meningeal-associated BMPRⅡ^(+)vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers,CD34/CD39.LIF immunostaining identified a population of somas concentrated in dorsal and ventral horn interneurons,mirroring the expression of LIF receptor/CD118.A combination of LIF supplementation and high-density culture maintained culture growth beyond 10 passages,while synergistically increasing the proportion of neurospheres with a stratified,cytoarchitecture.These neurospheres were characterized by BMPRⅡ^(+)/MAP2ab^(+/–)/βⅢ-tubulin^(+)/nestin^(–)/vimentin^(–)/GFAP^(–)/NeuN^(–)surface hNPCs surrounding a heterogeneous core ofβⅢ-tubulin^(+)/nestin^(+)/vimentin^(+)/GFAP^(+)/MAP2ab^(–)/NeuN^(–)multipotent precursors.Dissociated cultures from tripotential neurospheres contained neuronal(βⅢ-tubulin^(+)),astrocytic(GFAP+),and oligodendrocytic(O4+)lineage cells.Fluorescence-activated cell sorting-sorted BMPRⅡ^(+)hNPCs were MAP2ab^(+/–)/βⅢ-tubulin^(+)/GFAP^(–)/O4^(–)in culture.This is the first isolation of BMPRⅡ^(+)hNPCs identified and characterized in human fetal spinal cords.Our data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres,characterized by surface BMPRⅡ^(+)hNPCs.Our study has provided a new methodology for an in vitro model capable of amplifying human fetal spinal cord cell numbers for>10 passages.Investigations of the role BMPRⅡplays in spinal cord development have primarily relied upon mouse and rat models,with interpolations to human development being derived through inference.Because of significant species differences between murine biology and human,including anatomical dissimilarities in central nervous system(CNS)structure,the findings made in murine models cannot be presumed to apply to human spinal cord development.For these reasons,our human in vitro model offers a novel tool to better understand neurodevelopmental pathways,including BMP signaling,as well as spinal cord injury research and testing drug therapies.
文摘背景:课题组以往研究证实,骨形态发生蛋白4对生长发育期下颌骨的生长有促进作用,而骨形态发生蛋白2是否能与骨形态发生蛋白4相互促进下颌骨生长目前未见有相关报道。目的:检测生长发育高峰期骨性Ⅱ类错畸形患者血液中骨形态发生蛋白2和骨形态发生蛋白4的表达情况,以探究骨形态发生蛋白2和骨形态发生蛋白4的表达量与下颌骨生长的关系。方法:生长发育高峰期骨性Ⅰ类错畸形患者为Ⅰ组,以下颌后缩为主的骨性Ⅱ类错畸形患者为Ⅱ组,每组18人。采用实时荧光定量PCR(RT-PCR)分别检测两组血液骨形态发生蛋白2和骨形态发生蛋白4的表达。结果与结论:骨性Ⅱ类错畸形组中骨形态发生蛋白2 mR NA的表达量明显低于对照组骨性Ⅰ类错畸形组(P<0.05),骨性Ⅱ类错畸形组中骨形态发生蛋白4 m RNA的表达量明显低于对照组骨性Ⅰ类错畸形组(P<0.05),骨性Ⅱ组中骨形态发生蛋白2与骨形态发生蛋白4的表达有显著相关性。结果证实,骨形态发生蛋白2和骨形态发生蛋白4在生长发育高峰期的表达量均降低与下颌骨发育不足有密切关系,且骨形态发生蛋白2和骨形态发生蛋白4相互协同,共同参与调节下颌骨的生长。
文摘目的研究TGF-1β、BM P-2和typeⅡco llagen在退行性腰椎滑脱(degenerative lum bar spondy lo listhes is,DLS)和腰椎间盘突出症(lum bar d isc hern iation,LDH)黄韧带中的表达及其意义。方法37例手术切除的腰椎椎板间部黄韧带标本分为3组,第1组为退行性腰椎滑脱组(DLS)10例;第2组为腰椎间盘突出症组(LDH)17例,第3组为正常对照组10例,其中7例取自腰椎骨折手术病人,3例取自意外死亡者。应用EnV is ion二步免疫组化的方法检测其TGF-1β、BM P-2和typeⅡco llagen的表达情况,普通光镜观察,计算出各标本的表达阳性率和表达强度,数据以x-±s标准差及表达强度表示,结果分别用Spss统计软件和R id it进行分析。结果TGF-1β、BM P-2和typeⅡco llagen的阳性表达产物见于成纤维细胞、成软骨细胞和软骨细胞中,而Ⅱ型胶原染色还可同时见于基质。TGF-1β、BM P-2和typeⅡco llagen在DLS组中的表达明显高于LDH组和正常组(P<0.01或P<0.05),Ⅱ型胶原基质染色明显深于LDH组和对照组。LDH组的TGF-1β和typeⅡco llagen的表达阳性率和表达强度与正常组之间差异无显著性(P>0.05),而BM P-2的表达阳性率和表达强度在LDH组与正常组之间具有统计学意义(P<0.01)。结论黄韧带所受到的异常机械牵张力可以增加TGF-1β在黄韧带细胞中的合成,而TGF-1β则促进退行性腰椎滑脱黄韧带中的Ⅱ型胶原合成,导致黄韧带的退变和肥厚。BM P-2在退变黄韧带中的表达异常增高,可能与黄韧带的软骨化倾向有关。
