FEZ1在星形胶质细胞中的表达及其功能研究
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摘要
星形胶质细胞在中枢神经系统中数量最多、分布最广,在形态、受体的分布及胞质活性物质的含量等方面都存在明显的异质性,不同谱系发生的星形胶质细胞在中枢神经系统中所起的作用至今仍不明确。
目的:
(1)分离、培养获得不同谱系来源的两型星形胶质细胞(T1A和T2A),通过基因表达谱芯片技术构建星形胶质细胞基因表达谱,进行对比分析,筛选出细胞特异性差异表达基因。
(2)从获得的基因表达谱中,选定在胶质细胞中新发现的轴突成束和延伸相关基因FEZ1,分析FEZ1在培养的两型星形胶质细胞中的表达定位。
(3)分析FEZ1在大鼠脑组织发育过程中的表达变化。
(4)通过FEZ1基因克隆,瞬时转染及原核表达方法,初步探讨FEZ1对星形胶质细胞活性及神经元突起生长的影响。
方法:
第一部分(1)采用振荡法和差速贴壁法,从新生一天SD大鼠大脑皮层分离获得T1A和T2A,免疫荧光双标鉴定;(2)应用BiostarR-40s基因表达谱芯片技术对比分析在培养的T1A和T2A中细胞特异性差异表达基因情况。
第二部分(1)利用半定量RT-PCR、Realtime RT-PCR、western blot和细胞免疫化学的方法检测FEZ1基因在T1A和T2A中的表达及细胞定位情况,验证基因表达谱芯片结果。(2)取生后1天、3天、7天、2周、1个月、3个月的SD大鼠,灌注后取脑进行冰冻切片,并分别以FEZ1和GFAP或NF对星形胶质细胞和神经元进行免疫荧光双标,检测FEZ1在脑组织发育过程中的表达定位。
第三部分应用RT-PCR方法从大鼠大脑皮层克隆获得FEZ1全长序列,构建pEGFP-N1-FEZ1真核表达重组质粒和FEZ1 RNAi质粒,瞬时转染星形胶质细胞,MTT法检测细胞活力,初步观察FEZ1基因对星形胶质细胞生长的影响;构建pET-28a-FEZ1原核表达重组质粒,体外原核表达His-FEZ1融和蛋白,观察FEZ1对背根节神经元突起生长的影响。
结果:
(1)应用上海博星基因芯片公司星形胶质细胞基因表达谱芯片BiostarR-40s,四次芯片结果交集显示,在4096个检测点中,发现差异表达基因138条,99个为已知基因,其中42个在T1A中高表达,57个在T2A中高表达。与神经轴突传导相关基因均在T2A中表达上调,其中新发现的与轴突成束和延伸相关的FEZ1基因在胶质细胞中的表达及作用国内外尚未见相关研究报道。
(2)RT-PCR及western blot结果显示FEZ1在体外培养的两型星形胶质细胞中mRNA水平及蛋白水平均有表达,且在T2A中表达明显增加,与基因表达谱芯片结果一致。细胞免疫荧光双标结果显示,FEZ1蛋白在O2A祖细胞、T1A、T2A细胞中均有表达,分布于细胞胞浆及突起,在T1A及T2A中可与GFAP共定位,细胞核中没有表达。
(3)FEZ1在大鼠脑组织嗅球的僧帽细胞和颗粒细胞,海马齿状回的颗粒细胞,海马CA1-3区的锥体细胞,皮层锥体细胞也有表达,嗅球及海马齿状回星形胶质细胞有表达,生后7天FEZ1表达量达最高。
(4)构建的pEGFP-N1-FEZ1真核重组质粒和FEZ1 RNAi质粒经测序鉴定正确,瞬时转染星形胶质细胞,干扰FEZ1基因表达可降低细胞活力,抑制细胞生长。
(5)构建的pET-28a-FEZ1原核表达重组质粒经测序鉴定正确,在BL21大肠杆菌中经诱导可表达His-FEZ1融和蛋白,该蛋白对大鼠背根节突起生长有一定促进作用。
结论:
(1)不同谱系来源的两型星形胶质细胞基因表达谱存在明显差异,其中与神经轴突传导相关基因表达差异提示T1A和T2A可能在诱导轴突生长方面有不同功能。
(2)首次研究证实,FEZ1基因在体外培养的星形胶质谱系细胞中均有表达,但在T2A中表达明显增加。
(3)FEZ1在大鼠脑组织发育过程中嗅球、海马神经元及星形胶质细胞中有表达。
(4)成功构建了pEGFP-N1-FEZ1真核重组质粒、FEZ1 RNAi质粒以及pET-28a-FEZ1原核表达重组质粒,为进一步研究FEZ1对星形胶质细胞生物学特性的影响及在其中枢神经系统中的作用机制等奠定了实验基础。
Astrocytes play a more important role than simply providing physical support for neurons, however, the function(s) of type 1 and type 2 astrocytes (T1As, T2As), remains unclear.
Objective:
The aim of this study was (1) to establish and analyze gene expression profiles of cultured T1As and T2As, determine the possible biological function of these genes expressed in T1As and T2As; (2) to confirm the expression and location of FEZ1 in T1As and T2As. (3) to investigate the expression of FEZ1 in rat developing brain; (4) to evaluate the infuence of FEZ1 on cellular activity of astrocytes through transient transfection; (5) to establish FEZ1 fusion protein expression system in the E.coil and evaluate the influence of ectogenic FEZ1 on neurons.
Methods:
Part one (1) Based on the differential properties of developmental time-course and cellular adhesions in T1A cell linage and O-2A cell linage, Primary rat T1As and T2As were isolated from postnatal day 1 (P1) rat cortex. (2) A DNA microarray was used to identify gene expression in cultured T1As and T2As. Different gene expression profile of purified T1As and T2As were compared and analyzed.
Part two (1) Real-time reverse transcription-polymerase chain reactions (RT-PCR), western blots and immunocytochemistry were performed to demonstrate the expression of FEZ1 in cultured T1As and T2As. (2) The expression of FEZ1 in developing brain from rat postnatal 1day, 3 days, 7 days, 2 weeks, 1 month, 3 months were investigated using immunohistochemistry assays.
Part three RT-PCR was used to obtain the full length FEZ1 gene from rat cortex. The recombinant plasmids of pEGFP-N1-FEZ1 and FEZ1 RNAi were transfected into asrocyte respectively, and cell activities were examined by MTT. The recombinant protein of FEZ1 with His tag was expressed in prokaryotic cells and its function on dorsal root ganglia outgrowth was observed.
Results:
(1) Gene profiling, using the BiostarR-40 genechip array, created a transcriptome database of the expression levels of 4096 genes for T1A and T2A. Of the 4069 analyzed genes, a total of 138 were differentially expressed between T1A and T2A, 60 of which were highly expressed in T1As, and 78 in T2As. Approximately 28% (39 of 138) of the genes with altered expression were unknown genes. The remaining 99 genes were involved in metabolism, growth factors, structural molecules, signal transduction, neurite outgrowth, tumors, migration, cell adhesion and transporter activity. There were 57 up-regulated genes (Ratio>2.0) and 42 down-regulated genes (Ratio<0.5) among the 99 known ones. Four genes were found that, according to previous reports, may participate in neurite outgrowth. The four genes were related to the protein tyrosine Phosphatase receptor-type Z polypeptide 1 (PTPRZ1), the dihydropyrimidinase-like 2 (DPYSL2), the fasciculation and elongation protein zeta 1 (FEZ1) and the growth associated protein 43 (GAP-43). All of them were 3-6 fold more highly expressed in T2As compared with T1As. The fasciculation and elongation protein zeta-1 (FEZ1) gene was studied further because it has been suggested that it is not expressed by astrocytes.
(2) RT-PCR and Western blots confirmed the microarray data and showed that FEZ1 was present in T1As and T2As and is more highly expressed in T2As. Immunocytochemistry revealed that FEZ1 was located in the astrocytic cytoplasm and cell processes but not the nucleus.
(3) Immunohistochemistry assays were performed to demonstrate the expression of FEZ1 in developping rat brain. FEZ1 is preferentially expressed in the olfactory bulb, cortical and hippocampal neurons, and weaker in astrocytes. The highest expression of FEZ1 during the period examined was observed on P7.
(4) The recombinant of pEGFP-N1-FEZ1 and FEZ1 RNAi plasmid was constructed successfully. Transient transfection assays showed that inhibition of FEZ1 expression in astrocytes can decrease the cell growth.
(5) The recombinant of pET-28a-FEZ1 plasmid was constructed successfully and the fusion protein can promote axon outgrowth of dorsal root ganglia.
Conclusions:
(1) The present results provided a novel genomewide information for further investigations of T1A and T2A.
(2) It was confirmed for the first time that FEZ1 expressed in both types of astrocytes and upregulated in T2A.
(3) In rat developing brain, FEZ1 is expressed in the olfactory bulb, cortical and hippocampal neurons, and weaker in astrocytes.
(4) The presence of FEZ1 in astrocytes may affect cell behavior and make influence on neurons.
The results contribute to a clearer understanding of the expression and function of FEZ1 in the two types of astrocytes.
目的:
(1)分离、培养获得不同谱系来源的两型星形胶质细胞(T1A和T2A),通过基因表达谱芯片技术构建星形胶质细胞基因表达谱,进行对比分析,筛选出细胞特异性差异表达基因。
(2)从获得的基因表达谱中,选定在胶质细胞中新发现的轴突成束和延伸相关基因FEZ1,分析FEZ1在培养的两型星形胶质细胞中的表达定位。
(3)分析FEZ1在大鼠脑组织发育过程中的表达变化。
(4)通过FEZ1基因克隆,瞬时转染及原核表达方法,初步探讨FEZ1对星形胶质细胞活性及神经元突起生长的影响。
方法:
第一部分(1)采用振荡法和差速贴壁法,从新生一天SD大鼠大脑皮层分离获得T1A和T2A,免疫荧光双标鉴定;(2)应用BiostarR-40s基因表达谱芯片技术对比分析在培养的T1A和T2A中细胞特异性差异表达基因情况。
第二部分(1)利用半定量RT-PCR、Realtime RT-PCR、western blot和细胞免疫化学的方法检测FEZ1基因在T1A和T2A中的表达及细胞定位情况,验证基因表达谱芯片结果。(2)取生后1天、3天、7天、2周、1个月、3个月的SD大鼠,灌注后取脑进行冰冻切片,并分别以FEZ1和GFAP或NF对星形胶质细胞和神经元进行免疫荧光双标,检测FEZ1在脑组织发育过程中的表达定位。
第三部分应用RT-PCR方法从大鼠大脑皮层克隆获得FEZ1全长序列,构建pEGFP-N1-FEZ1真核表达重组质粒和FEZ1 RNAi质粒,瞬时转染星形胶质细胞,MTT法检测细胞活力,初步观察FEZ1基因对星形胶质细胞生长的影响;构建pET-28a-FEZ1原核表达重组质粒,体外原核表达His-FEZ1融和蛋白,观察FEZ1对背根节神经元突起生长的影响。
结果:
(1)应用上海博星基因芯片公司星形胶质细胞基因表达谱芯片BiostarR-40s,四次芯片结果交集显示,在4096个检测点中,发现差异表达基因138条,99个为已知基因,其中42个在T1A中高表达,57个在T2A中高表达。与神经轴突传导相关基因均在T2A中表达上调,其中新发现的与轴突成束和延伸相关的FEZ1基因在胶质细胞中的表达及作用国内外尚未见相关研究报道。
(2)RT-PCR及western blot结果显示FEZ1在体外培养的两型星形胶质细胞中mRNA水平及蛋白水平均有表达,且在T2A中表达明显增加,与基因表达谱芯片结果一致。细胞免疫荧光双标结果显示,FEZ1蛋白在O2A祖细胞、T1A、T2A细胞中均有表达,分布于细胞胞浆及突起,在T1A及T2A中可与GFAP共定位,细胞核中没有表达。
(3)FEZ1在大鼠脑组织嗅球的僧帽细胞和颗粒细胞,海马齿状回的颗粒细胞,海马CA1-3区的锥体细胞,皮层锥体细胞也有表达,嗅球及海马齿状回星形胶质细胞有表达,生后7天FEZ1表达量达最高。
(4)构建的pEGFP-N1-FEZ1真核重组质粒和FEZ1 RNAi质粒经测序鉴定正确,瞬时转染星形胶质细胞,干扰FEZ1基因表达可降低细胞活力,抑制细胞生长。
(5)构建的pET-28a-FEZ1原核表达重组质粒经测序鉴定正确,在BL21大肠杆菌中经诱导可表达His-FEZ1融和蛋白,该蛋白对大鼠背根节突起生长有一定促进作用。
结论:
(1)不同谱系来源的两型星形胶质细胞基因表达谱存在明显差异,其中与神经轴突传导相关基因表达差异提示T1A和T2A可能在诱导轴突生长方面有不同功能。
(2)首次研究证实,FEZ1基因在体外培养的星形胶质谱系细胞中均有表达,但在T2A中表达明显增加。
(3)FEZ1在大鼠脑组织发育过程中嗅球、海马神经元及星形胶质细胞中有表达。
(4)成功构建了pEGFP-N1-FEZ1真核重组质粒、FEZ1 RNAi质粒以及pET-28a-FEZ1原核表达重组质粒,为进一步研究FEZ1对星形胶质细胞生物学特性的影响及在其中枢神经系统中的作用机制等奠定了实验基础。
Astrocytes play a more important role than simply providing physical support for neurons, however, the function(s) of type 1 and type 2 astrocytes (T1As, T2As), remains unclear.
Objective:
The aim of this study was (1) to establish and analyze gene expression profiles of cultured T1As and T2As, determine the possible biological function of these genes expressed in T1As and T2As; (2) to confirm the expression and location of FEZ1 in T1As and T2As. (3) to investigate the expression of FEZ1 in rat developing brain; (4) to evaluate the infuence of FEZ1 on cellular activity of astrocytes through transient transfection; (5) to establish FEZ1 fusion protein expression system in the E.coil and evaluate the influence of ectogenic FEZ1 on neurons.
Methods:
Part one (1) Based on the differential properties of developmental time-course and cellular adhesions in T1A cell linage and O-2A cell linage, Primary rat T1As and T2As were isolated from postnatal day 1 (P1) rat cortex. (2) A DNA microarray was used to identify gene expression in cultured T1As and T2As. Different gene expression profile of purified T1As and T2As were compared and analyzed.
Part two (1) Real-time reverse transcription-polymerase chain reactions (RT-PCR), western blots and immunocytochemistry were performed to demonstrate the expression of FEZ1 in cultured T1As and T2As. (2) The expression of FEZ1 in developing brain from rat postnatal 1day, 3 days, 7 days, 2 weeks, 1 month, 3 months were investigated using immunohistochemistry assays.
Part three RT-PCR was used to obtain the full length FEZ1 gene from rat cortex. The recombinant plasmids of pEGFP-N1-FEZ1 and FEZ1 RNAi were transfected into asrocyte respectively, and cell activities were examined by MTT. The recombinant protein of FEZ1 with His tag was expressed in prokaryotic cells and its function on dorsal root ganglia outgrowth was observed.
Results:
(1) Gene profiling, using the BiostarR-40 genechip array, created a transcriptome database of the expression levels of 4096 genes for T1A and T2A. Of the 4069 analyzed genes, a total of 138 were differentially expressed between T1A and T2A, 60 of which were highly expressed in T1As, and 78 in T2As. Approximately 28% (39 of 138) of the genes with altered expression were unknown genes. The remaining 99 genes were involved in metabolism, growth factors, structural molecules, signal transduction, neurite outgrowth, tumors, migration, cell adhesion and transporter activity. There were 57 up-regulated genes (Ratio>2.0) and 42 down-regulated genes (Ratio<0.5) among the 99 known ones. Four genes were found that, according to previous reports, may participate in neurite outgrowth. The four genes were related to the protein tyrosine Phosphatase receptor-type Z polypeptide 1 (PTPRZ1), the dihydropyrimidinase-like 2 (DPYSL2), the fasciculation and elongation protein zeta 1 (FEZ1) and the growth associated protein 43 (GAP-43). All of them were 3-6 fold more highly expressed in T2As compared with T1As. The fasciculation and elongation protein zeta-1 (FEZ1) gene was studied further because it has been suggested that it is not expressed by astrocytes.
(2) RT-PCR and Western blots confirmed the microarray data and showed that FEZ1 was present in T1As and T2As and is more highly expressed in T2As. Immunocytochemistry revealed that FEZ1 was located in the astrocytic cytoplasm and cell processes but not the nucleus.
(3) Immunohistochemistry assays were performed to demonstrate the expression of FEZ1 in developping rat brain. FEZ1 is preferentially expressed in the olfactory bulb, cortical and hippocampal neurons, and weaker in astrocytes. The highest expression of FEZ1 during the period examined was observed on P7.
(4) The recombinant of pEGFP-N1-FEZ1 and FEZ1 RNAi plasmid was constructed successfully. Transient transfection assays showed that inhibition of FEZ1 expression in astrocytes can decrease the cell growth.
(5) The recombinant of pET-28a-FEZ1 plasmid was constructed successfully and the fusion protein can promote axon outgrowth of dorsal root ganglia.
Conclusions:
(1) The present results provided a novel genomewide information for further investigations of T1A and T2A.
(2) It was confirmed for the first time that FEZ1 expressed in both types of astrocytes and upregulated in T2A.
(3) In rat developing brain, FEZ1 is expressed in the olfactory bulb, cortical and hippocampal neurons, and weaker in astrocytes.
(4) The presence of FEZ1 in astrocytes may affect cell behavior and make influence on neurons.
The results contribute to a clearer understanding of the expression and function of FEZ1 in the two types of astrocytes.
引文
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