BDNF-TrkB通路对心脏微血管内皮细胞的影响
发布时间：2018-12-17 17:59
【摘要】： 目的：心脏微血管内皮细胞(CMECs)的在心肌血管新生过程中扮演重要的角色。最近研究表明,脑源性神经营养因子(BDNF)除了在神经系统中起着重要作用外,还在维持血管内皮细胞的存活,促进缺血部位内皮细胞的增殖中发挥重要的作用,是一种重要的血管再生因子。BDNF的受体是TrkB,目前对BDNF-TrkB通路在心脏微血管内皮细胞的功能角色及调控机制尚不清楚。本硕士论文对(1)BDNF对CMECs的增殖及复制性衰老对BDNF受体和对CMECs增殖的影响；(2)及BDNF受体TrkB三种亚型在CMECs表达及复制性衰老可能对三种亚型表达影响进行了研究；(3)传代对CMECs核型的影响进行了研究。 方法与结果：(1)对经分离CMECs随传代形态变化及核型分析,研究发现：CMECs在体外传代过程中,随着细胞代数的增加,染色体缺失率上升,染色体数目为42条的细胞比率从63.3%(P20)降至18.6%(P100)；(2)利用实时定量PCR分析,源白年轻大鼠TrkB三种亚型随传代表达量减少,且老年大鼠CMECs中TrkB-FL和TrkB-T2表达量很低或不表达,仅表达TrkB-T1；(3)使用流式细胞术结合AlamarBlue还原法测定细胞生长曲线证明：经BDNF处理的CMECs生长加快,处于分裂期的细胞数量增加,且不同浓度的BDNF刺激具有不同的增幅,其中年轻大鼠CMECs P6 50ng/mL BDNF处理组S期细胞由31.64±1.63%增多至36.27±1.74%(P0.01), CMECs P14 50ng/mL BDNF处理组S期细胞由25.95±0.39%增多至28.92±1.46%(P0.01),而老年大鼠CMECs P550ng／mL BDNF处理组S期细胞由30.38±1.02%增多至31.31±0.88%,该变化没有统计学意义(4)选取TrkB三种亚型及基因芯片结果中差异显著的增殖和移行相关基因,应用实时定量PCR分析经BDNF处理后,不同时间点CMECs基因表达的变化,处理8h时,上调1.5倍或以上的基因包括：Syndecan-4(1.78倍)、BGN(1.55倍)、Cav-1(1.56倍)、RTN-4(1.60倍)、MGP(1.69倍)、Hoxdl3(1.97倍)和ACTG-2(1.48倍),下调的基因包括：TrkB-FL(0.78倍)、Nisch(0.63倍)、FLNa(0.55倍)和Spna2(0.72倍)；(5)使用TrkB-shiRNA质粒对CMECs进行转染,运用实时定量PCR对TrkB-FL、TrkB-T1及TrkB-T2的mRNA相对表达进行检测,以确定对该三个亚型表达的影响,并对经转染CMECs增殖的影响进行研究,应用TrkB-shiRNA质粒转染CMECs后,TrkB三种亚型在转染后的四天内mRNA表达均呈下降,且经转染CMECs的生长变慢。 结论：(1)经分离CMECs在连续传代20代后染色体开始缺失,且随代数增高缺失增多,细胞形态有所变化；(2)源自年轻大鼠CMECs随着代数的增加,TrkB三种亚型表达量减少,而源自老年大鼠CMECs则只表达TrkB-T1;(3)不同浓度的BDNF均能促进CMECs的增殖,且BDNF会影响CMECs多个基因的表达；(4)利用TrkB-shiRNA质粒能成功沉默CMECs的TrkB-FL、TrkB-T1及TrkB-T2的表达,并初步揭示：抑制TrkB的表达可能会影响CMECs的增殖。
[Abstract]:Objective: (CMECs) plays an important role in myocardial angiogenesis. Recent studies have shown that brain-derived neurotrophic factor (BDNF) not only plays an important role in the nervous system, but also plays an important role in maintaining the survival of vascular endothelial cells and promoting the proliferation of endothelial cells in ischemic areas. BDNF receptor is an important vascular regeneration factor. The role of TrkB, in the function and regulation of BDNF-TrkB pathway in cardiac microvascular endothelial cells is unclear. The effects of BDNF on the proliferation of CMECs and the effects of replicative senescence on BDNF receptor and CMECs proliferation, and on the expression of TrkB and BDNF receptor TrkB in CMECs and the possible effects of replicative senescence on the expression of three subtypes were studied in this thesis. (3) the effect of passage on the karyotype of CMECs was studied. Methods and results: (1) according to the morphological changes and karyotype analysis of isolated CMECs with passage, it was found that the chromosome deletion rate increased with the increase of cell algebra during in vitro passage of CMECs. The percentage of cells with 42 chromosomes decreased from 63.3% (P20) to 18.6% (P100). (2) by using real-time quantitative PCR analysis, the three subtypes of TrkB of young white rats decreased with passage expression, and the expression of TrkB-FL and TrkB-T2 in CMECs of aged rats was very low or not, only TrkB-T1; was expressed. (3) flow cytometry combined with AlamarBlue reduction method was used to determine the cell growth curve. The results showed that CMECs treated with BDNF accelerated the growth, and the number of cells in mitotic phase increased, and different concentrations of BDNF stimulation had different growth rate. The S phase cells increased from 31.64 卤1.63% to 36.27 卤1.74% in young rats treated with CMECs P6 50ng/mL BDNF (P0.01), CMECs P14 50ng/mL BDNF increased from 25.95 卤0.39% to 28.92 卤1.46% (P0.01). However, the S phase cells increased from 30.38 卤1.02% to 31.31 卤0.88 in aged rats treated with CMECs P550ng/mL BDNF. There was no statistical significance in this change. (4) there were significant differences in proliferation and migration related genes among the three subtypes of TrkB and gene microarray. Real time quantitative PCR was used to analyze the changes of CMECs gene expression at different time points after BDNF treatment. After 8 h treatment, the up-regulated genes were Syndecan-4 (1.78 times), BGN (1.55 times) and Cav-1 (1.56 times). RTN-4 (1.60 times), MGP (1.69 times), Hoxdl3 (1.97 times) and ACTG-2 (1.48 times) were down-regulated by TrkB-FL (0.78 times), Nisch (0.63 times). FLNa (0.55 times) and Spna2 (0.72 times); (5) TrkB-shiRNA plasmid was used to transfect CMECs, and real-time quantitative PCR was used to detect the relative expression of TrkB-FL,TrkB-T1 and TrkB-T2 mRNA in order to determine the effect on the expression of the three subtypes. The effect of transfection on the proliferation of CMECs was studied. After transfection of CMECs with TrkB-shiRNA plasmid, the expression of mRNA in the three subtypes of TrkB decreased within four days after transfection, and the growth of transfected CMECs became slower. Conclusion: (1) chromosome deletion began after successive passage of CMECs for 20 generations, and the cell morphology changed with the increase of deletion. (2) the expression of TrkB subtypes decreased with the increase of CMECs algebra in young rats, while CMECs from aged rats only expressed TrkB-T1; (3) BDNF at different concentrations could promote the proliferation of CMECs, and BDNF could affect the expression of multiple CMECs genes. (4) the expression of TrkB-FL,TrkB-T1 and TrkB-T2 of CMECs could be silenced successfully by using TrkB-shiRNA plasmid, and it was suggested that inhibiting the expression of TrkB might affect the proliferation of CMECs.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R33
本文编号：2384595
[Abstract]:Objective: (CMECs) plays an important role in myocardial angiogenesis. Recent studies have shown that brain-derived neurotrophic factor (BDNF) not only plays an important role in the nervous system, but also plays an important role in maintaining the survival of vascular endothelial cells and promoting the proliferation of endothelial cells in ischemic areas. BDNF receptor is an important vascular regeneration factor. The role of TrkB, in the function and regulation of BDNF-TrkB pathway in cardiac microvascular endothelial cells is unclear. The effects of BDNF on the proliferation of CMECs and the effects of replicative senescence on BDNF receptor and CMECs proliferation, and on the expression of TrkB and BDNF receptor TrkB in CMECs and the possible effects of replicative senescence on the expression of three subtypes were studied in this thesis. (3) the effect of passage on the karyotype of CMECs was studied. Methods and results: (1) according to the morphological changes and karyotype analysis of isolated CMECs with passage, it was found that the chromosome deletion rate increased with the increase of cell algebra during in vitro passage of CMECs. The percentage of cells with 42 chromosomes decreased from 63.3% (P20) to 18.6% (P100). (2) by using real-time quantitative PCR analysis, the three subtypes of TrkB of young white rats decreased with passage expression, and the expression of TrkB-FL and TrkB-T2 in CMECs of aged rats was very low or not, only TrkB-T1; was expressed. (3) flow cytometry combined with AlamarBlue reduction method was used to determine the cell growth curve. The results showed that CMECs treated with BDNF accelerated the growth, and the number of cells in mitotic phase increased, and different concentrations of BDNF stimulation had different growth rate. The S phase cells increased from 31.64 卤1.63% to 36.27 卤1.74% in young rats treated with CMECs P6 50ng/mL BDNF (P0.01), CMECs P14 50ng/mL BDNF increased from 25.95 卤0.39% to 28.92 卤1.46% (P0.01). However, the S phase cells increased from 30.38 卤1.02% to 31.31 卤0.88 in aged rats treated with CMECs P550ng/mL BDNF. There was no statistical significance in this change. (4) there were significant differences in proliferation and migration related genes among the three subtypes of TrkB and gene microarray. Real time quantitative PCR was used to analyze the changes of CMECs gene expression at different time points after BDNF treatment. After 8 h treatment, the up-regulated genes were Syndecan-4 (1.78 times), BGN (1.55 times) and Cav-1 (1.56 times). RTN-4 (1.60 times), MGP (1.69 times), Hoxdl3 (1.97 times) and ACTG-2 (1.48 times) were down-regulated by TrkB-FL (0.78 times), Nisch (0.63 times). FLNa (0.55 times) and Spna2 (0.72 times); (5) TrkB-shiRNA plasmid was used to transfect CMECs, and real-time quantitative PCR was used to detect the relative expression of TrkB-FL,TrkB-T1 and TrkB-T2 mRNA in order to determine the effect on the expression of the three subtypes. The effect of transfection on the proliferation of CMECs was studied. After transfection of CMECs with TrkB-shiRNA plasmid, the expression of mRNA in the three subtypes of TrkB decreased within four days after transfection, and the growth of transfected CMECs became slower. Conclusion: (1) chromosome deletion began after successive passage of CMECs for 20 generations, and the cell morphology changed with the increase of deletion. (2) the expression of TrkB subtypes decreased with the increase of CMECs algebra in young rats, while CMECs from aged rats only expressed TrkB-T1; (3) BDNF at different concentrations could promote the proliferation of CMECs, and BDNF could affect the expression of multiple CMECs genes. (4) the expression of TrkB-FL,TrkB-T1 and TrkB-T2 of CMECs could be silenced successfully by using TrkB-shiRNA plasmid, and it was suggested that inhibiting the expression of TrkB might affect the proliferation of CMECs.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R33
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