【摘要】： 目的：心臟微血管內(nèi)皮細(xì)胞(CMECs)的在心肌血管新生過(guò)程中扮演重要的角色。最近研究表明,腦源性神經(jīng)營(yíng)養(yǎng)因子(BDNF)除了在神經(jīng)系統(tǒng)中起著重要作用外,還在維持血管內(nèi)皮細(xì)胞的存活,促進(jìn)缺血部位內(nèi)皮細(xì)胞的增殖中發(fā)揮重要的作用,是一種重要的血管再生因子。BDNF的受體是TrkB,目前對(duì)BDNF-TrkB通路在心臟微血管內(nèi)皮細(xì)胞的功能角色及調(diào)控機(jī)制尚不清楚。本碩士論文對(duì)(1)BDNF對(duì)CMECs的增殖及復(fù)制性衰老對(duì)BDNF受體和對(duì)CMECs增殖的影響；(2)及BDNF受體TrkB三種亞型在CMECs表達(dá)及復(fù)制性衰老可能對(duì)三種亞型表達(dá)影響進(jìn)行了研究；(3)傳代對(duì)CMECs核型的影響進(jìn)行了研究。 方法與結(jié)果：(1)對(duì)經(jīng)分離CMECs隨傳代形態(tài)變化及核型分析,研究發(fā)現(xiàn)：CMECs在體外傳代過(guò)程中,隨著細(xì)胞代數(shù)的增加,染色體缺失率上升,染色體數(shù)目為42條的細(xì)胞比率從63.3%(P20)降至18.6%(P100)；(2)利用實(shí)時(shí)定量PCR分析,源白年輕大鼠TrkB三種亞型隨傳代表達(dá)量減少,且老年大鼠CMECs中TrkB-FL和TrkB-T2表達(dá)量很低或不表達(dá),僅表達(dá)TrkB-T1；(3)使用流式細(xì)胞術(shù)結(jié)合AlamarBlue還原法測(cè)定細(xì)胞生長(zhǎng)曲線證明：經(jīng)BDNF處理的CMECs生長(zhǎng)加快,處于分裂期的細(xì)胞數(shù)量增加,且不同濃度的BDNF刺激具有不同的增幅,其中年輕大鼠CMECs P6 50ng/mL BDNF處理組S期細(xì)胞由31.64±1.63%增多至36.27±1.74%(P0.01), CMECs P14 50ng/mL BDNF處理組S期細(xì)胞由25.95±0.39%增多至28.92±1.46%(P0.01),而老年大鼠CMECs P550ng／mL BDNF處理組S期細(xì)胞由30.38±1.02%增多至31.31±0.88%,該變化沒(méi)有統(tǒng)計(jì)學(xué)意義(4)選取TrkB三種亞型及基因芯片結(jié)果中差異顯著的增殖和移行相關(guān)基因,應(yīng)用實(shí)時(shí)定量PCR分析經(jīng)BDNF處理后,不同時(shí)間點(diǎn)CMECs基因表達(dá)的變化,處理8h時(shí),上調(diào)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),下調(diào)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質(zhì)粒對(duì)CMECs進(jìn)行轉(zhuǎn)染,運(yùn)用實(shí)時(shí)定量PCR對(duì)TrkB-FL、TrkB-T1及TrkB-T2的mRNA相對(duì)表達(dá)進(jìn)行檢測(cè),以確定對(duì)該三個(gè)亞型表達(dá)的影響,并對(duì)經(jīng)轉(zhuǎn)染CMECs增殖的影響進(jìn)行研究,應(yīng)用TrkB-shiRNA質(zhì)粒轉(zhuǎn)染CMECs后,TrkB三種亞型在轉(zhuǎn)染后的四天內(nèi)mRNA表達(dá)均呈下降,且經(jīng)轉(zhuǎn)染CMECs的生長(zhǎng)變慢。 結(jié)論：(1)經(jīng)分離CMECs在連續(xù)傳代20代后染色體開(kāi)始缺失,且隨代數(shù)增高缺失增多,細(xì)胞形態(tài)有所變化；(2)源自年輕大鼠CMECs隨著代數(shù)的增加,TrkB三種亞型表達(dá)量減少,而源自老年大鼠CMECs則只表達(dá)TrkB-T1;(3)不同濃度的BDNF均能促進(jìn)CMECs的增殖,且BDNF會(huì)影響CMECs多個(gè)基因的表達(dá)；(4)利用TrkB-shiRNA質(zhì)粒能成功沉默CMECs的TrkB-FL、TrkB-T1及TrkB-T2的表達(dá),并初步揭示：抑制TrkB的表達(dá)可能會(huì)影響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 鹵1.63% to 36.27 鹵1.74% in young rats treated with CMECs P6 50ng/mL BDNF (P0.01), CMECs P14 50ng/mL BDNF increased from 25.95 鹵0.39% to 28.92 鹵1.46% (P0.01). However, the S phase cells increased from 30.38 鹵1.02% to 31.31 鹵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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R33

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