本文選題：內(nèi)皮祖細(xì)胞 + 神經(jīng)干細(xì)胞��； 參考：《南昌大學(xué)》2008年碩士論文

【摘要】： 目的和意義: 缺血性腦血管疾病已成為人類致死致殘的最主要的原因之一,它主要包括兩大病理損害,即局部血循環(huán)結(jié)構(gòu)的缺失和神經(jīng)元的損失。故而血供的再恢復(fù)及神經(jīng)修復(fù)是缺血性腦損傷臨床治療的兩個(gè)關(guān)鍵環(huán)節(jié)。 傳統(tǒng)觀念認(rèn)為,成年哺乳動(dòng)物中樞神經(jīng)系統(tǒng)不可能再生,如果出現(xiàn)神經(jīng)元丟失或缺損則難以修復(fù),神經(jīng)功能的重建被視為幾乎不可能。但上世紀(jì)90年代,神經(jīng)新生與神經(jīng)干細(xì)胞(neural stem cells,NSCs)的發(fā)現(xiàn)改變了腦損害后神經(jīng)不可修復(fù)的傳統(tǒng)觀念。近年亦發(fā)現(xiàn),各種腦損害如腦缺血、癲癇和腦創(chuàng)傷等能產(chǎn)生各種可溶性細(xì)胞損傷信號(hào)激活NSCs,通過神經(jīng)新生,選擇性補(bǔ)充腦皮層神經(jīng)元與神經(jīng)膠質(zhì)的丟失。有關(guān)NSCs的增殖及分化的調(diào)控機(jī)制隨之成為神經(jīng)科學(xué)的研究熱點(diǎn)�，F(xiàn)已知NSCs的增殖與分化的作用機(jī)制復(fù)雜而多樣,其中Notch信號(hào)通路是調(diào)節(jié)細(xì)胞增殖分化的一條古老的途徑,在神經(jīng)發(fā)生過程中,它決定著NSCs是選擇繼續(xù)增殖還是向神經(jīng)元分化,并與NSCs分化為神經(jīng)元與膠質(zhì)細(xì)胞的比例密切相關(guān)。 隨著血管內(nèi)皮祖細(xì)胞(endothelial progenitor cells, EPCs)參與體內(nèi)血管新生理論的建立,利用內(nèi)源性EPCs或移植EPCs促進(jìn)腦缺血區(qū)域血管重建也開始受到關(guān)注。研究發(fā)現(xiàn)缺血性腦損傷發(fā)生時(shí), EPCs可從骨髓動(dòng)員釋放到外周血循環(huán)或經(jīng)人為移植到體內(nèi),這些細(xì)胞遷移到缺血部位參與缺血組織的血管重建和損傷血管的再內(nèi)皮化過程,使缺血區(qū)域中毛細(xì)血管密度上調(diào),腦梗死面積縮小,神經(jīng)功能改善。值得重視的是,神經(jīng)發(fā)生與腦血管發(fā)生具有密切關(guān)系,血供的恢復(fù)是神經(jīng)修復(fù)的基礎(chǔ),EPCs不僅參與血供的恢復(fù),而且還可促進(jìn)內(nèi)源性NSCs遷入缺血腦組織參與修復(fù),提示EPCs移植或NSCs與EPCs共移植治療腦缺血性中風(fēng)具有良好前景。但EPCs對(duì)NSCs的具體作用及影響還不盡了解。因此我們應(yīng)用體外的共培養(yǎng)模型,研究在不同的共培養(yǎng)條件下,例如NSCs分別在SU5416及DAPT作用后與EPCs共培養(yǎng)等,觀察骨髓來源的EPCs對(duì)NSCs增殖分化的影響及對(duì)其Notch信號(hào)通路的影響,探討EPCs調(diào)控NSCs增殖分化的分子機(jī)制,為EPCs、NSCs移植或兩者聯(lián)合移植治療缺血性腦損傷等中樞神經(jīng)系統(tǒng)疾病奠定理論基礎(chǔ)。 研究?jī)?nèi)容和方法: 1.貼壁篩選法分離培養(yǎng)大鼠EPCs,形態(tài)學(xué)、免疫細(xì)胞染色法檢測(cè)VIII因子、FLK-1、CD133、CD34的表達(dá)及RT-PCR檢測(cè)eNOS、VIII因子、FLK-1、CD133、Tie-2、CD34的基因表達(dá)鑒定EPCs。 2.神經(jīng)球懸浮法培養(yǎng)小鼠NSCs,免疫細(xì)胞染色法檢測(cè)nestin的表達(dá)以鑒定NSCs。 3.建立transwell共培養(yǎng)體系,實(shí)驗(yàn)共分為5組:①EPCs與NSCs共培養(yǎng)組(EPCs+NSCs):實(shí)驗(yàn)所用EPCs為原代,NSCs為第3代,共培養(yǎng)時(shí),transwell上層皿為EPCs,下層皿為NSCs;②EPCs與經(jīng)SU5416處理后的NSCs共培養(yǎng)組[EPCs+(SU5416+NSCs)]:transwell下層皿的NSCs經(jīng)SU5416(10μM)作用6h后再與上層皿的EPCs共培養(yǎng);③EPCs與經(jīng)DAPT處理后的NSCs共培養(yǎng)組[EPCs+(DAPT+NSCs)]:transwell上層皿為EPCs,下層皿為已經(jīng)DAPT(10μM)作用6h后的NSCs;④經(jīng)DAPT處理后的NSCs對(duì)照組(DAPT+NSCs):transwell上層皿為混合培養(yǎng)培養(yǎng)基,下層皿為已經(jīng)DAPT(10μM)作用6h后的NSCs;⑤單純NSCs對(duì)照組(NSCs):transwell上層皿為混合培養(yǎng)培養(yǎng)基,下層皿為NSCs。 4.采用RT-PCR方法觀察各培養(yǎng)組共培養(yǎng)2h、6h、12h后notch信號(hào)通路相關(guān)基因notch1、Hes1、mash1以及NSCs增殖分化相關(guān)基因ki67、神經(jīng)巢蛋白(nestin)、β-微管蛋白(β-Tubulin)、神經(jīng)膠質(zhì)酸性蛋白(GFAP)的基因表達(dá)的變化。 結(jié)果: 1.EPCs呈典型的“鵝卵石”排列生長(zhǎng),并經(jīng)細(xì)胞化學(xué)染色結(jié)果顯示VIII因子、CD34、FLK-1和CD133陽性,RT-PCR檢測(cè)有eNOS、VIII因子、CD34、FLK-1、CD133和Tie-2的基因表達(dá),符合EPCs特征。 2.成球懸浮法培養(yǎng)的小鼠NSCs增殖旺盛,nestin呈陽性表達(dá)。 3.各共培養(yǎng)組NSCs增殖分化相關(guān)基因及notch信號(hào)通路相關(guān)基因的變化: (1)EPCs可促進(jìn)NSCs的ki67及nestin基因表達(dá)。(EPCs+NSCs)組與[EPCs+(SU5416+NSCs)]組比較,ki67及nestin基因表達(dá)明顯上調(diào),說明EPCs可以促進(jìn)NSCs的ki67、nestin基因表達(dá)且與EPCs分泌的內(nèi)皮生長(zhǎng)因子有關(guān)。 (2)EPCs使NSCs的β-Tubulin基因表達(dá)上調(diào),同時(shí)GFAP基因表達(dá)下降。(EPCs+NSCs)組、[EPCs+(SU5416+NSCs)]組及[EPCs+(DAPT+NSCs)]組的NSCs的向神經(jīng)元分化相關(guān)基因β-Tubulin表達(dá)均不同程度上調(diào),同時(shí)向膠質(zhì)細(xì)胞分化相關(guān)基因GFAP基因下調(diào)。其中尤以(EPCs+NSCs)明顯,說明EPCs可以促進(jìn)NSCs向神經(jīng)元方向轉(zhuǎn)化,并與Notch信號(hào)通路的調(diào)節(jié)及內(nèi)皮生長(zhǎng)因子有關(guān)。 (3)(EPCs+NSCs)組及[EPCs+(SU5416+NSCs)]組NSCs的hes1基因較其他組表達(dá)下調(diào),而mash1基因表達(dá)上調(diào)。 結(jié)論: EPCs可以促進(jìn)NSCs的增殖及其向神經(jīng)元方向的分化,這種作用與EPCs對(duì)NSCs的Notch信號(hào)通路相關(guān)基因的調(diào)節(jié)以及EPCs的內(nèi)皮生長(zhǎng)因子密切相關(guān)。
[Abstract]:Purpose and significance:
Ischemic cerebrovascular disease has become one of the most important causes of death and disability of human being. It mainly includes two major pathological damage, that is, the loss of local blood circulation structure and the loss of neurons. Therefore, the re recovery of blood supply and nerve repair are the two key links in the clinical treatment of ischemic brain injury.
Traditionally, the central nervous system of adult mammals is not possible to regenerate, and it is difficult to repair if neuron loss or defect occurs, and the reconstruction of neural function is considered almost impossible. In 90s last century, the discovery of neurogenesis and neural stem cells (neural stem cells, NSCs) changed the non repairable transmission of nerve after brain damage. In recent years, it is also found that all kinds of brain damage, such as cerebral ischemia, epilepsy and brain trauma, can produce a variety of soluble cell damage signals to activate NSCs, through neurogenesis, and selectively supplement the loss of cerebral cortex neurons and neuroglia. The regulatory mechanism related to the proliferation and differentiation of NSCs has become a hot topic in neuroscience. Now NS is known. The mechanism of proliferation and differentiation of Cs is complex and diverse, in which the Notch signaling pathway is an ancient way to regulate cell proliferation and differentiation. In the process of neurogenesis, it determines whether NSCs is selected to continue to proliferate or differentiate into neurons, and it is closely related to the proportion of NSCs into neurons and the proportion of gelatin cells.
As vascular endothelial progenitor cells (endothelial progenitor cells, EPCs) are involved in the establishment of angiogenesis theory in vivo, the use of endogenous EPCs or transplantation of EPCs to promote vascular reconstruction in ischemic regions is also concerned. The study found that when ischemic brain injury occurred, EPCs could be released from bone marrow mobilization to peripheral blood circulation or human transplantation. In vivo, these cells migrate to the ischemic site to participate in the vascular reconstruction of ischemic tissue and to damage the endothelialization of blood vessels. The capillary density is up-regulated in the ischemic region, the area of cerebral infarction is reduced, and the nerve function is improved. It is worth paying attention to that the neurogenesis is closely related to the occurrence of cerebral vasculogenesis, and the recovery of blood supply is the basis of nerve repair. EPCs not only participates in the recovery of blood supply, but also promotes the migration of endogenous NSCs into the ischemic brain tissue to participate in the repair. It is suggested that EPCs transplantation or NSCs and EPCs have good prospects for the treatment of cerebral ischemic stroke. But the specific effects and effects of EPCs on NSCs are not well understood. Therefore, we apply a co culture model in vitro to study in different ways. Under co culture conditions, such as NSCs and EPCs co culture after SU5416 and DAPT respectively, the effects of EPCs on the proliferation and differentiation of NSCs and its influence on the Notch signaling pathway were observed, and the molecular mechanism of EPCs regulating NSCs proliferation and differentiation was discussed, and EPCs, NSCs transplantation or combined transplantation for the treatment of ischemic brain injury and other central nervous systems The theoretical foundation of the disease is established.
Research contents and methods:
1. adherent wall screening method was used to isolate and culture EPCs, morphology, immune cell staining method was used to detect VIII factor, FLK-1, CD133, CD34 expression and RT-PCR detection eNOS, VIII factor, FLK-1, CD133, Tie-2, CD34 gene expression identification
2. mouse NSCs was cultured by neurosphere suspension. The expression of nestin was detected by immuno cell staining to identify NSCs..
3. the Transwell co culture system was established. The experiment was divided into 5 groups: (1) EPCs and NSCs co culture group (EPCs+NSCs): the experimental EPCs was the original generation and the NSCs was the third generation. When co culture, the upper Transwell Petri dish was EPCs, the lower layer was NSCs; (2) EPCs and SU5416 treated NSCs co culture group] EPCs co culture after the action of 6h; (3) EPCs and DAPT treated NSCs co culture group [EPCs+ (DAPT+NSCs)]: the upper layer of Transwell is EPCs, the lower layer is after DAPT (10 mu M) action 6h. 6h after NSCs; NSCs simple control group (NSCs): Transwell upper layer was mixed culture medium, and the lower layer was NSCs..
4. RT-PCR method was used to observe the gene expression of Notch1, Hes1, Mash1 and NSCs proliferation and differentiation related genes Notch1, Hes1, Mash1, and NSCs proliferation and differentiation genes, Notch1, Hes1, Mash1, and NSCs proliferation and differentiation related genes after 2h, 6h, 12h, and the gene expression of glial acidic protein (beta -Tubulin) and glial acidic protein.
Result:
1.EPCs showed a typical "cobblestone" arrangement, and the results of cytochemical staining showed that VIII, CD34, FLK-1 and CD133 were positive. RT-PCR detected the gene expression of eNOS, VIII factor, CD34, FLK-1, CD133 and Tie-2.
2. the proliferation of mouse NSCs cultured in suspension culture was strong and nestin was positive.
3. changes in NSCs proliferation and differentiation related genes and Notch signaling pathway related genes in co culture group:
(1) EPCs could promote the expression of Ki67 and nestin gene in NSCs. (EPCs+NSCs) the expression of Ki67 and nestin genes was obviously up-regulated compared with [EPCs+ (SU5416+NSCs) group, indicating that EPCs can promote NSCs Ki67, and it is related to endothelial growth factor secreted.
(2) EPCs increased the expression of NSCs's beta -Tubulin gene and decreased the expression of GFAP gene. (EPCs+NSCs), the beta -Tubulin expression of the neuron differentiation related genes in the [EPCs+ (SU5416+NSCs)] group and [EPCs+ (DAPT+NSCs)] group were up regulated in varying degrees, and the GFAP genes of the glial cell differentiation related genes were downregulated. It shows that EPCs can promote the transformation of NSCs into neurons, and is related to the regulation of Notch signaling pathway and endothelial growth factor.
(3) (EPCs+NSCs) and [EPCs+ (SU5416+NSCs)] group NSCs Hes1 gene expression was down regulated compared with other groups, while Mash1 gene expression was upregulated.
Conclusion:
EPCs can promote the proliferation of NSCs and its differentiation into the direction of neuron, which is closely related to the regulation of EPCs related genes related to the Notch signaling pathway of NSCs and the endothelial growth factor of EPCs.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

【引證文獻(xiàn)】

中國(guó)期刊全文數(shù)據(jù)庫 前1條

1 龔婷;賈素潔;張畢奎;;內(nèi)皮祖細(xì)胞生物學(xué)特性及其進(jìn)展[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2012年21期

中國(guó)碩士學(xué)位論文全文數(shù)據(jù)庫 前1條

1 龔婷;冠心病患者DDAH2啟動(dòng)子甲基化對(duì)內(nèi)皮祖細(xì)胞功能的調(diào)節(jié)及機(jī)制[D];中南大學(xué);2012年

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本文編號(hào)：1895278