【摘要】： O-2A祖細(xì)胞是雙潛能干細(xì)胞,既能分化為少突膠質(zhì)細(xì)胞(oligodendrocyte,OL),又能分化為2型星形膠質(zhì)細(xì)胞。從O-2A祖細(xì)胞發(fā)育到成熟的OL這個(gè)過程中往往伴隨著一些抗原的消失和出現(xiàn)。本文研究在OL發(fā)育過程中S-100β表達(dá)的變化。S-100β屬于S100家族的一個(gè)鈣離子結(jié)合蛋白,它廣泛的分布在脊椎動(dòng)物的神經(jīng)系統(tǒng),沉積在哺乳動(dòng)物的腦中S-100β,參與細(xì)胞類型的分化。本研究在分離、純化和體外培養(yǎng)O-2A祖細(xì)胞的基礎(chǔ)上,檢測(cè)S-100β在O-2A祖細(xì)胞分化至成熟OL的過程中S-100β表達(dá)的變化,探討S-100β是否在OL發(fā)生與分化過程中具有調(diào)控作用。腦室周圍白質(zhì)的軟化(Periventricular leukomalacia,PVL)主要是發(fā)生在早產(chǎn)兒的一種白質(zhì)的損傷,發(fā)育中的少突膠質(zhì)譜系細(xì)胞的損傷是PVL的一個(gè)標(biāo)志性特征。據(jù)臨床和動(dòng)物實(shí)驗(yàn)報(bào)道促炎癥因子TNF-α在PVL中發(fā)揮作用,因此研究其對(duì)發(fā)育中少突膠質(zhì)譜系細(xì)胞的作用非常重要。 第一部分少突膠質(zhì)譜系細(xì)胞發(fā)育過程中S-100β的表達(dá) 目的探討新生大鼠大腦皮質(zhì)來源的少突膠質(zhì)譜系細(xì)胞中S-100β是否表達(dá),如若表達(dá)始于少突膠質(zhì)譜系細(xì)胞發(fā)育過程中的何種階段。 方法取新生大鼠大腦皮質(zhì)進(jìn)行混合膠質(zhì)細(xì)胞的原代培養(yǎng),根據(jù)各種膠質(zhì)細(xì)胞黏附性及生長(zhǎng)時(shí)間的差異,通過機(jī)械振蕩法和差速貼壁法獲得純化的O-2A祖細(xì)胞,并結(jié)合使用生長(zhǎng)因子獲得一系列的少突膠質(zhì)譜系細(xì)胞,且通過細(xì)胞特異性抗體進(jìn)行鑒定。采用免疫熒光雙重標(biāo)記方法,檢測(cè)S-100β在少突膠質(zhì)譜系細(xì)胞中的表達(dá)情況。 結(jié)果純化的O-2A祖細(xì)胞在含有bFGF和PDGF的培養(yǎng)液中培養(yǎng)48h,不表達(dá)S-100β。換成含3′碘甲狀腺原氨酸(triiodothyronine,T3)培養(yǎng)液繼續(xù)培養(yǎng)24 h,開始表達(dá)S-100β。原少突膠質(zhì)細(xì)胞和成熟的OL中S-100β免疫反應(yīng)呈現(xiàn)陽性。 結(jié)論S-100β表達(dá)始于O-2A祖細(xì)胞分化為原少突膠質(zhì)細(xì)胞的階段,可能與O-2A祖細(xì)胞從雙潛能階段分化成OL過程中的形態(tài)學(xué)變化有關(guān)。 第二部分少突膠質(zhì)譜系細(xì)胞易損性的研究 目的研究TNF-α對(duì)少突膠質(zhì)譜系細(xì)胞的三個(gè)不同發(fā)育階段的易損性。 方法(1)5 ng/ml、50 ng/ml、500 ng/ml TNF-α分別刺激O-2A祖細(xì)、原少突膠質(zhì)細(xì)胞、未成熟的OL48 h,同時(shí)設(shè)立對(duì)照組(未加TNF-α處理)。利用MTT法以酶聯(lián)免疫檢測(cè)儀測(cè)定各孔光吸收值(OD),計(jì)算細(xì)胞的存活率。存活率((?)±s)%=實(shí)驗(yàn)組細(xì)胞OD/對(duì)照組細(xì)胞OD×100%。(2)50 ng/ml TNF-α刺激O-2A祖細(xì)、原少突膠質(zhì)細(xì)胞、未成熟的OL12 h、24 h、48 h,以未加TNF-α處理的為對(duì)照組,利用MTT法計(jì)算細(xì)胞的存活率。存活率((?)±s)%=實(shí)驗(yàn)組細(xì)胞OD/對(duì)照組細(xì)胞OD×100%。(3)50 ng/ml TNF-α刺激O-2A祖細(xì)、原少突膠質(zhì)細(xì)胞、未成熟的OL48 h,caspase-3的免疫細(xì)胞化學(xué)染色檢測(cè)細(xì)胞的凋亡。 結(jié)果(1)TNF-α降低細(xì)胞的存活率具有劑量依賴性和時(shí)間依賴性,隨著TNF-α劑量的增加和刺激時(shí)間的延長(zhǎng)O-2A祖細(xì)胞、原少突膠質(zhì)細(xì)胞、未成熟OL的存活率降低。(2)隨著TNF-α劑量的增加,O-2A祖細(xì)胞與未成熟OL相比較細(xì)胞存活率明顯降低(P＜0.01),原少突膠質(zhì)細(xì)胞與未成熟OL相比較細(xì)胞存活率也是降低的(P＜0.05),O-2A祖細(xì)胞存活率與原少突膠質(zhì)細(xì)胞存活率相比較差別沒有統(tǒng)計(jì)學(xué)意義;50 ng/ml TNF-α刺激少突膠質(zhì)譜系細(xì)胞48 h以內(nèi),0.2A祖細(xì)胞的存活率與未成熟OL存活率相比較差別沒有統(tǒng)計(jì)學(xué)意義,O-2A祖細(xì)胞的存活率與原少突膠質(zhì)細(xì)胞的存活率相比較也不存在差別,而原少突膠質(zhì)細(xì)胞的存活率與未成熟OL存活率相比較細(xì)胞的存活率明顯降低(P＜0.05)。(3)50 ng/ml TNF-α分別刺激O-2A祖細(xì)胞、原少突膠質(zhì)細(xì)胞、未成熟OL48 h,caspase-3免疫細(xì)胞化學(xué)染色顯示在這些細(xì)胞的胞漿和胞核有大量的特異性棕黃色的著色而對(duì)照組這三個(gè)發(fā)育階段的細(xì)胞未見陽性表達(dá)。 結(jié)論TNF-α降低少突膠質(zhì)譜系細(xì)胞的存活率具有細(xì)胞成熟程度的依賴性;caspase-3參與TNF-α誘導(dǎo)的O-2A祖細(xì)胞、原少突膠質(zhì)細(xì)胞及未成熟OL的凋亡。
[Abstract]:The O-2A progenitor cells are potent stem cells that can differentiate into oligodendrocyte (OL) and differentiate into 2 astrocytes. It is often accompanied by the disappearance and appearance of some antigens from the development of O-2A progenitor cells to mature OL. In this paper, the changes of S-100 gene expression during the development of OL were studied. S-100 is a calcium ion binding protein belonging to the S100 family, which is widely distributed in the nervous system of vertebrates, deposited in the brain of a mammal, S-100 kcal, participating in the differentiation of cell types. On the basis of isolation, purification and in vitro culture of O-2A progenitors, this study examined the changes of S-100 gene expression in the process of differentiation of O-2A progenitors into mature OL, and discussed the regulation of S-100 CCR5 in the development and differentiation of OL-2A progenitor cells. Periventricular leukomalacia (PVL) is mainly a white matter damage occurring in premature infants, and the damage of oligodendrocyte in development is a characteristic feature of PVL. According to clinical and animal experiments, we reported the role of pro-inflammatory factor TNF-7721 in PVL, and therefore it was very important to study its role in the development of oligodendrocyte. S-100 in the development of oligodendrocytes in the first part Objective To investigate the expression of S-100 gene in oligodendrocyte of newborn rat cerebral cortex, if the expression begins with oligodendrocyte development. Methods: The primary culture of mixed glial cells in the cerebral cortex of neonatal rats was carried out by means of mechanical oscillation method and differential attachment method. The purified O-2A progenitor cells are obtained and a series of oligodendrocyte lineage cells are obtained using growth factors, and by Cell-specific antibodies were identified. Immunofluorescence double labeling was used to detect S-100 in oligodendrocytes. Expression of purified O-2A progenitor cells in a culture medium containing bFGF and PDGF It was cultured for 48h without expression of S-100 kcal. It was replaced with triiodothyronine (T3). 24 h, starting to express S-100 kcal. oligodendrocytes and mature OL Conclusion The expression of S-100 may begin with the differentiation of O-2A progenitor cells into oligodendrocytes, which may be related to the potential of O-2A progenitors from double potential. Morphological Changes in the Process of Energy Differentiation into OL A study on the study of the cytoskeleton of oligodendrocytes in the second part Methods (1) 5ng/ ml, 50ng/ ml, 500ng/ ml TNF-gal stimulated O-2A progenitor cells and oligodendrocytes, respectively. Cells, immature OL48 h, and control group (not treated with TNF-MAA). Use MT T-method is free from enzyme-linked The light absorption value (OD) of each hole was measured by the disease detector and the survival rate of cells was calculated. The survival rate ((?)% s)% = OD of the OD/ control group of the experimental group was 100%. (2) 50 ng/ ml of TNF-MAA stimulated the O-2A progenitor thin, oligodendrocyte, immature OL12h, 24h, 48h in the absence of TN The survival rate of cells was calculated by MTT method. The survival rate ((?)% s)% = OD of the OD/ control group of the experimental group was 100%. (3) 50 ng/ ml of TNF-MAA stimulated the O-2A progenitor cell thin, oligodendrocyte and immature. The results showed that (1) TNF-7721 decreased the survival rate of cells with dose-dependent and time-dependent, and increased with the increase of TNF-tau dose. At the same time, the survival rate of O-2A progenitor cells and immature L-cells decreased with the increase of the dose of TNF-7721, and the survival rate of O-2A progenitor cells compared with the immature OL decreased significantly (P <0.01). Compared with the immature OL, the survival rate was also decreased (P <0.05). The survival rate of O-2A progenitor cells was significantly different from that of oligodendrocyte. The survival rate of O-2A progenitor cells was not statistically significant compared with the survival rate of oligodendrocytes, but the survival rate of O-2A progenitors did not differ from the survival rate of oligodendrocytes. The survival rate of the cells was significantly lower than that of the immature OL (P <0.05). (3) 50 ng/ ml of TNF-7721 stimulated O-2A progenitors, oligodendrocytes, immature OL48 h, caspase-3 immune cells chemically stained the cytoplasm of these cells. There was a large number of specific brown-yellow coloration in the nucleus, and no positive expression was found in the three developmental stages of the control group. Conclusion TNF-7721 reduced the survival rate of oligodendrocytes with the dependence on the degree of cell maturation; casp
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

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相關(guān)期刊論文 前1條

1 李春鵬;張曄;夏春林;沈慧;張靜;陸志方;;巢蛋白和階段特異性胚胎抗原-1在大鼠2型星形膠質(zhì)細(xì)胞中的表達(dá)[J];解剖學(xué)報(bào);2007年02期

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本文編號(hào)：2283201