【摘要】：運(yùn)動(dòng)神經(jīng)元損傷可導(dǎo)致軀體肌肉喪失神經(jīng)支配,這種疾病一直以來被認(rèn)為是無法治愈的。通過干細(xì)胞誘導(dǎo)分化成運(yùn)動(dòng)神經(jīng)元來替代受損的運(yùn)動(dòng)神經(jīng)元將是一種比較可行的治療手段。近來,已有報(bào)道胚胎干細(xì)胞可以誘導(dǎo)分化成運(yùn)動(dòng)神經(jīng)元。然而,胚胎干細(xì)胞存在免疫排斥和高成瘤等問題。理想的運(yùn)動(dòng)神經(jīng)元來源是從患者自身細(xì)胞誘導(dǎo)得來的。我們以前的研究結(jié)果表明,在損傷的條件下成體的星形膠質(zhì)細(xì)胞去分化成神經(jīng)干細(xì)胞。本文旨在探討星形膠質(zhì)細(xì)胞來源的神經(jīng)干細(xì)胞是否可以被誘導(dǎo)分化成為運(yùn)動(dòng)神經(jīng)元。 本文應(yīng)用體外限制性培養(yǎng)基選擇培養(yǎng)出生后15-20 d的大鼠脊髓星形膠質(zhì)細(xì)胞。通過免疫熒光染色、流式細(xì)胞分析、蛋白免疫印跡、細(xì)胞轉(zhuǎn)染及Time-lapse熒光追蹤等實(shí)驗(yàn)方法,研究了星形膠質(zhì)細(xì)胞在體外條件下去分化的機(jī)制。通過表皮生長(zhǎng)因子(EGF)和堿性成纖維生長(zhǎng)因子(bFGF)的神經(jīng)球分析體系,研究去分化后的星形膠質(zhì)細(xì)胞的干細(xì)胞特征。利用維甲酸(RA)和音猬因子(Shh)胞外誘導(dǎo)信號(hào)分子,誘導(dǎo)干細(xì)胞向運(yùn)動(dòng)神經(jīng)元分化。 本文結(jié)果顯示,大鼠脊髓星形膠質(zhì)細(xì)胞在體外選擇培養(yǎng)的條件下去分化表達(dá)神經(jīng)干細(xì)胞的標(biāo)記物巢蛋白(Nestin)。在EGF和bFGF存在的條件下可以長(zhǎng)出神經(jīng)球。神經(jīng)球細(xì)胞可以大量增殖并且可以傳16代以上。神經(jīng)球不僅可以自我更新(Self-renew),還可以分化成神經(jīng)元和膠質(zhì)細(xì)胞。結(jié)果表明,在體外培養(yǎng)條件下星形膠質(zhì)細(xì)胞可以去分化成為神經(jīng)干細(xì)胞。進(jìn)一步的研究發(fā)現(xiàn),這種神經(jīng)干細(xì)胞在RA和Shh信號(hào)分子誘導(dǎo)5 d后,97.7 %±0.5 %的細(xì)胞是表達(dá)Olig2的運(yùn)動(dòng)神經(jīng)元祖細(xì)胞;97.6 %±0.5 %的細(xì)胞是表達(dá)βIII-tubulin的神經(jīng)元,且Olig2和βIII-tubulin在這種誘導(dǎo)分化后的細(xì)胞均為共表達(dá)。在加入腦源性神經(jīng)營(yíng)養(yǎng)因子(BDNF)、睫狀神經(jīng)營(yíng)養(yǎng)因子(CNTF)、膠質(zhì)源性神經(jīng)營(yíng)養(yǎng)因子(GDNF)和神經(jīng)營(yíng)養(yǎng)因子-3 (NT-3)繼續(xù)分化成熟2周后,24.4 %±1.1 %的細(xì)胞是表達(dá)HB9的運(yùn)動(dòng)神經(jīng)元,這些細(xì)胞也是βIII-tubulin和ChAT陽性的。在撤除RA和Shh信號(hào)分子后,運(yùn)動(dòng)神經(jīng)元的分化比例顯著的下降。結(jié)果表明,RA和Shh可以顯著地誘導(dǎo)星形膠質(zhì)細(xì)胞去分化來源的神經(jīng)干細(xì)胞分化成為運(yùn)動(dòng)神經(jīng)元。 上述結(jié)果表明,星形膠質(zhì)細(xì)胞源的神經(jīng)干細(xì)胞可以被高效地誘導(dǎo)分化運(yùn)動(dòng)神經(jīng)元的祖細(xì)胞,進(jìn)而可以分化成運(yùn)動(dòng)神經(jīng)元。臨床通過組織活檢很容易從患者自身獲得星形膠質(zhì)細(xì)胞,結(jié)合本研究結(jié)果,提示自體星形膠質(zhì)細(xì)胞體外培養(yǎng)誘導(dǎo)分化為運(yùn)動(dòng)神經(jīng)元再移植將可能成為運(yùn)動(dòng)神經(jīng)元損傷治療的一種有效方法。
[Abstract]:Motor neuron damage can lead to loss of muscle innervation, a disease that has long been considered incurable. Differentiation of motor neurons by stem cells in place of injured motor neurons will be a feasible treatment. Recently, embryonic stem cells have been reported to differentiate into motor neurons. However, embryonic stem cells have immune rejection and Gao Cheng tumor problems. The ideal source of motor neurons is derived from the patient's own cells. Our previous studies have shown that adult astrocytes dedifferentiate into neural stem cells under damaged conditions. This paper aims to investigate whether neural stem cells derived from astrocytes can be induced to differentiate into motor neurons. The spinal cord astrocytes from 15 to 20 days after birth were cultured in a restricted medium in vitro. The mechanism of dedifferentiation of astrocytes in vitro was studied by immunofluorescence staining, flow cytometry, Western blot, cell transfection and Time-lapse fluorescence tracing. The stem cell characteristics of dedifferentiated astrocytes were studied by using the neurosphere analysis system of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Retinoic acid (RA) and Hedgehog factor (Shh) were used to induce extracellular signaling molecules to induce stem cells to differentiate into motor neurons. The results showed that rat spinal astrocytes dedifferentiated and expressed nestin (Nestin)., a marker of neural stem cells, under the condition of selective culture in vitro. In the presence of EGF and bFGF, neurospheres can be grown. Neuroglobular cells can proliferate in large numbers and can be passed through more than 16 generations. Neurospheres can not only self-renew (Self-renew), but also differentiate into neurons and glial cells. The results showed that astrocytes could dedifferentiate into neural stem cells in vitro. Further studies showed that 97.7% 鹵0.5% of the neural stem cells were motor neuron progenitor cells expressing Olig2 and 97.6% 鹵0.5% of the cells expressing Olig2 were 尾 III-tubulin expressing neurons after 5 days of induction by RA and Shh signaling molecules. Both Olig2 and 尾 III-tubulin were co-expressed in the differentiated cells. After the addition of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial derived neurotrophic factor (GDNF) and neurotrophic factor -3 (NT-3), 24.4% 鹵1.1% of the cells were motoneurons expressing HB9. These cells are also 尾 III-tubulin and ChAT positive. After the removal of RA and Shh signaling molecules, the differentiation ratio of motor neurons decreased significantly. The results showed that RA and Shh could significantly induce neural stem cells derived from dedifferentiation of astrocytes to differentiate into motor neurons. These results suggest that astrocyte-derived neural stem cells can be efficiently induced to differentiate into motor neuron progenitor cells and then to differentiate into motor neurons. It is easy to obtain astrocytes from patients themselves by clinical biopsies, combined with the results of this study. These results suggest that autologous astrocytes can be induced to differentiate into motor neurons in vitro and then transplanted into motor neurons, which may be an effective method for the treatment of motor neuron injury.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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