本文關(guān)鍵詞：Notch1信號在法舒地爾誘導(dǎo)MSCs向神經(jīng)元樣細(xì)胞分化過程中的作用　出處：《鄭州大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

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【摘要】： 背景與目的 間充質(zhì)干細(xì)胞具有較強(qiáng)的增殖、自我更新及分化的能力,可以從成熟骨髓、脂肪組織及臍帶血等多種組織分離出來。它不但可以向中胚層細(xì)胞如骨骼肌細(xì)胞、心肌細(xì)胞及脂肪細(xì)胞分化,而且還可以向外胚層細(xì)胞如神經(jīng)細(xì)胞、上皮細(xì)胞分化。骨髓間充質(zhì)干細(xì)胞的發(fā)現(xiàn),為各種神經(jīng)系統(tǒng)變性病、中樞神經(jīng)損傷及腦卒中等疾病的治療帶來新的手段,給許多神經(jīng)系統(tǒng)疾病的患者帶來新的希望,具有廣泛的臨床運(yùn)用前景。因而對骨髓間充質(zhì)干細(xì)胞生物學(xué)特性、分離培養(yǎng)、誘導(dǎo)分化、向神經(jīng)細(xì)胞分化的機(jī)制及移植治療神經(jīng)系統(tǒng)疾病等研究已經(jīng)成為神經(jīng)科學(xué)研究的重要領(lǐng)域。目前,體外誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向神經(jīng)細(xì)胞分化仍然存在分化效率低及分化機(jī)制不明確等不足。因此,骨髓間充質(zhì)干細(xì)胞向神經(jīng)細(xì)胞分化誘導(dǎo)方法和分化機(jī)制尚需進(jìn)一步研究。 Notch信號通路是生物體內(nèi)的一條重要細(xì)胞信號傳導(dǎo)通路,參與胚胎神經(jīng)系統(tǒng)的發(fā)育,調(diào)控干細(xì)胞的增殖、分化和凋亡等生物活動。研究表明骨髓間充質(zhì)干細(xì)胞向神經(jīng)細(xì)胞分化過程中Notch信號分子表達(dá)下調(diào),抑制Notch信號有利于其向神經(jīng)元的分化,而Hes1蛋白在這過程中的作用仍未十分明確。近年研究發(fā)現(xiàn),CoCl2聯(lián)合Rho激酶抑制劑可以誘導(dǎo)骨髓間充質(zhì)干細(xì)胞分化為神經(jīng)細(xì)胞,這提示我們RhoA/Rho激酶信號通路可能參與調(diào)控骨髓間充質(zhì)干細(xì)胞分化為神經(jīng)元樣細(xì)胞過程。 因此,本研究旨在探討法舒地爾在骨髓間充質(zhì)干細(xì)胞分化為神經(jīng)元樣細(xì)胞的作用,并深入探討Notch信號抑制后是否有利于促進(jìn)骨髓間充質(zhì)干細(xì)胞向神經(jīng)元樣細(xì)胞分化。這對于深入研究骨髓間充質(zhì)干細(xì)胞向神經(jīng)元樣細(xì)胞分化機(jī)制,將來臨床應(yīng)用骨髓間充質(zhì)干細(xì)胞移植聯(lián)合法舒地爾治療相關(guān)神經(jīng)系統(tǒng)疾病提供依據(jù)等具有重要的意義。 材料和方法 以Wistar大鼠為實(shí)驗(yàn)動物,鼠齡2-3月,體重150-200g,雌雄不限,以10代以上的MSCs傳代培養(yǎng),進(jìn)行細(xì)胞實(shí)驗(yàn)。實(shí)驗(yàn)將培養(yǎng)的MSCs分為未轉(zhuǎn)染組、轉(zhuǎn)染組(轉(zhuǎn)染Rn-Notch1-siRNA)、陽性對照組(轉(zhuǎn)染Rn-MAPK-1 Control siRNA)及陰性對照組(轉(zhuǎn)染Negative Control siRNA)等4組。采用鹽酸法舒地爾誘導(dǎo)大鼠MSCs分化為神經(jīng)細(xì)胞。倒置熒光顯微鏡下觀察MSCs轉(zhuǎn)染后熒光表達(dá)情況；RT-PCR檢測Notch1、Hes1和MAPK1 mRNA的表達(dá)變化；免疫細(xì)胞化學(xué)法檢測Notch1、神經(jīng)元巢蛋白(Nestin)、神經(jīng)微絲蛋白亞單位(NF-M)和膠質(zhì)纖維酸性蛋白(GFAP)的表達(dá)變化；MTT方法檢測細(xì)胞存活率。 結(jié)果 1. siRNA轉(zhuǎn)染72h,MSCs熒光表達(dá)最強(qiáng),轉(zhuǎn)染率可達(dá)0.914±0.04；同時(shí),轉(zhuǎn)染組MSCs的Notch1和Hes1 mRNA轉(zhuǎn)錄下降(P0.05)；MTT提示轉(zhuǎn)染組細(xì)胞存活率也顯著減少(P0.05),與對照組差異有統(tǒng)計(jì)學(xué)意義。 2.鹽酸法舒地爾可以誘導(dǎo)MSCs向神經(jīng)細(xì)胞分化；其中以轉(zhuǎn)染組誘導(dǎo)效果最佳,Nestin、NF-M的表達(dá)率顯著的高于其它各組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論 1.鹽酸法舒地爾可以誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向神經(jīng)元樣細(xì)胞分化。 2.抑制Notch信號,有利于鹽酸法舒地爾高效誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向神經(jīng)元樣細(xì)胞分化, 3.細(xì)胞內(nèi)可能存在Notch信號通路與RhoA/Rho激酶信號通路協(xié)同促進(jìn)骨髓間 充質(zhì)干細(xì)胞向神經(jīng)元樣細(xì)胞分化。
[Abstract]:Background and purpose
Mesenchymal stem cells have strong proliferation ability of self-renewal and differentiation, from mature bone marrow tissues, adipose tissue and cord blood separated. It can not only to mesodermal cells such as skeletal muscle cells, myocardial cells and adipocyte differentiation, but also to ectoderm cells such as nerve cells, epithelial cells differentiation of bone marrow mesenchymal stem cells, for a variety of neurodegenerative disease, bring new means of treatment of CNS injury and stroke, to many nervous system disease patients new hope, has broad clinical application prospect. The biological characteristics of bone marrow mesenchymal stem cells separation. Cultivation, differentiation, to the mechanisms of neural differentiation and transplantation for the treatment of diseases of the nervous system research has become an important field of neuroscience research. At present, bone marrow mesenchymal induction in vitro The differentiation of stem cells into neural cells still has the disadvantages of low differentiation efficiency and unclear differentiation mechanism. Therefore, the differentiation and induction mechanism of bone marrow mesenchymal stem cells into neural cells need further study.
Notch signal pathway is an important signal transduction pathway in living organisms, involved in embryonic development of the nervous system, the regulation of stem cell proliferation, differentiation and apoptosis. The results show that the biological activity of bone marrow mesenchymal stem cells to form Notch signaling molecules during neural differentiation were reduced, inhibition of the Notch signal is conducive to neuron the role of Hes1 protein and differentiation, not in this process is very clear. Recent studies have found that CoCl2 and Rho kinase inhibitors can induce differentiation of bone marrow mesenchymal stem cells into neural cells. This suggests that RhoA/Rho kinase signaling pathway may be involved in the regulation of bone marrow mesenchymal stem cells to differentiate into neuron like cells.
Therefore, the purpose of this study was to investigate the effects of fasudil on differentiation of bone marrow mesenchymal stem cells into neuron like cells, and explore whether inhibition of Notch signaling is conducive to the promotion of bone marrow mesenchymal stem cells to differentiate into neuron like cells. In the study of bone marrow mesenchymal stem cells into neuron like cell differentiation mechanism, the future clinical application of bone marrow mesenchymal stem cell transplantation and fasudil plays an important role in the treatment of diseases of the nervous system to provide related evidence.
Materials and methods
The Wistar rats were used as experimental animal, rats aged 2-3 months, weight 150-200g, male or female, with more than 10 generations of MSCs cells were subcultured. The experiment cultured MSCs were divided into non transfection group, transfection group (transfected with Rn-Notch1-siRNA), positive control group (transfected with Rn-MAPK-1 Control siRNA) and negative control group (Negative Control siRNA 4 transfection) group. Rat MSCs induced differentiation into neural cells fasudil hydrochloride. Inverted fluorescence microscope MSCs after transfection, the expression of fluorescence detection; RT-PCR Notch1, the expression of Hes1 and MAPK1 mRNA detection; Notch1 immunocytochemistry, nestin neurons (Nestin), neurofilament protein unit (NF-M) and glial fibrillary acidic protein (GFAP) expression; MTT method to detect cell survival rate.
Result
1. siRNA transfected 72h, MSCs showed the strongest fluorescence expression, and the transfection rate reached 0.914 + 0.04. Meanwhile, the transcription of Notch1 and Hes1 mRNA decreased (P0.05) in the transfection group, and MTT indicated that the cell survival rate of transfection group was also significantly reduced (P0.05), and the difference between the transfection group and the control group was statistically significant (MSCs).
2. fasudil hydrochloride can induce MSCs to differentiate into neural cells; the transfection group was the best, Nestin, the expression rate of NF-M was significantly higher than other groups, the difference was statistically significant (P0.05).
conclusion
1. fasudil hydrochloride can induce bone marrow mesenchymal stem cells to differentiate into neuron like cells.
2. inhibition of the Notch signal, is conducive to the efficiency of fasudil hydrochloride induced bone marrow mesenchymal stem cells to differentiate into neuron like cells,
3. cells may have Notch signaling pathway and RhoA/Rho kinase signaling pathway to promote bone marrow
The mesenchymal stem cells differentiate into neuron like cells.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 王明闖;賈延R，

本文編號：1438283